Class 1 Oxidoreductases XII: EC 1.14.15 - 1.97 (Springer Handbook of Enzymes) [2nd ed.] 354026583X, 9783540265832

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Dietmar Schomburg and Ida Schomburg (Eds.)


      Volume 27 Class 1  Oxidoreductases XII EC 1.14.15±1.97 coedited by Antje Chang

Second Edition

13

Professor Dietmar Schomburg e-mail: [email protected] Dr. Ida Schomburg e-mail: [email protected]

University to Cologne Institute for Biochemistry Zülpicher Strasse 47 50674 Cologne Germany

Dr. Antje Chang e-mail: [email protected]

Library of Congress Control Number: 2005928338

ISBN-10 3-540-26583-X

2nd Edition Springer Berlin Heidelberg New York

ISBN-13 978-3-540-26583-2

2nd Edition Springer Berlin Heidelberg New York

The first edition was published as Volume 10 (ISBN 3-540-59494-9) of the ªEnzyme Handbookº.

This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilm or in any other way, and storage in data banks. Duplication of this publication or parts thereof is permitted only under the provisions of the German Copyright Law of September 9, 1965, in its current version, and permission for use must always be obtained from Springer. Violations are liable to prosecution under the German Copyright Law. Springer is a part of Springer Science+Business Media springeronline.com # Springer-Verlag Berlin Heidelberg 2006 Printed in Germany The use of general descriptive names, registered names, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and free for general use. The publisher cannot assume any legal responsibility for given data, especially as far as directions for the use and the handling of chemicals and biological material are concerned. This information can be obtained from the instructions on safe laboratory practice and from the manufacturers of chemicals and laboratory equipment. Cover design: Erich Kirchner, Heidelberg Typesetting: medionet AG, Berlin Printed on acid-free paper 2/3141m-5 4 3 2 1 0

      
     

Information on this handbook can be found on the internet at http://www.springeronline.com choosing ªChemistryº and then ªReference Worksº. A complete list of all enzyme entries either as an alphabetical Name Index or as the EC-Number Index is available at the above mentioned URL. You can download and print them free of charge. A complete list of all synonyms (> 25,000 entries) used for the enzymes is available in print form (ISBN 3-540-41830-X).

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Today, as the full information about the genome is becoming available for a rapidly increasing number of organisms and transcriptome and proteome analyses are beginning to provide us with a much wider image of protein regulation and function, it is obvious that there are limitations to our ability to access functional data for the gene products ± the proteins and, in particular, for enzymes. Those data are inherently very difficult to collect, interpret and standardize as they are widely distributed among journals from different fields and are often subject to experimental conditions. Nevertheless a systematic collection is essential for our interpretation of genome information and more so for applications of this knowledge in the fields of medicine, agriculture, etc. Progress on enzyme immobilisation, enzyme production, enzyme inhibition, coenzyme regeneration and enzyme engineering has opened up fascinating new fields for the potential application of enzymes in a wide range of different areas. The development of the enzyme data information system BRENDAwas started in 1987 at the German National Research Centre for Biotechnology in Braunschweig (GBF) and is now continuing at the University at Cologne, Institute of Biochemistry. The present book ªSpringer Handbook of Enzymesº represents the printed version of this data bank. The information system has been developed into a full metabolic database. The enzymes in this Handbook are arranged according to the Enzyme Commission list of enzymes. Some 3,700 ªdifferentº enzymes are covered. Frequently enzymes with very different properties are included under the same EC-number. Although we intend to give a representative overview on the characteristics and variability of each enzyme, the Handbook is not a compendium. The reader will have to go to the primary literature for more detailed information. Naturally it is not possible to cover all the numerous literature references for each enzyme (for some enzymes up to 40,000) if the data representation is to be concise as is intended. It should be mentioned here that the data have been extracted from the literature and critically evaluated by qualified scientists. On the other hand, the original authors' nomenclature for enzyme forms and subunits is retained. In order to keep the tables concise, redundant information is avoided as far as possible (e.g. if Km values are measured in the presence of an obvious cosubstrate, only the name of the cosubstrate is given in parentheses as a commentary without reference to its specific role). The authors are grateful to the following biologists and chemists for invaluable help in the compilation of data: Cornelia Munaretto and Dr. Antje Chang. Cologne October 2005

Dietmar Schomburg, Ida Schomburg

VII

!  "  

A Ac ADP Ala All Alt AMP Ara Arg Asn Asp ATP Bicine C cal CDP CDTA CMP CoA CTP Cys d dDFP DNA DPN DTNB DTT EC E. coli EDTA EGTA ER Et EXAFS FAD FMN Fru Fuc G Gal

adenine acetyl adenosine 5'-diphosphate alanine allose altrose adenosine 5'-monophosphate arabinose arginine asparagine aspartic acid adenosine 5'-triphosphate N,N'-bis(2-hydroxyethyl)glycine cytosine calorie cytidine 5'-diphosphate trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetic acid cytidine 5'-monophosphate coenzyme A cytidine 5'-triphosphate cysteine deoxy(and l-) prefixes indicating configuration diisopropyl fluorophosphate deoxyribonucleic acid diphosphopyridinium nucleotide (now NAD+ ) 5,5'-dithiobis(2-nitrobenzoate) dithiothreitol (i.e. Cleland's reagent) number of enzyme in Enzyme Commission's system Escherichia coli ethylene diaminetetraacetate ethylene glycol bis(-aminoethyl ether) tetraacetate endoplasmic reticulum ethyl extended X-ray absorption fine structure flavin-adenine dinucleotide flavin mononucleotide (riboflavin 5'-monophosphate) fructose fucose guanine galactose

IX

List of Abbreviations

GDP Glc GlcN GlcNAc Gln Glu Gly GMP GSH GSSG GTP Gul h H4 HEPES His HPLC Hyl Hyp IAA Ig Ile Ido IDP IMP ITP Km lLeu Lys Lyx M mM mMan MES Met min MOPS Mur MW NAD+ NADH NADP+ NADPH NAD(P)H NBS

X

guanosine 5'-diphosphate glucose glucosamine N-acetylglucosamine glutamine glutamic acid glycine guanosine 5'-monophosphate glutathione oxidized glutathione guanosine 5'-triphosphate gulose hour tetrahydro 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid histidine high performance liquid chromatography hydroxylysine hydroxyproline iodoacetamide immunoglobulin isoleucine idose inosine 5'-diphosphate inosine 5'-monophosphate inosine 5'-triphosphate Michaelis constant (and d-) prefixes indicating configuration leucine lysine lyxose mol/l millimol/l metamannose 2-(N-morpholino)ethane sulfonate methionine minute 3-(N-morpholino)propane sulfonate muramic acid molecular weight nicotinamide-adenine dinucleotide reduced NAD NAD phosphate reduced NADP indicates either NADH or NADPH N-bromosuccinimide

List of Abbreviations

NDP NEM Neu NMN NMP NTP oOrn pPBS PCMB PEP pH Ph Phe PHMB PIXE PMSF p-NPP Pro Q10 Rha Rib RNA mRNA rRNA tRNA Sar SDS-PAGE Ser T tH Tal TDP TEA Thr TLCK Tm TMP TosTPN Tris Trp TTP Tyr U U/mg

nucleoside 5'-diphosphate N-ethylmaleimide neuraminic acid nicotinamide mononucleotide nucleoside 5'-monophosphate nucleoside 5'-triphosphate orthoornithine paraphosphate-buffered saline p-chloromercuribenzoate phosphoenolpyruvate -log10[H+ ] phenyl phenylalanine p-hydroxymercuribenzoate proton-induced X-ray emission phenylmethane-sulfonylfluoride p-nitrophenyl phosphate proline factor for the change in reaction rate for a 10  C temperature increase rhamnose ribose ribonucleic acid messenger RNA ribosomal RNA transfer RNA N-methylglycine (sarcosine) sodium dodecyl sulfate polyacrylamide gel electrophoresis serine thymine time for half-completion of reaction talose thymidine 5'-diphosphate triethanolamine threonine Na-p-tosyl-l-lysine chloromethyl ketone melting temperature thymidine 5'-monophosphate tosyl-(p-toluenesulfonyl-) triphosphopyridinium nucleotide (now NADP+ ) tris(hydroxymethyl)-aminomethane tryptophan thymidine 5'-triphosphate tyrosine uridine mmol/(mg*min)

XI

List of Abbreviations

UDP UMP UTP Val Xaa XAS Xyl

XII

uridine 5'-diphosphate uridine 5'-monophosphate uridine 5'-triphosphate valine symbol for an amino acid of unknown constitution in peptide formula X-ray absorption spectroscopy xylose

!  #  $  

Since its foundation in 1956 the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB) has continually revised and updated the list of enzymes. Entries for new enzymes have been added, others have been deleted completely, or transferred to another EC number in the original class or to different EC classes, catalyzing other types of chemical reactions. The old numbers have not been allotted to new enzymes; instead the place has been left vacant or cross-references given to the changes in nomenclature. Deleted and Transferred Enzymes For EC class 1.14.15±1.97 these changes are: Recommended name

Old EC number Alteration

4-coumarate 3-monooxygenase

1.14.17.2

3-hydroxybenzoate 4-monooxygenase methylsterol monooxygenase glyceryl-ether monooxygenase CMP-N-acetylneuraminate monooxygenase linoleoyl-CoA desaturase stearoyl-CoA desaturase acyl-[acyl-carrier-protein] desaturase arene monooxygenase (epoxidizing) hydrogenase superoxide reductase hydrogenase arsenate reductase (glutaredoxin) arsenate reductase (donor) methylarsonate reductase

1.14.99.13

deleted, included in EC 1.14.18.1 transferred to EC 1.14.13.23

1.14.99.16 1.14.99.17 1.14.99.18

transferred to EC 1.14.13.72 transferred to EC 1.14.16.5 deleted

1.14.99.25 1.14.99.5 1.14.99.6

transferred to EC 1.14.19.3 transferred to EC 1.14.19.1 transferred to EC 1.14.19.2

1.14.99.8

deleted, included in EC 1.14.14.1 transferred to EC 1.18.99.1 transferred to EC 1.15.1.2 transferred to EC 1.12.7.2 transferred to EC 1.20.4.1 transferred to EC1.20.99.1 transferred to EC 1.20.4.2

1.18.3.1 1.18.96.1 1.18.99.1 1.97.1.5 1.97.1.6 1.97.1.7

XIII

% &  '    ( 

EC-No.

Recommended Name

1.16.1.8 1.14.99.6 1.14.19.2 1.14.15.3 1.14.17.4 1.14.99.12 1.14.16.3 1.16.1.3 1.16.1.5 1.14.99.8 1.20.98.1 1.97.1.6 1.20.99.1 1.97.1.5 1.20.4.1 1.14.21.3 1.21.4.4 1.14.15.2 1.14.15.1 1.14.21.5 1.14.99.36 1.14.99.30 1.17.1.1 1.14.21.2 1.97.1.1 1.14.15.6 1.14.15.7 1.14.99.18 1.16.1.4 1.21.3.2 1.14.15.5 1.14.17.2 1.17.99.1 1.16.1.6 1.21.99.1 1.14.20.1 1.14.99.33 1.14.99.29 1.16.1.2 1.14.17.1 1.14.99.22 1.14.99.11 1.17.99.2 1.18.1.3 1.18.1.2

[methionine synthase] reductase . . . . . . . . . . . . . . . . . acyl-[acyl-carrier-protein] desaturase (transferred to EC 1.14.19.2) . . acyl-[acyl-carrier-protein] desaturase . . . . . . . . . . . . . . . alkane 1-monooxygenase . . . . . . . . . . . . . . . . . . . . aminocyclopropanecarboxylate oxidase . . . . . . . . . . . . . . androst-4-ene-3,17-dione monooxygenase . . . . . . . . . . . . . anthranilate 3-monooxygenase . . . . . . . . . . . . . . . . . . aquacobalamin reductase . . . . . . . . . . . . . . . . . . . . aquacobalamin reductase (NADPH) . . . . . . . . . . . . . . . arene monooxygenase (epoxidizing) (deleted, included in EC 1.14.14.1) arsenate reductase (azurin) . . . . . . . . . . . . . . . . . . . arsenate reductase (donor) (transferred to EC1.20.99.1) . . . . . . . arsenate reductase (donor) . . . . . . . . . . . . . . . . . . . arsenate reductase (glutaredoxin) (transferred to EC 1.20.4.1). . . . . arsenate reductase (glutaredoxin). . . . . . . . . . . . . . . . . berbamunine synthase . . . . . . . . . . . . . . . . . . . . . betaine reductase. . . . . . . . . . . . . . . . . . . . . . . . camphor 1,2-monooxygenase . . . . . . . . . . . . . . . . . . camphor 5-monooxygenase . . . . . . . . . . . . . . . . . . . (S)-canadine synthase . . . . . . . . . . . . . . . . . . . . . b-carotene 15,15'-monooxygenase . . . . . . . . . . . . . . . . carotene 7,8-desaturase . . . . . . . . . . . . . . . . . . . . . CDP-4-dehydro-6-deoxyglucose reductase . . . . . . . . . . . . . (S)-cheilanthifoline synthase . . . . . . . . . . . . . . . . . . . chlorate reductase . . . . . . . . . . . . . . . . . . . . . . . cholesterol monooxygenase (side-chain-cleaving). . . . . . . . . . choline monooxygenase . . . . . . . . . . . . . . . . . . . . . CMP-N-acetylneuraminate monooxygenase . . . . . . . . . . . . cob(II)alamin reductase . . . . . . . . . . . . . . . . . . . . . columbamine oxidase . . . . . . . . . . . . . . . . . . . . . . corticosterone 18-monooxygenase . . . . . . . . . . . . . . . . 4-coumarate 3-monooxygenase (deleted, included in EC 1.14.18.1) . . . 4-cresol dehydrogenase (hydroxylating) . . . . . . . . . . . . . . cyanocobalamin reductase (cyanide-eliminating) . . . . . . . . . . b-cyclopiazonate dehydrogenase . . . . . . . . . . . . . . . . . deacetoxycephalosporin-C synthase. . . . . . . . . . . . . . . . D12 -fatty acid dehydrogenase . . . . . . . . . . . . . . . . . . deoxyhypusine monooxygenase . . . . . . . . . . . . . . . . . diferric-transferrin reductase . . . . . . . . . . . . . . . . . . dopamine b-monooxygenase. . . . . . . . . . . . . . . . . . . ecdysone 20-monooxygenase . . . . . . . . . . . . . . . . . . estradiol 6b-monooxygenase. . . . . . . . . . . . . . . . . . . ethylbenzene hydroxylase . . . . . . . . . . . . . . . . . . . . ferredoxin-NAD+ reductase . . . . . . . . . . . . . . . . . . . ferredoxin-NADP+ reductase. . . . . . . . . . . . . . . . . . .

Page 463 279 208 16 154 310 95 444 451 289 598 659 601 658 594 237 633 9 1 243 388 375 481 235 638 44 56 329 449 611 41 139 527 458 635 223 382 370 441 126 349 308 535 559 543

XV

Index of Recommended Enzyme Names

1.16.1.7 1.16.3.1 1.97.1.4 1.14.16.5 1.14.99.17 1.21.4.2 1.14.99.3 1.18.99.1 1.18.3.1 1.17.4.3 1.17.1.2 1.14.99.23 1.14.99.13 1.14.99.26 1.21.3.1 1.14.99.27 1.14.99.2 1.14.99.21 1.17.1.3 1.14.99.28 1.14.19.3 1.14.99.25 1.14.16.6 1.16.1.1 1.14.99.15 1.97.1.7 1.20.4.2 1.14.99.16 1.14.18.1 1.14.99.34 1.14.99.31 1.14.99.32 1.18.6.1 1.19.6.1 1.14.17.3 1.14.16.1 1.20.1.1 1.14.99.20 1.14.99.19 1.14.99.14 1.14.99.4 1.21.4.1 1.14.99.1 1.17.3.1 1.97.1.2 1.21.3.3 1.17.4.1 1.17.4.2 1.18.1.4 1.18.1.1 1.14.21.4 1.21.4.3 1.14.99.7

XVI

ferric-chelate reductase . . . . . . . . . . . . . . . . . . . ferroxidase . . . . . . . . . . . . . . . . . . . . . . . . . formate acetyltransferase activating enzyme . . . . . . . . . . glyceryl-ether monooxygenase . . . . . . . . . . . . . . . . glyceryl-ether monooxygenase (transferred to EC 1.14.16.5) . . . . glycine reductase . . . . . . . . . . . . . . . . . . . . . . heme oxygenase (decyclizing) . . . . . . . . . . . . . . . . hydrogenase (transferred to EC 1.12.7.2) . . . . . . . . . . . . hydrogenase (transferred to EC 1.18.99.1). . . . . . . . . . . . 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase . . . . . 4-hydroxy-3-methylbut-2-enyl diphosphate reductase . . . . . . 3-hydroxybenzoate 2-monooxygenase . . . . . . . . . . . . . 3-hydroxybenzoate 4-monooxygenase (transferred to EC 1.14.13.23) 2-hydroxypyridine 5-monooxygenase . . . . . . . . . . . . . isopenicillin-N synthase . . . . . . . . . . . . . . . . . . . juglone 3-monooxygenase . . . . . . . . . . . . . . . . . . kynurenine 7,8-hydroxylase . . . . . . . . . . . . . . . . . Latia-luciferin monooxygenase (demethylating) . . . . . . . . . leucoanthocyanidin reductase . . . . . . . . . . . . . . . . linalool 8-monooxygenase . . . . . . . . . . . . . . . . . . linoleoyl-CoA desaturase. . . . . . . . . . . . . . . . . . . linoleoyl-CoA desaturase (transferred to EC 1.14.19.3) . . . . . . mandelate 4-monooxygenase . . . . . . . . . . . . . . . . . mercury(II) reductase . . . . . . . . . . . . . . . . . . . . 4-methoxybenzoate monooxygenase (O-demethylating) . . . . . methylarsonate reductase (transferred to EC 1.20.4.2) . . . . . . methylarsonate reductase . . . . . . . . . . . . . . . . . . methylsterol monooxygenase (transferred to EC 1.14.13.72) . . . . monophenol monooxygenase . . . . . . . . . . . . . . . . . monoprenyl isoflavone epoxidase . . . . . . . . . . . . . . . myristoyl-CoA 11-(E) desaturase . . . . . . . . . . . . . . . myristoyl-CoA 11-(Z) desaturase . . . . . . . . . . . . . . . nitrogenase . . . . . . . . . . . . . . . . . . . . . . . . nitrogenase (flavodoxin) . . . . . . . . . . . . . . . . . . . peptidylglycine monooxygenase. . . . . . . . . . . . . . . . phenylalanine 4-monooxygenase . . . . . . . . . . . . . . . phosphonate dehydrogenase . . . . . . . . . . . . . . . . . phylloquinone monooxygenase (2,3-epoxidizing) . . . . . . . . plasmanylethanolamine desaturase . . . . . . . . . . . . . . progesterone 11a-monooxygenase. . . . . . . . . . . . . . . progesterone monooxygenase. . . . . . . . . . . . . . . . . D-proline reductase (dithiol) . . . . . . . . . . . . . . . . . prostaglandin-endoperoxide synthase . . . . . . . . . . . . . pteridine oxidase . . . . . . . . . . . . . . . . . . . . . . pyrogallol hydroxytransferase . . . . . . . . . . . . . . . . reticuline oxidase . . . . . . . . . . . . . . . . . . . . . . ribonucleoside-diphosphate reductase . . . . . . . . . . . . . ribonucleoside-triphosphate reductase . . . . . . . . . . . . . rubredoxin-NAD(P)+ reductase . . . . . . . . . . . . . . . . rubredoxin-NAD+ reductase . . . . . . . . . . . . . . . . . salutaridine synthase . . . . . . . . . . . . . . . . . . . . sarcosine reductase . . . . . . . . . . . . . . . . . . . . . squalene monooxygenase . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

460 466 654 111 328 629 261 586 568 526 485 355 313 362 602 364 258 347 486 367 217 361 123 431 318 660 596 327 156 384 378 380 569 587 140 60 591 342 338 314 273 624 246 487 642 613 489 515 565 538 240 631 280

Index of Recommended Enzyme Names

1.14.19.1 1.14.99.5 1.14.15.4 1.14.99.9 1.14.99.10 1.14.99.24 1.14.21.1 1.97.1.3 1.21.3.5 1.21.3.4 1.15.1.1 1.15.1.2 1.18.96.1 1.14.99.37 1.97.1.8 1.14.99.35 1.14.16.4 1.14.16.2

stearoyl-CoA 9-desaturase . . . . . . . . . . . . stearoyl-CoA desaturase (transferred to EC 1.14.19.1) steroid 11b-monooxygenase . . . . . . . . . . . steroid 17a-monooxygenase . . . . . . . . . . . steroid 21-monooxygenase . . . . . . . . . . . steroid 9a-monooxygenase . . . . . . . . . . . (S)-stylopine synthase . . . . . . . . . . . . . sulfur reductase . . . . . . . . . . . . . . . . sulochrin oxidase [(-)-bisdechlorogeodin-forming] . sulochrin oxidase [(+)-bisdechlorogeodin-forming]. superoxide dismutase . . . . . . . . . . . . . . superoxide reductase . . . . . . . . . . . . . . superoxide reductase (transferred to EC 1.15.1.2) . . taxadiene 5a-hydroxylase . . . . . . . . . . . . tetrachloroethene reductive dehalogenase . . . . . thiophene-2-carbonyl-CoA monooxygenase . . . . tryptophan 5-monooxygenase . . . . . . . . . . tyrosine 3-monooxygenase . . . . . . . . . . .

. . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . .

194 278 26 290 302 357 233 647 621 617 399 426 585 396 661 386 98 81

XVII

Description of Data Fields

#    #  ) 

All information except the nomenclature of the enzymes (which is based on the recommendations of the Nomenclature Committee of IUBMB (International Union of Biochemistry and Molecular Biology) and IUPAC (International Union of Pure and Applied Chemistry) is extracted from original literature (or reviews for very well characterized enzymes). The quality and reliability of the data depends on the method of determination, and for older literature on the techniques available at that time. This is especially true for the fields Molecular Weight and Subunits. The general structure of the fields is: Information ± Organism ± Commentary ± Literature The information can be found in the form of numerical values (temperature, pH, Km etc.) or as text (cofactors, inhibitors etc.). Sometimes data are classified as Additional Information. Here you may find data that cannot be recalculated to the units required for a field or also general information being valid for all values. For example, for Inhibitors, Additional Information may contain a list of compounds that are not inhibitory. The detailed structure and contents of each field is described below. If one of these fields is missing for a particular enzyme, this means that for this field, no data are available.

* (    +  The number is as given by the IUBMB, classes of enzymes and subclasses defined according to the reaction catalyzed.
    This is the name as given by the IUBMB/IUPAC Nomenclature Committee '     This is the name as given by the IUBMB/IUPAC Nomenclature Committee
  Synonyms which are found in other databases or in the literature, abbreviations, names of commercially available products. If identical names are frequently used for different enzymes, these will be mentioned here, cross references are given. If another EC number has been included in this entry, it is mentioned here.

XIX

Description of Data Fields

+
  The majority of enzymes have a single chemical abstract (CAS) number. Some have no number at all, some have two or more numbers. Sometimes two enzymes share a common number. When this occurs, it is mentioned in the commentary.


  ,  For listing organisms their systematic name is preferred. If these are not mentioned in the literature, the names from the respective literature are used. For example if an enzyme from yeast is described without being specified further, yeast will be the entry. This field defines the code numbers for the organisms in which the enzyme with the respective EC number is found. These code numbers (form ) are displayed together with each entry in all fields of Brenda where organism-specific information is given.

- '   
   +      The reaction as defined by the IUBMB. The commentary gives information on the mechanism, the stereochemistry, or on thermodynamic data of the reaction. '    According to the enzyme class a type can be attributed. These can be oxidation, reduction, elimination, addition, or a name (e.g. Knorr reaction) (         These are substrates and products which are metabolized in vivo. A natural substrate is only given if it is mentioned in the literature. The commentary gives information on the pathways for which this enzyme is important. If the enzyme is induced by a specific compound or growth conditions, this will be included in the commentary. In Additional information you will find comments on the metabolic role, sometimes only assumptions can be found in the references or the natural substrates are unknown. In the listings, each natural substrate (indicated by a bold S) is followed by its respective product (indicated by a bold P). Products are given with organisms and references included only if the respective authors were able to demonstrate the formation of the specific product. If only the disappearance of the substrate was observed, the product is included without organisms of references. In cases with unclear product formation only a ? as a dummy is given.
      All natural or synthetic substrates are listed (not in stoichiometric quantities). The commentary gives information on the reversibility of the reaction,

XX

Description of Data Fields

on isomers accepted as substrates and it compares the efficiency of substrates. If a specific substrate is accepted by only one of several isozymes, this will be stated here. The field Additional Information summarizes compounds that are not accepted as substrates or general comments which are valid for all substrates. In the listings, each substrate (indicated by a bold S) is followed by its respective product (indicated by a bold P). Products are given with organisms and references included if the respective authors demonstrated the formation of the specific product. If only the disappearance of the substrate was observed, the product will be included without organisms or references. In cases with unclear product formation only a ? as a dummy is given. %  Compounds found to be inhibitory are listed. The commentary may explain experimental conditions, the concentration yielding a specific degree of inhibition or the inhibition constant. If a substance is activating at a specific concentration but inhibiting at a higher or lower value, the commentary will explain this. +   .     This field contains cofactors which participate in the reaction but are not bound to the enzyme, and prosthetic groups being tightly bound. The commentary explains the function or, if known, the stereochemistry, or whether the cofactor can be replaced by a similar compound with higher or lower efficiency.  "  +    This field lists compounds with a positive effect on the activity. The enzyme may be inactive in the absence of certain compounds or may require activating molecules like sulfhydryl compounds, chelating agents, or lipids. If a substance is activating at a specific concentration but inhibiting at a higher or lower value, the commentary will explain this. / .   This field lists all metals or ions that have activating effects. The commentary explains the role each of the cited metal has, being either bound e.g. as Fe-S centers or being required in solution. If an ion plays a dual role, activating at a certain concentration but inhibiting at a higher or lower concentration, this will be given in the commentary. $ "  0- *1 The kcat is given in the unit min-1 . The commentary lists the names of the substrates, sometimes with information on the reaction conditions or the type of reaction if the enzyme is capable of catalyzing different reactions with a single substrate. For cases where it is impossible to give the turnover number in the defined unit (e.g., substrates without a defined molecular weight, or an undefined amount of protein) this is summarized in Additional Information.

XXI

Description of Data Fields


  " 021 The unit is micromol/minute/milligram of protein. The commentary may contain information on specific assay conditions or if another than the natural substrate was used in the assay. Entries in Additional Information are included if the units of the activity are missing in the literature or are not calculable to the obligatory unit. Information on literature with a detailed description of the assay method may also be found. 34  0/1 The unit is mM. Each value is connected to a substrate name. The commentary gives, if available, information on specific reaction condition, isozymes or presence of activators. The references for values which cannot be expressed in mM (e.g. for macromolecular, not precisely defined substrates) are given in Additional Information. In this field we also cite literature with detailed kinetic analyses. 34  0/1 The unit of the inhibition constant is mM. Each value is connected to an inhibitor name. The commentary gives, if available, the type of inhibition (e.g. competitive, non-competitive) and the reaction conditions (pH-value and the temperature). Values which cannot be expressed in the requested unit and references for detailed inhibition studies are summerized under Additional information. 4, The value is given to one decimal place. The commentary may contain information on specific assay conditions, such as temperature, presence of activators or if this optimum is valid for only one of several isozymes. If the enzyme has a second optimum, this will be mentioned here. 4'  Mostly given as a range e.g. 4.0±7.0 with an added commentary explaining the activity in this range. Sometimes, not a range but a single value indicating the upper or lower limit of enzyme activity is given. In this case, the commentary is obligatory. $   0 +1 Sometimes, if no temperature optimum is found in the literature, the temperature of the assay is given instead. This is always mentioned in the commentary. $    0 +1 This is the range over which the enzyme is active. The commentary may give the percentage of activity at the outer limits. Also commentaries on specific assay conditions, additives etc.

XXII

Description of Data Fields

5 
  /    6 This field gives the molecular weight of the holoenzyme. For monomeric enzymes it is identical to the value given for subunits. As the accuracy depends on the method of determination this is given in the commentary if provided in the literature. Some enzymes are only active as multienzyme complexes for which the names and/or EC numbers of all participating enzymes are given in the commentary.
 The tertiary structure of the active species is described. The enzyme can be active as a monomer a dimer, trimer and so on. The stoichiometry of subunit composition is given. Some enzymes can be active in more than one state of complexation with differing effectivities. The analytical method is included.           The main entries in this field may be proteolytic modification, or side-chain modification, or no modification. The commentary will give details of the modifications e.g.: ± proteolytic modification (, propeptide Name) [1]; ± side-chain modification (, N-glycosylated, 12% mannose) [2]; ± no modification [3]

7 %    2     2 /   2   
  2  For multicellular organisms, the tissue used for isolation of the enzyme or the tissue in which the enzyme is present is given. Cell-lines may also be a source of enzymes. !    The subcellular localization is described. Typical entries are: cytoplasm, nucleus, extracellular, membrane.    The field consists of an organism and a reference. Only references with a detailed description of the purification procedure are cited. '    Commentary on denaturant or renaturation procedure. +    The literature is cited which describes the procedure of crystallization, or the X-ray structure.

XXIII

Description of Data Fields

+  Lists of organisms and references, sometimes a commentary about expression or gene structure.  The properties of modified proteins are described.    Actual or possible applications in the fields of pharmacology, medicine, synthesis, analysis, agriculture, nutrition are described.

8
  4
  This field can either give a range in which the enzyme is stable or a single value. In the latter case the commentary is obligatory and explains the conditions and stability at this value. $    This field can either give a range in which the enzyme is stable or a single value. In the latter case the commentary is obligatory and explains the conditions and stability at this value. ,&     Stability in the presence of oxidizing agents, e.g. O2, H2 O2, especially important for enzymes which are only active under anaerobic conditions. ,   "   The stability in the presence of organic solvents is described. 9        This field summarizes general information on stability, e.g., increased stability of immobilized enzymes, stabilization by SH-reagents, detergents, glycerol or albumins etc.
     Storage conditions and reported stability or loss of activity during storage.

'  Authors, Title, Journal, Volume, Pages, Year.

XXIV


     



   
 1.14.15.1    (+)-camphor,reduced putidaredoxin:oxygen oxidoreductase (5-hydroxylating)    camphor 5-monooxygenase    2-bornanone 5-exo-hydroxylase camphor 5-monooxygenase d-camphor-exo-hydroxylase P450cam bornanone 5-exo-hydroxylase camphor 5-exo-hydroxylase camphor 5-exo-methylene hydroxylase camphor 5-exohydroxylase camphor hydroxylase camphor methylene hydroxylase cytochrome p450cam d-camphor monooxygenase haem mono-oxygenase CYP101 methylene hydroxylase methylene monooxygenase oxygenase, camphor 5-mono
    9030-82-4

     Pseudomonas putida (Swiss-Prot Accession Number PC00183; strain C1 [1, 9, 10]; PpG786, mutant derived from PpG1 [2-5, 8]; strain PpG1 [11]; CYP101 [15]) [1-24] Pseudomonas sp. (soil isolate, strain C5) [1]

1

Camphor 5-monooxygenase

1.14.15.1

!    "
 # 

(+)-camphor + putidaredoxin + O2 = (+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2 O (multi-component mixed function oxidase, consisting of putidaredoxin reductase, putidaredoxin and cytochrome m P450cam, heme-thiolate protein, [2])    oxidation redox reaction reduction       (+)-camphor + putidaredoxin + O2 [1-6, 8, 12, 13, 15, 16, 17, 18, 19, 21, 24] $ (R)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2 O (i.e. (+)-exo-5-hydroxycamphor) [1-6, 8, 12, 13, 15, 16, 17, 18, 19, 21, 24]      (+)-camphor + putidaredoxin + O2 ( when deuterated at either 5-exo- or 5-endo-position, only 5-exo-hydroxycamphor is the product [8]; the (+)- and (-)-enantiomers serve as substrates [2]) (Reversibility: ? [1-24]) [1-6, 8, 12, 13, 15, 16, 17, 18, 19, 21, 24] $ (R)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2 O (i.e. (+)-exo-5-hydroxycamphor) [1-6, 8, 12, 13, 15, 16, 17, 18, 19, 21, 24]  (R)-3-ethylhexanol + putidaredoxin + O2 (Reversibility: ? [18]) [18] $ 2-ethylhexanoic acid + 2-ethyl-1,2-hexanediol + 2-ethyl-1,3-hexanediol + 2-ethyl-1,4-hexanediol + oxidized putidaredoxin + H2 O ( ratio: 50:13:15:8 [18]) [18]  (S)-3-ethylhexanol + putidaredoxin + O2 ( the (S)-isomer is turned over 1.4times faster than the (R)-isomer [18]) (Reversibility: ? [18]) [18] $ 2-ethylhexanoic aicd + 2-ethyl-1,2-hexanediol + 2-ethyl-1,3-hexanediol + 2-ethyl-1,4-hexanediol + oxidized putidaredoxin + H2 O ( ratio: 15:53:28:10 [18]) [18]  1,2,4,5-tetrachlorobenzene + putidaredoxin + O2 (Reversibility: ? [19]) [19] $ 2,3,5,6-tetrachlorophenol + oxidized putidaredoxin + H2 O [19]  1,2-campholide + putidaredoxin + O2 (Reversibility: ? [1, 2]) [1, 2] $ 5-exo-hydroxy-1,2-campholide + oxidized putidaredoxin + H2 O [1, 2]  1,2-dichlorobenzene + putidaredoxin + O2 (Reversibility: ? [19]) [19] $ 2,3-dichlorophenol + 3,4-dichlorophenol + oxidized putidaredoxin + H2 O [19]

2

1.14.15.1

Camphor 5-monooxygenase

 1,3,5-trichlorobenzene + putidaredoxin + O2 (Reversibility: ? [19]) [19] $ 2,4,6-trichlorophenol + oxidized putidaredoxin + H2 O [19]  1,3-dichlorobenzene + putidaredoxin + O2 (Reversibility: ? [19]) [19] $ 2,6-dichlorophenol + 2,4-dichlorophenol + 2,5-dichlorophenol + 2,3-dichlorophenol + oxidized putidaredoxin + H2 O [19]  1,4-dichlorobenzene + putidaredoxin + O2 (Reversibility: ? [19]) [19] $ 2,5-dichlorophenol + oxidized putidaredoxin + H2 O [19]  1-dehydrocamphor + putidaredoxin + O2 (Reversibility: ? [5]) [5] $ exo-5,6-epoxycamphor + oxidized putidaredoxin + H2 O [5]  5,5-difluorocamphor + putidaredoxin + O2 (Reversibility: ? [6]) [6] $ 5,5-difluoro-9-hydroxycamphor + oxidized putidaredoxin + H2 O [6]  5-exo-bromocamphor + putidaredoxin + O2 ( (+)- and (-)-enantiomer [4]) (Reversibility: ? [4]) [4] $ 5-ketocamphor + oxidized putidaredoxin + H2 O [4]  benzo[a]pyrene + putidaredoxin + O2 (Reversibility: ? [20]) [20] $ 3-hydroxybenzo[a]pyrene + oxidized putidaredoxin + H2 O [19]  ethylbenzene + putidaredoxin + O2 ( at 5% of the reaction with (+)-camphor [13]) (Reversibility: ? [13]) [13] $ 1-phenylethanol + oxidized putidaredoxin + H2 O ( ratio of (R)to (S)-1-phenylethanol produced depends on mutant form [13]) [13]  fluoranthene + putidaredoxin + O2 (Reversibility: ? [20]) [20] $ 3-fluoranthol + oxidized putidaredoxin + H2 O [19]  phenanthrene + putidaredoxin + O2 (Reversibility: ? [20]) [20] $ 1-phenanthrol + 2-phenanthrol + 3-phenanthrol + 4-phenanthrol + oxidized putidaredoxin + H2 O [19]  pyrene + putidaredoxin + O2 (Reversibility: ? [20]) [20] $ 1-pyrenol + 2-pyrenol + 1,6-pyrenequinone + 1,8-pyrenequinone + oxidized putidaredoxin + H2 O [19]  Additional information [2, 6] $ ?
"%  FAD ( increase of activity, can replace FMN [9]) [9] FMN ( requirement, prosthetic group of putidaredoxin reductase [9]) [1-3, 9, 10] NADH ( requirement, reduces reductase flavoprotein and putidaredoxin, but not P450cam , in the absence of camphor [2]) [2-6, 8, 10, 11, 13, 19, 24] NADPH ( requirement, instead of NADH, with campholide as substrate, Pseudomonas sp. C5 [1]) [1, 9, 10] cytochrome m ( cytochrome P-450cam , essential, b-type heme-thiolate protein of P-450-class [3]) [2-11]

3

Camphor 5-monooxygenase

1.14.15.1

putidaredoxin ( essential, iron-sulfur redox protein, Fe2 S2 *Cys4class, e-transfer agent to and effector of cytochrome P450cam [3, 4]; cannot be replaced by other FeS-proteins or the phospholipid of the hepatic microsomal P450 system [2]; the putidaredoxin-binding site is only minimally affected by cytochrome b5 [16]) [2-4, 6, 8-11, 16] &

  tetrahydrofuran ( activation [1]) [1] '(   Fe2+ ( requirement, enzyme complex with two FeS-protein components [2-11]) [2-11] K+ ( increase of activity, selective effector of enhanced substrate affinity to cytochrome m [3]) [3] ) &  * +, 50 (5,5-difluorocamphor, cytochrome P-450 [6]) [6] 120 (putidaredoxin, constituent protein of the multi-component oxygenase [3]) [3] 150 (camphor, cytochrome P-450 [6]) [6] 2040 (cytochrome m, P-450cam , constituent part of the multi-component enzyme [3]) [3] " & *-% , 0.016 [1] 30.26 ( putidaredoxin reductase [3]) [3] . / *', 0.0016 ((+)-camphor) [18] 0.068 ((S)-2-ethylhexanol) [18] 0.086 ((R)-2-ethylhexanol) [18] 0  7 ( assay at [3-5, 8]) [3-5, 8] 7.4 ( assay at [2, 5, 6, 9-11]) [2, 5, 6, 9-11] )  *
, 20 ( assay at [5]) [5] 25 ( assay at [2, 3, 11]) [2, 3, 11]

  #   ' 1  Additional information ( multi-component mixed function oxidase, consisting of cytochrome P-450, MW 40000, putidaredoxin and putidaredoxin reductase, slightly smaller than cytochrome P-450, Pseudomonas C1, gel filtration [10]; putidaredoxin reductase: MW 43000, putidaredoxin: MW 12500, cytochrome P450cam : MW 45000, Pseudomonas putida PpG786, ultracentrifugal, diffusion and amino acid analysis [2]; putidaredoxin

4

1.14.15.1

Camphor 5-monooxygenase

reductase, MW 43500, putidaredoxin, MW 11594, cytochrome m, MW 45000, Pseudomonas putida PpG786, analytical data [3]; FMN-containing NADH-reductase, MW 43500, putidaredoxin, MW 12500, cytochrome P450cam , MW 45000, Pseudomonas putida C1 , ultracentrifugal analysis, gel filtration, end group analysis and quantification of prosthetic group material [9]; putidaredoxin reductase: MW 45000, putidaredoxin: MW 11000, cytochrome m: MW 44000, Pseudomonas putida PpG786, gel filtration [3]) [2, 3, 9, 10]   Additional information ( multi-component mixed function monooxygenase consisting of putidaredoxin reductase, MW 47000, putidaredoxin, MW 11700, cytochrome m, MW 50500, Pseudomonas putida PpG786, SDSPAGE [3]; cytochrome P450cam dimerizes via the formation of an intermolecular disulfide bond [17]) [3, 17] $   "

glycoprotein ( small amount of carbohydrate in putidaredoxin and in cytochrome P-450cam [9]) [9]

 2 %$ %' %

3#

cytoplasm ( activity sediments after 14 h centrifugation of crude extract at 140000 * g, not after 2 h at 100000 * g, Pseudomonas sp. C5 [1]) [111] $"

(PpG786 [2, 3]; C1 [9, 10]; PpG1 [11]) [2, 3, 9-11]
 #

[7, 18, 23] (Pseudomonas putida PpG786, cytochrome m [3]; Pseudomonas sp., ternary complex [7]) [3, 7] (space group P212121, structure at 2 A resolution [14]) [14] (structure of the ferrous dioxygen adduct at 0.91 A resolution [15]) [15]
 

(Pseudomonas putida, cam operon has been isolated, cloned and expressed in Escherichia coli, review) [7] 

 

C136A ( altered NADH turnover rate [24]) [24] C148A ( altered NADH turnover rate [24]) [24] C334A ( identical with the wild-type monomer in terms of optical spectra, camphor-binding and turnover [17]) [17] C357H ( no activity [21]) [21] C58A ( altered NADH turnover rate [24]) [24] C85A ( altered NADH turnover rate [24]) [24] 5

Camphor 5-monooxygenase

1.14.15.1

F87A/Y96F ( altered product spectrum [20]) [20] F87L/Y96F ( altered product spectrum [20]) [20] K344C ( altered NADH turnover rate [24]) [24] L358P ( stereo- and regioselectivity for d-camphor hydroxylation unchanged [12]) [12] R112C ( altered NADH turnover rate [24]) [24] R364C ( altered NADH turnover rate [24]) [24] R72C ( altered NADH turnover rate [24]) [24] T101M ( ratio of (R)- to (S)-1-phenylethanol produced from ethylbenzene is 89:11 [13]) [13] T101M/T185F/V247M ( ratio of (R)- to (S)-1-phenylethanol produced from ethylbenzene is 87:13 [13]) [13] T185F ( ratio of (R)- to (S)-1-phenylethanol produced from ethylbenzene is 78:22 [13]) [13] T185L ( ratio of (R)- to (S)-1-phenylethanol produced from ethylbenzene is 80:20 [13]) [13] T185V ( ratio of (R)- to (S)-1-phenylethanol produced from ethylbenzene is 74:26 [13]) [13] T252I ( 10% of wild-type activity [14]) [14] V247A ( ratio of (R)- to (S)-1-phenylethanol produced from ethylbenzene is 87:13 [13]) [13] V247L ( increased turnover rate for NADH [19]) [19] V247M ( ratio of (R)- to (S)-1-phenylethanol produced from ethylbenzene is 83:17 [13]) [13] V295I ( ratio of (R)- to (S)-1-phenylethanol produced from ethylbenzene is 76:24 [13]) [13] Y96F ( 100fold increase of activity [19]) [19] Y96F ( altered product spectrum [20]) [20] Y96Y ( altered product spectrum [20]) [20]

4    , under aerobic conditions, cytochrome P450cam decays at 25 C with t1=2 of 180 min to cytochrome P420, not rapidly in the presence of camphor [2] 5    "

, 2-mercaptoethanol retards loss of FeS-chromophore from putidaredoxin during purification [3] , freeze-thawing, less than 5% loss of activity of cytochrome P450cam in the presence of camphor [2] , glycerol, minimizes loss of FMN during purification of putidaredoxin reductase [3] , multiple freezing and thawing, at -20 C, cytochrome m accumulates an equally active, heme-containing component of higher molecular weight, DTT reconverts it to native cytochrome m at 25 C [3]

6

1.14.15.1

Camphor 5-monooxygenase

, putidaredoxin suffers degradation by repeated cycles of freezing and thawing [2] , dialysis, loss of activity in crude extract of Pseudomonas sp. C5 , restorable by NADPH-addition [1]    , -196 C, no loss of activity of putidaredoxin reductase after repeated freeze-thaw cycles [3] , 0 C, putidaredoxin slowly loses its prosthetic group, 2-mercaptoethanol retards apoprotein formation [3] , 4 C, monomeric cytochrome P450cam can be stored at low protein concentrations for several days without appreciable accumulation of the dimer [17] , 0-4 C, after 24-96 h, the resuspended pellet of 140000*g sedimentation of Pseudomonas sp. C5 loses hydroxylation capacity, restorable with NADPH and THF [1]

"  [1] Hedegaard, J.; Gunsalus, I.C.: Mixed function oxidation. IV. An induced methylene hydroxylase in camphor oxidation. J. Biol. Chem., 240, 40384043 (1965) [2] Tyson, C.T.; Lipscomb, J.D.; Gunsalus, I.C.: The role of putidaredoxin and P450 cam in methylene hydroxylation. J. Biol. Chem., 247, 5777-5784 (1972) [3] Gunsalus, I.C.; Wagner, G.C.: Bacterial P-450cam methylene monooxygenase components: cytochrome m, putidaredoxin, and putidaredoxin reductase. Methods Enzymol., 52, 166-188 (1978) [4] Gould, P.V.; Gelb, M.H.; Sligar, S.G.: Interaction of 5-bromocamphor with cytochrome P-450 cam. Production of 5-ketocamphor from a mixed spin state hemoprotein. J. Biol. Chem., 256, 6686-6691 (1981) [5] Gelb, M.H.; Mälkönen, P.; Sligar, S.G.: Cytochrome P450cam catalyzed epoxidation of dehydrocamphor. Biochem. Biophys. Res. Commun., 104, 853-858 (1982) [6] Smith Eble, K.; Dawson, J.H.: Novel reactivity of cytochrome P-450-CAM. Methyl hydroxylation of 5,5-difluorocamphor. J. Biol. Chem., 259, 1438914393 (1984) [7] Sligar, S.G.; Filipovic, D.; Stayton, P.S.: Mutagenesis of cytochromes P450cam and b5. Methods Enzymol., 206, 31-49 (1991) [8] Gelb, M.H.; Heimbrook, D.C.; Mälkönen, P.; Sligar, S.G.: Stereochemistry and deuterium isotope effects in camphor hydroxylation by the cytochrome P450cam monoxygenase system. Biochemistry, 21, 370-377 (1982) [9] Tsai, R.L.; Gunsalus, I.C.; Dus, K.: Composition and structure of camphor hydroxylase components and homology between putidaredoxin and adrenodoxin. Biochem. Biophys. Res. Commun., 45, 1300-1306 (1971) [10] Katagiri, M.; Ganguli, B.N.; Gunsalus, I.C.: Reduction of palmitoyl dihydroxyacetone phosphate by mitochondria. J. Biol. Chem., 243, 3542-3555 (1968)

7

Camphor 5-monooxygenase

1.14.15.1

[11] Peterson, J.A.; Ishimura, Y.; Griffith, B.W.: Pseudomonas putida cytochrome P-450: characterization of an oxygenated form of the hemoprotein. Arch. Biochem. Biophys., 149, 197-208 (1972) [12] Yoshioka, S.; Takahashi, S.; Ishimori, K.; Morishima, I.: Roles of the axial push effect in cytochrome P450cam studied with the site-directed mutagenesis at the heme proximal site. J. Inorg. Biochem., 81, 141-151 (2000) [13] Loida, P.J.; Sligar, S.G.: Engineering cytochrome P-450cam to increase the stereospecificity and coupling of aliphatic hydroxylation. Protein Eng., 6, 207-212. (1993) [14] Hishiki, T.; Shimada, H.; Nagano, S.; Egawa, T.; Kanamori, Y.; Makino, R.; Park, S.Y.; Adachi, S.I.; Shiro, Y.; Ishimura, Y.: X-ray crystal structure and catalytic properties of Thr252Ile mutant of cytochrome P450cam: roles of Thr252 and water in the active center. J. Biochem., 128, 965-974 (2000) [15] Schlichting, I.; Berendzen, J.; Chu, K.; Stock, A.M.; Maves, S.A.; Benson, D.E.; Sweet, R.M.; Ringe, D.; Petsko, G.A.; Sligar, S.G.: The catalytic pathway of cytochrome P450cam at atomic resolution. Science, 287, 1615-1622 (2000) [16] Unno, M.; Christian, J.F.; Sjodin, T.; Benson, D.E.; Macdonald, I.D.G.; Sligar, S.G.; Champion, P.M.: Complex formation of cytochrome P450cam with putidaredoxin: evidence for protein-specific interactions involving the proximal thiolate ligand. J. Biol. Chem., 277, 2547-2553 (2002) [17] Nickerson, D.P.; Wong, L.L.: The dimerization of Pseudomonas putida cytochrome P450cam: practical consequences and engineering of a monomeric enzyme. Protein Eng., 10, 1357-1361 (1997) [18] French, K.J.; Strickler, M.D.; Rock, D.A.; Rock, D.A.; Bennett, G.A.; Wahlstrom, J.L.; Goldstein, B.M.; Jones, J.P.: Benign synthesis of 2-ethylhexanoic acid by cytochrome P450cam: enzymatic, crystallographic, and theoretical studies. Biochemistry, 40, 9532-9538 (2001) [19] Jones, J.P.; O'Hare, E.J.; Wong, L.L.: Oxidation of polychlorinated benzenes by genetically engineered CYP101 (cytochrome P450cam ). Eur. J. Biochem., 268, 1460-1467 (2001) [20] Harford-Cross, C.F.; Carmichael, A.B.; Allan, F.K.; England, P.A.; Rouch, D.A.; Wong, L.L.: Protein engineering of cytochrome p450cam (CYP101) for the oxidation of polycyclic aromatic hydrocarbons. Protein Eng., 13, 121128 (2000) [21] Yoshioka, S.; Takahashi, S.; Hori, H.; Ishimori, K.; Morishima, I.: Proximal cysteine residue is essential for the enzymatic activities of cytochrome P450cam. Eur. J. Biochem., 268, 252-259 (2001) [22] Sibbesen, O.; Zhang, Z.; Ortiz de Montellano, P.R.: Cytochrome P450cam substrate specificity: relationship between structure and catalytic oxidation of alkylbenzenes. Arch. Biochem. Biophys., 353, 285-296 (1998) [23] Lee, D.S; Park, S.Y.; Yamane, K.; Obayashi, E.; Hori, H.; Shiro, Y.: Structural characterization of n-butyl-isocyanide complexes of cytochromes p450nor and P450cam . Biochemistry, 40, 2669-2677 (2001) [24] Lo, K.K.; Wong, L.L.; Hill, A.O.: Surface-modified mutants of cytochrome p450cam enzymatic properties and electrochemistry. FEBS Lett., 451, 342346 (1999) 8


 (    



   
 1.14.15.2    (+)-camphor,reduced-rubredoxin:oxygen oxidoreductase (1,2-lactonizing)    camphor 1,2-monooxygenase    2,5-DKCMO [7] 2,5-diketocamphane 1,2-monooxygenase ( higher regio- and enatioselectivity than isoform 3,6-diketocamphane-1,6-monooxygenase [11]) [7, 8, 11, 11] 2,5-diketocamphane lactonizing enzyme 3,6-diketocamphane 1,6-monooxygenase ( isoform, which is probably a different enzyme, i.e. 3,6-DKCMO [7]; isozyme, enantiocomplementary and isofunctional isozymes 2,5-DKCMO EC 1.14.15.2 and 3,6DKCMO EC 1.14.15.? [7]; different enzyme with similar biochemical properties, immunological cross-reactivity [8]) [7, 8, 11] camphor ketolactonase I ketolactonase I oxygenase, camphor 1,2-mono Additional information ( 2 diketocamphane monooxygenases in Pseudomonas putida [10]) [10]
    37256-81-8

     Pseudomonas putida (inducible by (+)-camphor [8]; strain NCIMB 10007 [7, 9, 11]; 2 isozymes termed 2,5-diketocamphane-1,2-monooxygenase and 3,6-diketocamphane-1,6-monooxygenase [7, 11]; strain C1 ATCC 17453 [3, 4, 8]; wild type strain C1B (PpG1) [1, 5, 6]; strain PpG786, mutant derived from strain PpG1 [2]) [1-11]

9

Camphor 1,2-monooxygenase

1.14.15.2

!    "
 # 

(+)-bornane-2,5-dione + reduced rubredoxin + O2 = 5-oxo-1,2-campholide + oxidized rubredoxin + H2 O (requires Fe2+ ; higher activity in lactonization of ketones than in oxidation of sulfides to the corresponding sulfoxides [7]; catalysis of 2 mechanistically different types of biochemical reactions within the confines of the same active site [7]; cubic space active site model, topography [7]; belongs to group of Bayer-Villiger monooxygenases of the NADH plus FMN-dependent type 2 enzymes [7,9]; mechanism [1,4,6,7,9])    Bayer-Villiger reaction [7-9,11] oxidation redox reaction reduction       (+)-bornane-2,5-dione + NADH + O2 ( inducible by growth on (+)-camphor [11]; inducible by growth on racemic camphor [7,9]; i.e. 2,5-diketocamphane, reaction in camphor catabolism of Pseudomonas putida [2]) (Reversibility: ? [2, 7, 9, 11]) [2, 7, 9, 11] $ ?  Additional information ( catalyzes stereoselective electrophilic biooxidation of a wide range of prochiral organic sulfoxides to the corresponding chiral sulfoxides as well as the nucleophilic biooxidation of ketones to lactones with different enantio- and stereoselectivity, overview [7]) [7] $ ?      (+)-bornane-2,5-dione + NADH + O2 ( i.e. 2,5-diketocamphane [1-9,11]; flavin reduction during reaction is reversible [6]) (Reversibility: ? [1-9, 11]) [1-9] $ 3,4,4-trimethyl-5-carboxy-methyl-D2 -cyclopentenone + NAD+ + H2 O ( product is an unstable lactone-intermediate and forms spontaneously 2-oxo-D3 -4,5,5-trimethylcyclopentenyl acetic acid [9]; i.e. cyclopentenoic acid [1-6]) [1-7, 9]  (+)-bornanone + NADH + O2 ( (+)-camphor [3,4,6-8]) (Reversibility: ? [3, 4, 6-9]) [3, 4, 6-9] $ 1,2-campholide + NAD+ + H2 O [3, 4, 6-9]  1,3,3-trimethyl-2-oxabicyclo-(2,2,2)octane + NADH + O2 ( 6oxocineole [4]) (Reversibility: ? [4]) [4] $ 1,6,6-trimethyl-2,7-dioxa(3,2,2)bicyclononan-3-one + NAD+ + H2 O [4]  6-oxocineole + NADH + O2 (Reversibility: ? [4]) [4] $ 3-(1-hydroxy-1-methylethyl)-6-oxoheptanoic acid + NAD+ + H2 O [4]

10

1.14.15.2

Camphor 1,2-monooxygenase

 Additional information ( no activity with (-)-camphor and other ketocamphanes that do not possess a keto group at position 2 [8]; several sulfides and bicyclo[3,2,0]hept-2-en-6-one are enantioselectively oxidized to the corresponding sulfoxides and oxa lactones, respectively [10,11]; enzyme is associated with a NADH oxidase [7,8]; rubredoxin not mentioned [1-9]; catalyzes stereoselective electrophilic biooxidation of a wide range of prochiral organic sulfoxides to the corresponding chiral sulfoxides as well as the nucleophilic biooxidation of ketones to lactones with different enantio- and stereoselectivity, overview [7]) [1-11] $ ? 2  2,2'-bipyridine ( inhibition of the whole enzyme complex, but not of the components alone [1,5]; reactivation by Fe2+ , not by Co2+, Cu2+ , Mn2+ , Mg2+ , Zn2+ , Fe3+ [1]; dissociates FMN from oxygenating enzyme component [3]) [1, 3, 5] DTNB ( mild inhibition, dehydrogenase [5]) [5] H2 O2 ( mild inhibition, dehydrogenase [5]) [5] KI ( dehydrogenase [5]) [5] NEM ( partial inhibition, dehydrogenase [5]) [5] methylene blue ( dehydrogenase [5]) [5] p-hydroxymercuribenzoate ( oxygenase coupled to NADH oxidase [3]; incomplete inhibition, dehydrogenase, reversible by GSH or DTT [5]) [3, 5] sodium arsenite ( mild inhibition, dehydrogenase [5]) [5]
"%  FAD ( 2 mol per mol of component E3 [2]; activation, 30% activity of FMN [1]; 2 mol will bind reversibly at the binding site of the apoprotein of the dehydrogenase, in the presence of FMN only 1 mol [5]) [1, 2, 5, 6] FMN ( binds reversibly [8]; 0.4 mol per mol of component E1 , 0.7 mol per mol of component E2 [2]; both enzymatic steps require FMN, not replaceable by GSH, Cys, sodium borohydride or ascorbate [1]; components E1 and E2 bind FMN reversibly [2]; dialyzable, not covalently bound [3]) [1-8] NADH ( interaction of inducible NADH dehydrogenase component via bound FMN with oxygenating component [8]; usage of immobilized macromolecular cofactor in a membrane reactor [10]; requirement [18]) [1-10] &

  dichlorophenolindophenol ( activation, enhances NADH-oxidation [5]) [5] Additional information ( boiled cell-free extract, activation, replaces FMN in the assay [1]) [1]

11

Camphor 1,2-monooxygenase

1.14.15.2

'(   Fe2+ ( requirement [1]; no Fe2+ in dehydrogenase [1,3,6]) [1, 3, 6] ) &  * +, 1200 (NADH, component E3 [2]) [2] 1900 (NADH, component E2 [2]) [2] 3200 (NADH, component E1 [2]) [2] 19000 (NADH) [5] 39000 (FAD, component E2 [5]) [5] Additional information [5, 6] " & *-% , 0.335 ( substrate (+)-camphor [8]) [8] 0.43 ( substrate (+)-camphor, purified, reconstituted multimeric enzyme complex [6]) [6] 0.81 ( substrate diketocamphane, purified enzyme [6]) [6] 1.67 ( component E2 , lactonizing [1]) [1] 3.56 ( oxygenating component [3]) [3] 10-20 ( component E3 , ketolactonizing [2]) [2] 24 ( component E2 [2]) [2] 38.3 ( component E1 , FMN-reductase [1]) [1] 417 ( purified component E1 NADH-dehydrogenase [5]) [5] 420 ( purified component E1 , FMN-coupled NADH-dehydrogenase [2]) [2] Additional information ( no enzyme assay possible in crude extract due to competition with other enzymes for NADH [8]) [8] . / *', 0.0004 (FMN, component E1 [2]) [2] 0.001 (FAD, component E1 [2]) [2] 0.003 (FMN) [5] 0.019 (FAD) [5] 0.1 (NADH, interaction with apoenzyme/FMN-complex [5]) [5] 0.21 (NADH, interation with apoenzyme/2FAD-complex [5]) [5] 0  5 ( NADH-dehydrogenase [5]) [5] 7.2 [1-3, 6] 0 

 6.5-8.5 ( 80% of activity maximum at pH 6.5 and pH 8.5 [2]) [2] 7-8 ( 70% of activity maximum at pH 7 and pH 8, NADH-dehydrogenase [5]) [5] )  *
, 25 ( assay at [5,6]) [5, 6] 30 ( assay at [1-3]) [1-3]

12

1.14.15.2

Camphor 1,2-monooxygenase

  #   ' 1  36000 ( NADH dehydrogenase component, SDS-PAGE [9]; reductase component E2 , low speed sedimentation without reaching equilibrium [5]) [5, 9] 39000-40000 ( oxygenating component, gel filtration [9]) [9] 40000 ( component E1 , analytical centrifugation [2]) [2] 44000 ( about, oxygenating component, native PAGE [8]) [8] 76000 ( oxygenating component E1 , equilibrium ultracentrifugation [3]) [3] 80000 ( component E2 , analytical centrifugation [2]) [2] 120000 ( component E3 , analytical centrifugation [2]) [2, 3, 5] Additional information ( enzyme components form a very loose complex [8]; multi-component enzyme, consists of FMN-reductase and oxygenase compound [6]; enzyme consists of 2 components: 1 oxygenating and 1 NADH dehydrogenase [8,9]; multi-component enzyme, consisting of at least two components: a reductase E2 and an oxygenating component E1 [5]; 2 electrophoretic components, PAGE at pH 8.9 and higher [3]) [3, 5, 6, 8, 9]   dimer ( 2 * 37000, oxygenating component E1 , SDS-PAGE [3]) [3] Additional information ( enzyme components form a very loose complex [8]; enzyme consists of 2 components: 1 oxygenating and 1 NADH dehydrogenase [8,9]; multi-component enzyme, consisting of at least two components: a reductase E2 and an oxygenating component E1 [5]; 2 electrophoretic components, PAGE at pH 8.9 and higher [3]) [3, 5, 8, 9]

 2 %$ %' %

3#

cytoplasm ( NADH dehydrogenase component forming a loose complex with the oxygenating component [8]) [1-3, 5, 6, 8] $"

(both components [8,9]; component E1 , FMN-reductase [1,5]; component E2 , lactonizing enzyme [1]; separation of components E1 , E2 , E3 [2]) [19]  

(inactive enzyme after dialysis can be reactivated by Fe2+ at 0.1 mM, Fe3+ , Co2+, Zn2+ , Mn2+ , Mg2+ , and Cu2+ are not usefull for reactivation [1]) [1]

13

Camphor 1,2-monooxygenase

1.14.15.2


 #

(crystal clusters, grown in 0.2 M CaCl2 , 0.1 M HEPES and 30% PEG 400, X-ray analysis [9]) [9] 

synthesis ( production of useful chiral synthons for chemoenzymatic synthesis [7]) [7]

4  )  50 ( 10 min, component E2 retains full activity, component E3 loses 50%, component E3 loses 90% in the presence of substrate or component E1 [2]) [2]    , -15 C, in 0.05 M Tris/HCl or potassium phosphate buffer, pH 7.2, the dehydrogenase is stable for months [5] , -20 C, ammonium sulfate precipitates as frozen pastes stable over a long period of time [2] , 0 C, in buffer, the dehydrogenase is stable for a week, in very dilute solutions, the dehydrogenase is stable without protective agents [5]

"  [1] Conrad, H.E.; DuBus, R.; Gunsalus, I.C.: An enzyme system for cyclic ketone lactonization. Biochem. Biophys. Res. Commun., 6, 293-297 (1961) [2] Yu, C.A.; Gunsalus, I.C.: Monoxygenases. VII. Camphor ketolactonase I and the role of three protein components. J. Biol. Chem., 244, 6149-6152 (1969) [3] Taylor, D.G.; Trudgill, P.W.: Camphor revisited: studies of 2,5-diketocamphane 1,2-monooxygenase from Pseudomonas putida ATCC 17453. J. Bacteriol., 165, 489-497 (1986) [4] Williams, D.R.; Trudgill, P.W.; Taylor, D.G.: Metabolism of 1,8-cineole by a Rhodococcus species: ring cleavage reactions. J. Gen. Microbiol., 135, 19571967 (1989) [5] Trudgill, P.W.; DuBus, R.; Gunsalus, I.C.: Mixed function oxidation. V. Flavin interaction with a reduced diphosphopyridine nucleotide dehydrogenase, one of the enzymes participating in camphor lactonization. J. Biol. Chem., 241, 1194-1205 (1966) [6] Trudgill, P.W.; DuBus, R.; Gunsalus, I.C.: Mixed function oxidation. VI. Purification of a tightly coupled electron transport complex in camphor lactonization. J. Biol. Chem., 241, 4288-4290 (1966) [7] Beecher, J.; Willetts, A.: Biotransformation of organic sulfides. Predictive active site models for sulfoxidation catalyzed by 2,5-diketocamphane 1,2monooxygenase and 3,6-diketocamphane 1,6-monooxygenase, enantiocom-

14

1.14.15.2

[8] [9]

[10]

[11]

Camphor 1,2-monooxygenase

plementary enzymes from Pseudomonas putida NCIMB 10007. Tetrahedron, 9, 1899-1916 (1998) Jones, K.H.; Smith, R.T.; Trudgill, P.W.: Diketocamphane enantiomer-specific 'Baeyer-Villiger' monooxygenases from camphor-grown Pseudomonas putida ATCC 17453. J. Gen. Microbiol., 139, 797-805 (1993) McGhie, E.J.; Littlechild, J.A.: The purification and crystallization of 2,5-diketocamphane 1,2-monooxygenase and 3,6-diketocamphane 1,6-monooxygenase from Pseudomonas putida NCIMB 10007. Biochem. Soc. Trans., 24, 29S (1996) Pasta, P.; Carrea, G.; Gaggero, N.; Grogan, G.; Willetts, A.: Enantioselective oxidations catalyzed by diketocamphane monooxygenase from Pseudomonas putida with macromolecular NAD in a membrane reactor. Biotechnol. Lett., 18, 1123-1128 (1996) Beecher, J.; Grogan, G.; Roberts, S.; Willetts, A.: Enantioselective oxidations by the diketocamphane monooxygenase isoenzymes from Pseudomonas putida. Biotechnol. Lett., 18, 571-576 (1996)

15

6      

   
 1.14.15.3    alkane,reduced-rubredoxin:oxygen 1-oxidoreductase    alkane 1-monooxygenase    1-hydroxylase CYP4AII CYPIVA1 CYPIVA11 CYPIVA2 CYPIVA3 CYPIVA5 CYPIVA6 CYPIVA7 fatty acid w-hydroxylase lauric acid w-hydroxylase P-450 HK w P450 -HL-w P452 alkane 1-hydrolase alkane hydroxylase alkane monooxygenase fatty acid w-hydrolase w-hydrolase
    9059-16-9

    



16

Pseudomonas oleovorans [1, 3-6, 10] Corynebacterium sp. (strain 7E1C [2]) [2] Pseudomonas aeruginosa (strain S7B1 [7]) [7] Lodderomyces elongisporus (strain EH15D [8,9]) [8, 9]

!

1.14.15.3







Alkane 1-monooxygenase

Oryctolagus cuniculus (rabbit [11,16,17,24]) [11, 16, 17, 24] Homo sapiens [12, 19, 21-23] Fusarium oxysporum (strain MT-811 [15]) [13, 15] Candida maltosa (strain EH15 [14]) [14, 18] Vicia sativa [16] Cladosporium resinae (strain ATCC22711 [20]) [20] Rattus norvegicus [22] Nocardioides sp. (strain CF8 [25]) [25]

!    "
 # 

octane + reduced rubredoxin + O2 = 1-octanol + oxidized rubredoxin + H2 O    hydroxylation oxidation redox reaction reduction       arachidonic acid + NADPH + H+ + O2 [12, 19, 21, 22, 24] $ 20-hydroxyeicosatetraenoic acid + NADP+ + H2 O  n-alkane + NAD(P)H + H+ + O2 [1-22] $ n-alkanol + NAD(P)+ +H2 O  Additional information ( 20-hydroxyeicosatetraenoic acid modulates renal transport activities [22]) [22] $ ?      (n-1)-alkanoate + NADPH + H+ + O2 ( chain length C10 -C16 [11]) (Reversibility: ? [11, 12, 15, 17]) [11, 12, 15, 17] $ (w-1)-hydroxy-n-alkanoate + NADP+ + H2 O  1,7-octadiene + NADH + H+ + O2 ( epoxidation of simple, aliphatic terminal olefins [5,6,10]) (Reversibility: ? [5, 6, 10]) [5, 6, 10] $ 1,2-epoxy-7-octene + NAD+ + H2 O  2,5-dimethylhexane + NADH + H+ + O2 (Reversibility: ? [3, 6]) [3, 6] $ ?  arachidonic acid + NAD(P)H + H+ + O2 (Reversibility: ? [11, 12, 17, 22-24]) [11, 12, 17, 22-24] $ 20-hydroxyicosa-5,8,11,14-tetraenoic acid + NAD(P)+ + H2 O  cyclohexane + NADH + H+ + O2 (Reversibility: ? [3, 6]) [3, 6] $ cyclohexanol + NAD+ + H2 O  fatty acid + NAD(P)H + H+ + O2 ( chain length C6 -C11 , maximal activity with heptanoate [3]; chain length C6 -C14 [6]; chain length C10 -C19 [11]; NADPH-P-450 reductase and cytochrome

17

Alkane 1-monooxygenase

$  $  $  $  $ 

$  $  $  $  $  $  $  $

18

1.14.15.3

b5 are part of the enzyme system [11,17,22-24]; pig liver NADPH-P450 reductase and cytochrome b5 are part of the system [12]; NADPH-P-450 reductase and w-hydrolase are 1 protein [13]; protein from CYP52A3 has a higher affinity for fatty acids than for alkanes [14]; high affinity for short chain fatty acids [18]; 3D-structure analysis, substrate pocket determination [21]; chain length C12 -C16 , arachidonic acid and oleic acid [22]; medium-chain fatty acids [23]; laurate and arachidonic acid [24]) (Reversibility: ? [3-6, 11-18, 20-24]) [3-6, 11-18, 20-24] w-hydroxy fatty acid + NAD(P)+ + H2 O lauric acid + NAD(P)H + H+ + O2 (Reversibility: ? [3, 11-19, 21-24]) [3, 11-19, 21-24] 12-hydroxydodecanoic acid + NAD+ + H2 O lecithin + NADH + H+ + O2 (Reversibility: ? [3]) [3] ? methylcyclohexane + NADH + H+ + O2 (Reversibility: ? [3, 6]) [3, 6] ? monoolein + NADH + H+ + O2 (Reversibility: ? [3]) [3] 18-hydroxyoctadec-9-enoic acid 2,3-dihydroxypropyl ester + NAD+ + H2 O n-alkane + NAD(P)H + H+ + O2 ( rubredoxin is electron carrier [1, 3-6, 10]; chain length C6 -C16 , maximal activity with n-octane [3]; chain length C6 -C14 [6]; protein from CYP52A3 has a higher affinity for alkanes than for fatty acids [14]; alkanes above C6 [25]) (Reversibility: ? [1, 3-10, 14, 15, 20, 25]) [1, 3-10, 14, 15, 20, 25] n-alkanol + NAD(P)+ + H2 O n-hexadecane + NADPH + H+ + O2 ( [7]; NADPH-cyt P450-reductase [8, 9]) (Reversibility: ? [7-9]) [7-9] n-hexadecanol + NADP+ + H2 O n-octane + NADH + H+ + O2 ( cytochrome P-450 is electron carrier [2]) (Reversibility: ? [2]) [2] n-octanol + NAD+ + H2 O n-octane + reduced rubredoxin + O2 (Reversibility: ? [1]) [1] n-octanol + oxidized rubredoxin + H2 O palmitic acid + NAD(P)H + H+ + O2 (Reversibility: ? [11, 12-15, 17, 18, 23]) [11, 12-15, 17, 18, 23] 16-hydroxyhexadecanoic acid + NAD(P)+ + H2 O phosphatidylethanolamine + NADH + H+ + O2 (Reversibility: ? [3]) [3] ? phosphatidylserine + NADH + H+ + O2 (Reversibility: ? [3]) [3] ? prostaglandin A1 + NADPH + O2 (Reversibility: ? [11, 12, 17]) [11, 12, 17] ?

1.14.15.3

Alkane 1-monooxygenase

prostaglandin A2 + NADPH + O2 (Reversibility: ? [11, 17]) [11, 17] ? prostaglandin E1 + NADPH + O2 (Reversibility: ? [24]) [24] ? Additional information ( the enzyme also catalyzes the oxygenative O-demethylation of ethers, the sulfoxidation of methyl sulfides and the stereoselective epoxidation of terminal olefins [10]) [10] $ ?  $  $ 

2  11-dodecynoic acid ( completely inhibited lauric acid w-hydroxylation [16]) [16] 8-hydroxyquinoline ( 1 mM, 2 min 100% inhibition [2]; no inhibition [13]) [2, 13] CO ( 50:50 100% inhibition, 10:90 91% inhibition [2]) [2, 13] Cu2+ ( 0.1 mM concentration, 100% inhibition [2]) [2] Fe2+ ( in 0.5 mM concentration, 100% inhibition [2]) [2] Hg2+ ( 0.1 mM concentration, 100% inhibition [2]) [2] KCN ( 1 mM, 2 min 32% inhibition [2]; reversible inhibitor [5,6]) [2, 5, 6, 13] diethyldithiocarbamate ( reduces activity to 88% [13]) [13] menadione [13] p-chloromercuribenzoate ( 0.1 mM concentration, 51% inhibition [2]) [2] Additional information ( no effect: FAD and FMN [2]) [2] Additional information ( no inhibition by metyrapone [13]) [13]
"%  FAD ( 1 mol per 1 mol enzyme [9]) [9, 13, 15] FMN ( 1 mol per 1 mol enzyme [9]) [9, 13, 15] NAD+ ( very little activity at 1 mM level [2]) [2] NADH ( hydroxylation needs also a NADH-dependent bacterial reductase [1, 3-6]; maximal activity at 1 mM concentration [2]) [1-6, 10, 20] NADPH ( very little activity at 1 mM level [2]) [2, 7-9, 11-16, 2124] &

  Emulgen 911 ( needs detergent 0.3% [14]) [14] Emulgen 913 ( needs detergent 0.1% [13]) [13] Triton X-100 ( needs detergent, 0.04% [9]) [9] dilauroylphosphatidylcholine ( needs detergent [12]) [12] phospholipid ( enhances enzyme activity [4]) [4] '(   Cu ( 0.03 atoms per 42000 g/mol peptide [5]) [5] Fe ( non-heme iron protein [1,3,4]; heme protein [2]; 1 atom Fe per 42000 g/mol peptide [4-6]; addition of hemine enhances activity to 240% [13]) [1-6, 13, 14, 24, 25] 19

Alkane 1-monooxygenase

1.14.15.3

) &  * +, 0.08 (PGA1, w-hydroxylation [12]) [12] 0.15 (arachidonic acid, (w-1)-hydroxylation [12]) [12] 0.51 (lauric acid, (w-1)-hydroxylation [12]) [12] 0.55 (arachidonic acid, w-hydroxylation [12]) [12] 0.63 (palmitic acid, (w-1)-hydroxylation [12]) [12] 0.78 (palmitate, w-hydroxylation [23]) [23] 1.6 (laurate, CYP4A7, R90W-mutant, w-hydroxylation [24]) [24] 2.2 (palmitic acid, w-hydroxylation [12]) [12] 7 (laurate, CYP4A4wt, w-hydroxylation [24]) [24] 9.8 (lauric acid, w-hydroxylation [12]) [12] 10 (arachidonic acid, CYP4A7, R90W-mutant, w-hydroxylation [24]) [24] 11 (prostaglandin E1 , CYP4A7wt, w-hydroxylation [24]) [24] 12 (laurate, CYP4A7, R90W/W93S-mutant, w-hydroxylation [24]) [24] 13 (arachidonic acid, CYP4A7, R90W/W93S-mutant, w-hydroxylation [24]) [24] 14.7 (laurate, w-hydroxylation [23]) [23] 15 (prostaglandin E1 , CYP4A7, R90W/W93S-mutant, w-hydroxylation [24]) [24] 17 (prostaglandin E1 , CYP4A7, R90W-mutant, w-hydroxylation [24]) [24] 18 (prostaglandin E1 , CYP4A7, H206Y/S255F-mutant, w-hydroxylation [24]) [24] 18 (prostaglandin E1 , CYP4A7, S255F-mutant, w-hydroxylation [24]) [24] 18 (prostaglandin E1 , CYP4A7, W93S-mutant, w-hydroxylation [24]) [24] 23 (prostaglandin E1 , CYP4A4wt, w-hydroxylation [24]) [24] 25 (prostaglandin E1 , CYP4A7, H206Y-mutant, w-hydroxylation [24]) [24] 27 (arachidonic acid, CYP4A7, W93S-mutant, w-hydroxylation [24]) [24] 30 (laurate, CYP4A7, W93S-mutant, w-hydroxylation [24]) [24] 43 (arachidonic acid, CYP4A7wt, w-hydroxylation [24]) [24] 46 (laurate, CYP4A7wt, w-hydroxylation [24]) [24] 47 (arachidonic acid, CYP4A4wt, w-hydroxylation [24]) [24] 129 (arachidonic acid, CYP4A7, S255F-mutant, w-hydroxylation [24]) [24] 130 (arachidonic acid, CYP4A7, H206Y/S255F-mutant, w-hydroxylation [24]) [24] 134 (laurate, CYP4A7, H206Y-mutant, w-hydroxylation [24]) [24] 140 (arachidonic acid, CYP4A7, H206Y-mutant, w-hydroxylation [24]) [24] 147 (laurate, CYP4A7, H206Y/S255F-mutant, w-hydroxylation [24]) [24] 160 (laurate, CYP4A7, S255F-mutant, w-hydroxylation [24]) [24] 20

1.14.15.3

Alkane 1-monooxygenase

" & *-% , 0.59 [4] 1.23 ( hydroxylation of octane [5]) [5] 1.75 ( epoxidation of 1,7-octadiene [5]) [5] 2 [6] 4.26 [2] 6.5 ( hydroxylation of lauric acid [15]) [15] 596 [3] . / *', 0.029 (lauric acid, D323E-mutant [19]) [19] 0.031 (lauric acid, E320A/D323E-mutant [19]) [19] 0.032 (laurate) [3] 0.036 (lauric acid, E320A-mutant [19]) [19] 0.057 (laurate, gene CYP4A11 [23]) [23] 0.15 (lauric acid) [15] 0.16 (lauric acid) [19] 0.58 (decane) [1] 0.69 (nonanoate) [3] 0.77 (octane) [1] 5.2 (heptanoate) [3] 6 (hexane) [1] 22 (hexanoate) [3] 0  6.5 ( assay conditions [15]) [15] 7 ( Tris buffer [11]) [11, 13] 7.4 ( assay conditions [2,6,14]) [2, 6, 14] 7.5 ( potassium phosphate buffer [1]; Tris buffer [3]) [1, 3] 8 ( potassium phosphate buffer [11]) [11] 0 

 6.7-7.5 ( laurate hydroxylation in Tris buffer [3]) [3] )  *
, 25 ( assay conditions [15]) [15] 30 ( assay conditions [2,4,6]) [2, 4, 6] 37 ( assay conditions [12]) [12]

  #   ' 1  53000 ( SDS-PAGE [17]) [17] 55000 ( gene expression CYP52A3, SDS-PAGE [14]) [14] 56500 ( gene expression CYP52A4, SDS-PAGE [14]) [14] 59800 ( gene expression CYP52A3, amino acid composition [14]) [14]

21

Alkane 1-monooxygenase

1.14.15.3

61800 ( gene expression CYP52A4, amino acid composition [14]) [14] 79000 ( SDS-PAGE [8]) [8] 118000 ( SDS-PAGE [15]) [15] 800000 ( gel filtration [7]) [7] 2000000 ( gel filtration [3]) [3, 4]   ? ( x * 40100, amino acid composition [5]) [5] ? ( x * 42000, SDS-PAGE [3,4]) [3, 4] $   "

glycoprotein ( 5% carbohydrate [5]) [5]

 2 %$ %' %

% cell suspension culture [2] liver [22] renal cortex [11, 12, 16, 22] small intestine [17] 3#

cell membrane ( membrane-bound enzyme, needs 20 molecules phospholipid per polypeptide chain [5,6]; membrane-bound enzyme [8,9]) [5, 6, 8, 9] cytoplasm [20] microsome ( integral membrane protein [14]) [11, 13-17] $"

[3, 5, 10] [2] [8] [24] [15] [14]  

(addition of dilauroylglyceryl-3-phosphorylcholine restores initial activity after treatment with sodium cholate and subsequent precipitation with ammonium sulfate [4]) [4] (ferrous ions restore activity after treatment with EDTA and dithionite [5,6]) [5, 6]
 

(expressed in Escherichia coli strain XL1 and SCS1 [24]) [24] (expressed in E. coli, human gene CYP4AII [12]; expressed in Escherichia coli XL-1 blue, human gene CYP4A1 [19]; expressed in Escherichia coli, human gene CYP4A11 [22,23]) [12, 19, 22, 23] 22

1.14.15.3

Alkane 1-monooxygenase

(expressed in Saccharomyces cerevisiae strain GRF18 [14]) [14] (expressed in Escherichia coli, genes CYP4A1, CYP4A2, CYP4A3 and CYP4A8 [22]) [22] 

 

D323E ( gene CYP4A1 [19]) [19] E320A ( gene CYP4A1 [19]) [19] E320A/D323E ( gene CYP4A1 [19]) [19] H206Y ( gene CYP4A7 [24]) [24] H206Y/S255F ( gene CYP4A7 [24]) [24] R90W ( gene CYP4A7 [24]) [24] R90W/W93S ( gene CYP4A7 [24]) [24] S255F ( gene CYP4A7 [24]) [24] W93S ( gene CYP4A7 [24]) [24]

4  5    "

, after 24 h 15 mM EDTA 60% activity and iron retained [5] , after 4 h 15 mM EDTA and 10 mM dithionite 10% activity and iron retained [5] , treatment with 0.1 M EDTA and 0.1 M dithionite reduces activity to 10% [4] , treatment with sodium cholate and subsequent precipitation with ammonium sulfate reduces activity to 40% [4]    , -20 C, 1 month, under 50% activity retained [6] , -20 C, loss of 30% activity by freezing and subsequent thawing [10] , -70 C, 12 days, over 90% activity retained [6] , -70 C, 6 months, over 50% activity retained [6] , 4 C, 5 days, under 50% activity retained [6] , -30 C, 1 month, loss of 50% activity towards prostaglandin A1 [11] , -30 C, 3 years, no loss of activity towards laurate [11]

"  [1] Peterson, J.A.; Kusunose, M.; Kusunose, E.; Coon, M.J.: Enzymatic w-oxidation. II. Function of rubredoxin as the electron carrier in w-hydroxylation. J. Biol. Chem., 242, 4334-4340 (1967) [2] Cardini, G.; Jurtshuk, P.: The enzymatic hydroxylation of n-octane by Corybacterium sp. strain 7E1C. J. Biol. Chem., 245, 2789-2796 (1970) [3] McKenna, E.J.; Coon, M.J.: Enzymatic w-oxidation. IV. Purification and properties of the w-hydroxylase of Pseudomonas oleovorans. J. Biol. Chem., 245, 3882-3889 (1970)

23

Alkane 1-monooxygenase

1.14.15.3

[4] Ruettiger, R.T.; Olson, S.T.; Boyer, R.F.; Coon, M.J.: Identification of the whydroxylase of Pseudomonas oleovorans as a nonheme iron protein requiring phospholipid for catalytic activity. Biochem. Biophys. Res. Commun., 57, 1011-1017 (1974) [5] Ruettinger, R.T.; Griffith, G.R.; Coon, M.J.: Characterization of the w-hydroxylase of Pseudomonas oleovorans as a nonheme iron protein. Arch. Biochem. Biophys., 183, 528-537 (1977) [6] Griffith, G.R.; Ruettinger, R.T.; McKenna, E.J.; Coon, M.J.: Fatty acid w-hydroxylase (alkane hydroxylase) from Pseudomonas oleovorans. Methods Enzymol., 53, 356-360 (1978) [7] Matsuyama, H.; Nakahara, T.; Minoda, Y.: A new n-alkane oxidation system from Pseudomonas aeruginosa S7B1. Agric. Biol. Chem., 45, 9-14 (1981) [8] Honeck, H.; Schunck, W.H.; Riege, P.; Muller, H.G.: The cytochrome P-450 alkane monooxygenase system of the yeast Lodderomyces elongisporus: purification and some properties of the NADPH-cytochrome P-450 reductase. Biochem. Biophys. Res. Commun., 106, 1318-1324 (1982) [9] Schnuck, W.H.; Riege, P.; Honeck, H.; Muller, H.G.: Isolierung und Rekonstitution des Alkan-Monooxygenase-Systems der Hefe Lodderomyces elongisporus. Z. Allg. Mikrobiol., 23, 653-660 (1983) [10] May, S.W.; Katopodis, A.G.: Hydrocarbon monooxygenase system of Pseudomonas oleovorans. Methods Enzymol., 188, 3-9 (1990) [11] Sawamura, A.; Kusunose, E.; Satouchi, K.; Kusunose, M.: Catalytic properties of rabbit kidney fatty acid w-hydroxylase cytochrome P-450ka2 (CYP4A7). Biochim. Biophys. Acta, 1168, 30-36 (1993) [12] Palmer, C.N.A.; Richardson, T.H.; Griffin, K.J.; Hsu, M.H.; Muerhoff, A.S.; Clark, J.E.; Johnson, E.F.: Characterization of a cDNA encoding a human kidney cytochrome P-450 4A fatty acid w-hydroxylase and the cognate enzyme expressed in Escherichia coli. Biochim. Biophys. Acta, 1172, 161-166 (1993) [13] Nakayama, N.; Shoun, H.: Fatty acid hydroxylase of the fungus Fusarium oxysporum is possibly a fused protein of cytochrome P-450 and its reductase. Biochem. Biophys. Res. Commun., 202, 586-590. (1994) [14] Scheller, U.; Zimmer, T.; Kaergel, E.; Schunck, W.H.: Characterization of the n-alkane and fatty acid hydroxylating cytochrome P450 forms 52A3 and 52A4. Arch. Biochem. Biophys., 328, 245-254 (1996) [15] Nakayama, N.; Takemae, A.; Shoun, H.: Cytochrome P450foxy, a catalytically self-sufficient fatty acid hydroxylase of the fungus Fusarium oxysporum. J. Biochem., 119, 435-440 (1996) [16] Helvig, C.; Alayrac, C.; Mioskowski, C.; Koop, D.; Poullain, D.; Durst, F.; Salaun, J.P.: Suicide inactivation of cytochrome P450 by midchain and terminal acetylenes. A mechanistic study of inactivation of a plant lauric acid w-hydroxylase. J. Biol. Chem., 272, 414-421 (1997) [17] Koike, K.; Kusunose, E.; Nishikawa, Y.; Ichihara, K.; Inagaki, S.; Takagi, H.; Kikuta, Y.; Kusunose, M.: Purification and characterization of rabbit small intestinal cytochromes P450 belonging to CYP2J and CYP4A subfamilies. Biochem. Biophys. Res. Commun., 232, 643-647 (1997)

24

1.14.15.3

Alkane 1-monooxygenase

[18] Zimmer, T.; Iida, T.; Schunck, W.H.; Yoshida, Y.; Ohta, A.; Takagi, M.: Relation between evolutionary distance and enzymic properties among the members of the CYP52A subfamily of Candida maltosa. Biochem. Biophys. Res. Commun., 251, 244-247 (1998) [19] Dierks, E.A.; Davis, S.C.; Ortiz de Montellano, P.R.: Glu-325 and Asp-328 are determinants of the cyp4A1 hydroxylation regiospecificity and resistance to inactivation by 1-aminobenzotriazole. Biochemistry, 37, 18391847 (1998) [20] Goswami, P.; Cooney, J.J.: Subcellular location of enzymes involved in oxidation of n-alkane by Cladosporium resinae. Appl. Microbiol. Biotechnol., 51, 860-864 (1999) [21] Chang, Y.T.; Loew, G.H.: Homology modeling and substrate binding study of human CYP4A11 enzyme. Proteins, 34, 403-415 (1999) [22] Hoch, U.; Zhang, Z.; Kroetz, D.L.; Ortiz de Montellano, P.R.: Structural determination of the substrate specificities and regioselectivities of the rat and human fatty acid w-hydroxylases. Arch. Biochem. Biophys., 373, 63-71 (2000) [23] Kawashima, H.; Naganuma, T.; Kusunose, E.; Kono, T.; Yasumoto, R.; Sugimura, K.; Kishimoto, T.: Human fatty acid w-hydroxylase, CYP4A11: determination of complete genomic sequence and characterization of purified recombinant protein. Arch. Biochem. Biophys., 378, 333-339 (2000) [24] Loughran, P.A.; Roman, L.J.; Aitken, A.E.; Miller, R.T.; Masters, B.S.: Identification of unique amino acids that modulate CYP4A7 activity. Biochemistry, 39, 15110-15120 (2000) [25] Hamamura, N.; Yeager, C.M.; Arp, D.J.: Two distinct monooxygenases for alkane oxidation in Nocardioides sp. strain CF8. Appl. Environ. Microbiol., 67, 4992-4998 (2001)

25

 b    



   
 1.14.15.4    steroid,reduced-adrenal-ferredoxin:oxygen oxidoreductase (11b-hydroxylating)    steroid 11b-monooxygenase    ALDOS aldosterone synthase aldosterone-synthesizing enzyme C450XIB2 CYPXIB CYPXIB1 CYPXIB2 CYPXIB3 EC 1.14.1.6 (formerly) EC 1.99.1.7 (formerly) P-450(11 b,aldo) P-450Aldo P-450C18 P-450XIB1 [20] P-450c11 P450(11 b)-DS P450C11 steroid 11-b-hydroxylase steroid 18-hydroxylase cytochrome P-45011-b [11, 20] steroid 11b-hydroxylase steroid 11b-monooxygenase steroid 11b/18-hydroxylase
    9029-66-7

26

1.14.15.4

Steroid 11b-monooxygenase

    







Bos taurus (calf [1]; ox [2]) [1, 2, 4-6, 8-11, 13-15, 17-20] Sus scrofa [10] Oryctolagus cuniculus [1] Rattus norvegicus (male, Sprague-Dawley, low sodium, high potassium treated [7]; male, Zur:SIV low sodium, high potassium or high sodium, low potassium treated for stimulation or supprssion, respectively [12]; male Wistar [21]; neonatal and adult [25]) [7, 12, 21, 25, 26] Curvularia lunata (Boedijn NRRL 2380 [3, 24]) [3, 24] Ovis aries [16] Homo sapiens [22] Rana catesbeiana [23]

!    "
 # 

a steroid + reduced adrenal ferredoxin + O2 = an 11b-hydroxysteroid + oxidized adrenal ferredoxin + H2 O (a heme-thiolate protein (P-450). Also hydroxylates steroids at the 18-position, and converts 18-hydroxycorticosterone into aldosterone. Formerly EC 1.14.1.6 and 1.99.1.7 both hydroxylation activities seem to occur in a single enzyme [3-5]; heme protein of socalled P-450 family [5]; P-45011b responsible for two hydroxylase reactions [6]; 2 step reduction: reduction of P-450 with non-specific donor and reduction which is specifically dependent on reduced iron-sulfur protein [8]; 11b-/18-hydroxylation and aldosterone synthesis are catalyzed by 2 distinct forms of cytochrome P-45011b [11, 12]; cytochrome P45011b is implicated in mineralocorticoid, synthesized and secreted from zona glomerulosa, as well as glucocorticoid, secreted primarily in zonae fasciculate and reticularis, biosynthesis with multiple catalytic activities, 11b-/18-/19-hydroxylase and 18-/19-hydroxysteroid oxidase activities [13]; ferric cytochrome P-45011b-deoxycorticosterone complex classified as a pentacoordinated species, H2 O molecule binds ferric heme iron [14]; for synthesis of aldosterone 3 molecules P450 must be reduced or one molecule must be reduced 3 times [15]; superfamily of heme-containing enzyme, 11-/18-/ 19-hydroxylations of 11-deoxycorticosterone, 11-deoxycortisol and androstenedione [20]; single enzyme, 11b-/18-hydroxylation and aldosterone synthetic activities [23]; one bifunctional cytochrome P-450, 11b-/14ahydroxylation [24])    hydroxylation oxidation redox reaction reduction

27

Steroid 11b-monooxygenase

1.14.15.4

      11-deoxycorticosterone + reduced adrenal ferredoxin + O2 ( 11bhydroxylation, last 3 steps of pathway for aldosterone biosynthesis [16]) (Reversibility: ? [16]) [16] $ corticosterone + oxidized adrenal ferredoxin + H2 O [16]  18-hydroxy-corticosterone + reduced adrenal ferredoxin + O2 ( 18-hydroxylation, last 3 steps of pathway for aldosterone biosynthesis [16]) (Reversibility: ? [16]) [16] $ aldosterone + oxidized adrenal ferredoxin + H2 O [16]  corticosterone + reduced adrenal ferredoxin + O2 ( 18-hydroxylation, last 3 steps of pathway for aldosterone biosynthesis [16]) (Reversibility: ? [16]) [16] $ 18-hydroxy-corticosterone + oxidized adrenal ferredoxin + H2 O [16]  Additional information ( corticosterone generally believed to be preferred substrate for 18-hydroxylase [4]; corticosterone normal substrate in vivo [5]) [4, 5] $ ?      11-deoxycorticosterone + reduced adrenal ferredoxin + O2 ( addition of adrenodoxin and adrenodoxin reductase [4, 5]; containing adrenal ferredoxin, adrenal ferredoxin reductase and P-45011b, highly active hydroxylation, 11b-/18-hydroxylase activity [6]; by 49.5 kDa protein from capsular portion, 11b-/18-hydroxylation by 51.5 kDa protein [7]; 11b-hydroxylation [8, 9, 19]; 11b-hydroxylation [16, 17]; adrenal [21]; 11b-hydroxylation [22]; 18-/11bhydroxylation [25]) (Reversibility: ? [4-9, 16, 17, 19, 21, 22, 25]) [4-9, 16, 17, 19, 21, 22, 25] $ corticosterone + oxidized adrenal ferredoxin + H2 O ( 51.5 kDa protein [7]) [4-9, 16, 17, 19, 21, 22, 25]  11-deoxycorticosterone + reduced adrenal ferredoxin + O2 ( containing adrenal ferredoxin, adrenal ferredoxin reductase and P45011b, highly active hydroxylation, 11b-/18-hydroxylase activity [6]; by 49.5 kDa protein from capsular portion, 11b-/18-hydroxylation by 51.5 kDa protein [7]; 18-/11b-hydroxylation [25]) (Reversibility: ? [6, 7, 25]) [6, 7, 25] $ 18-hydroxycorticosterone + oxidized adrenal ferredoxin + H2 O [6, 7, 25]  11-deoxycorticosterone + reduced adrenal ferredoxin + O2 ( by 49.5 kDa protein from capsular portion, 11b-/18-hydroxylation by 51.5 kDa protein [7]; supported by adrenodoxin and adrenodoxin reductase, 3 step-hydroxylation with corticosterone and 18-hydroxycorticosterone as intermediates, 11b-/18-hydroxylase [10]; 11b-hydroxylase and 18-hydroxylase activities, after replacement of Tween 20 by phosphatidylcholine catalytic activity for aldosterone activity is exhibited, form 1 higher active in aldosterone and 18-hydroxycorticosterone production and less active in the production of corticosterone and 18-hydroxydeoxy-

28

1.14.15.4

$ 

$  $ 

$ 

$  $

 $ 

$

Steroid 11b-monooxygenase

corticosterone from deoxycorticosterone as form 2 [11]; 49 kDa protein, exclusively in zona glomerulosa, 11b-hydroxylation, as well as 18hydroxylation and 18-hydroxydehydrogenation of corticosterone with 18hydroxydeoxycorticosterone and 18-hydroxycorticosterone as intermediates; 51 kDa protein, zona fasciculata and glomerulosa, high sodium and low potassium, 11b-/18-hydroxylation [12]; 11b-/18-hydroxylation [14]; 3 steps in glomerulosa catalysed by 1 enzyme, 11b-hydroxylation to corticosterone, 18-hydroxylation to 18-hydroxycorticosterone and aldehyde synthesis to aldosterone [15]; 3 step 11b-/18-hydroxylation to aldosterone [18]) (Reversibility: ? [7, 10-12, 14, 15, 18]) [7, 1012, 14, 15, 18] aldosterone + oxidized adrenal ferredoxin + H2 O ( product of 49 kDa protein [12]) [7, 10-12, 14, 15, 18] 11-deoxycortisol + reduced adrenal ferredoxin + O2 ( cytochrome P-45011b-linked monoxygenase [20]; 11b-hydroxylation [22]; 11b-hydroxylase activity [24]) (Reversibility: ? [20, 22, 24]) [20, 22, 24] cortisol + oxidized adrenal ferredoxin + H2 O [20, 22, 24] 11-deoxycortisol + reduced adrenal ferredoxin + O2 ( 11a-hydroxylase activity [24]) (Reversibility: ? [24]) [24] 14a-hydroxy-11-deoxycortisol + oxidized adrenal ferredoxin + H2 O [24] 17a,21-dihydroxy-pregn-4-ene-3,20-dione + reduced adrenal ferredoxin + O2 ( i.e. Reichstein's compound, inducer [3]) (Reversibility: ? [3]) [3] 11b,17a,21-trihydroxy-pregn-4-ene-3,20-dione + oxidized adrenal ferredoxin + H2 O ( hydrocortisone, 60%, 11b-hydroxylation [3]) [3] 17a,21-dihydroxy-pregn-4-ene-3,20-dione + reduced adrenal ferredoxin + O2 ( i.e. Reichstein's compound, inducer [3]) (Reversibility: ? [3]) [3] 14a,17a,21-trihydroxy-pregn-4-ene-3,20-dione + oxidized adrenal ferredoxin + H2 O ( 25%, 14a hydroxylation [3]) [3] 18-hydroxycorticosterone + reduced adrenal ferredoxin + O2 (Reversibility: ? [13, 16, 17]) [13, 16, 17] aldosterone + oxidized adrenal ferredoxin + H2 O ( aldosterone production exclusively in the zona glomerulosa [16]; 18-hydroxylation [16,17]) [13, 16, 17] 19-hydroxy-11-deoxycorticosterone + reduced adrenal ferredoxin + O2 (Reversibility: ? [13]) [13] 19-oxo-11-deoxycorticosterone + oxidized adrenal ferredoxin + H2 O [13] 4-androstene-3,17-dione + reduced adrenal ferredoxin + O2 ( i.e. testosterone, 11b-/19-hydroxylase activity, good sustrate [6]) (Reversibility: ? [6]) [6] 11b-hydroxy-4-androstene-3,17-dione + oxidized adrenal ferredoxin + H2 O [6]

29

Steroid 11b-monooxygenase

1.14.15.4

 4-androstene-3,17-dione + reduced adrenal ferredoxin + O2 ( i.e. testosterone, 11b-/19-hydroxylase activity, good sustrate [6]) (Reversibility: ? [6]) [6] $ 19-hydroxy-4-androstene-3,17-dione + oxidized adrenal ferredoxin + H2 O [6]  4-pregnen-21-ol-3,20-dione + reduced adrenal ferredoxin + O2 ( 11-hydroxylation [1,3]; 11-deoxycorticosterone, fumarate required in mitochondria treated with hypotonic solutions of electrolytes [2]) (Reversibility: ? [1]) [1, 2] $ 4-pregnene-11,21-diol-3,20-dione + oxidized adrenal ferredoxin + H2 O [1]  4-pregnene-17,21-diol-3,20-dione + reduced adrenal ferredoxin + O2 ( 11-hydroxylation [1]) (Reversibility: ? [1]) [1] $ 4-pregnene-11,17,21-triol-3,20-dione + oxidized adrenal ferredoxin + H2 O [1]  corticosterone + reduced adrenal ferredoxin + O2 ( 18-hydroxycorticosterone as intermediate of 11b-hydroxylation [10]; 18-hydroxylation [19]) (Reversibility: ? [10, 19]) [10, 19] $ aldosterone + oxidized adrenal ferredoxin + H2 O [10, 19]  corticosterone + reduced adrenal ferredoxin + O2 ( addition of adrenodoxin and adrenodoxin reductase [4]; 18-hydroxylation [16,17]) (Reversibility: ? [4, 16, 17]) [4, 16, 17] $ 18-hydroxycorticosterone + oxidized adrenal ferredoxin + H2 O [4, 16, 17]  corticosterone + reduced adrenal ferredoxin + O2 ( addition of adrenodoxin and adrenodoxin reductase [5]) (Reversibility: ? [5]) [5] $ 18-hydroxy-11-deoxycorticosterone + oxidized adrenal ferredoxin + H2 O [5]  cortisol + reduced adrenal ferredoxin + O2 (Reversibility: ? [13]) [13] $ cortisone + oxidized adrenal ferredoxin + H2 O [13]  metyrapone + ? ( plasma, product-stereoselective reductive metabolism [21]) (Reversibility: r [21]) [21] $ metyropol + ? ( enantiomers [21]) [21] 2  18-ethynylprogesterone ( weaker than 18-vinylprogesterone, inhibitor of aldosterone synthesis for both activities, stronger for 18-hydroxylation inhibition [17]; mechanism-based inhibition [17,18]; progesterone analog, time-dependent pseudo-first-order inactivation, concentration dependent [18]; suicide-substrate of aldosterone biosythesis, inhibits more strongly the 18-hydroxylation step [19]) [17-19] 18-vinyldeoxycorticosterone ( deoxycorticosterone analog, very strong and reversible inhibitor for deoxycorticosterone and corticosterone oxidation, 0.001 mM leads to decrease in corticosterone production with 30% of total activity after 1 min, 100fold more efficient than 18-vinylproges-

30

1.14.15.4

Steroid 11b-monooxygenase

terone for inhibition of 11b-hydroxylation step, only 6fold of more inhibition of 18-hydroxylation step [19]) [19] 18-vinylprogesterone ( competitive, potent inhibitor of aldosterone synthesis for both activities, 18-hydroxylation more effected, 65% inhibition of corticosterone and 11-deoxy-18-hydroxycorticosterone production by 0.03 mM [17]; mechanism-based inhibition [17,18]; progesterone analog, with NADPH, time and concentration dependent, irreversible, pseudo-first-order process, covalent binding to and destruction of prosthetic heme group, 5fold more effective than its acetylenic analog 18-ethynylprogesterone [18]; with 0.001 mM no inhibition detectable, with 0.01 mM 23% decrease in corticosterone production, better suicide-substrate of aldosterone biosythesis than 18-ethynylprogesterone, inhibits more strongly the 18-hydroxylation step [19]) [17-19] CO ( affects 11b-/19-hydroxylase reaction [6]; strongly [24]) [4, 5, 6, 24] EDTA ( 10 mM [2]) [2] HgCl2 ( 0.2 mM, 50% inhibition [24]) [24] KCN ( slight inhibitor [2]) [2] KCl ( total inactivation [5]) [5] PCMB ( 0.1 mM, 50% inhibition [24]) [24] SKF 525A ( little inhibition of the purified 11b-hydroxylase, 18-hydroxylation and aldosterone synthesis of corticosterone are inhibited [10]) [10] acetone ( 10%, v/v, complete inhibition of hydroxylation [5]) [5] anti-11b-hydroxylase IgG ( polyclonal, raised in rabbits, inhibition of 11-b/18-hydroxylation and aldosterone synthesis of corticosterone [10]; cross-reaction with 51 kDa protein [12]) [10, 12] antiserum ( produced by rabbits [5]) [5] ascorbate ( without NADH, inhibition of aldosterone synthetase [15]) [15] clotrimazole ( azole derivative, antimycotic drug, strong dose-dependent inhibition [22]) [22] corticosterone ( competitive inhibition of 11b-hydroxylase activity [17]) [17] deoxycorticosterone ( competitive substrate inhibition [5]) [5] diphosphatidyl glycerol ( cardiolipin, dipalmitoyl phasphatidylcholine vesicles, 50% inhibition with 4-5 mol%, complete inhibition at 15 mol% [13]) [13] ethanol ( 10%, v/v, complete inhibition of hydroxylation [5]) [5] iron-sulfur protein ( adrenodoxin, at high concentrations, 25% inhibition [8]) [8] ketoconazole ( azole derivative, antimycotic drug, strong dosedependent inhibition [22]) [22, 24] methanol ( 10%, v/v, complete inhibition of hydroxylation [5]) [5] methyltrienolone ( synthetic androgen, used as photoaffinity ligand and substrate analog, covalent binding, 0.1 mM inhibits cortisol synthesis, during photolabeling radioactivity incorporation via radioactive methyltrie31

Steroid 11b-monooxygenase

1.14.15.4

nolone is blocked by 11-deoxycorticosterone, so it binds to the conserved substrate binding region Trp428-Leu429-Asp430-Arg431 between b3 -sheet and the L-helix analysed by trypsin digest [20]) [20] metyrapol ( 11b-hydroxylase inhibition, 40.8% by racemate, 38.1% by (+)-enantiomer and 33.8% by (-)-enantiomer, each 0.4 mM [21]) [21] metyrapone ( 2 mM [3]; competitive substrate inhibition [5]; competitive with substrate, affects strongly 11b-/18-hydroxylation and 11b-/19-hydroxylation [6]; inhibition of the purified 11b-hydroxylase, 18-hydroxylation and aldosterone synthesis of corticosterone are inhibited [10]; 11b-hydroxylase, 39.7% inhibition by 0.4 mM [21]; diagnostic inhibitor, strong inhibition, 0.02 mM complete inhibition [22]) [3, 5, 6, 10, 21, 22, 24] miconazole ( azole derivative, antimycotic drug, strong dose-dependent inhibition [22]) [22] norharman ( b-carboline, high affinity type II ligand to both cytochromes, progesterone binding to CYP17 competitively inhibited [26]) [26] p-chloromercuribenzoate ( 1 mM [3]) [3] phenazine methosulfate ( 1 mM, crude extract [3]) [3] phosphate [2] phosphatidylcholine ( unsaturated increasing dioleoyl/diphytanoyl phosphatidylcholine [13]) [13] stilbestrol [24] sodium cholate (0.2% effect nearly 20% inhibition [24]) [24] spironolactone ( diuretic and antihypertensive drug, competitive aldosterone antagonist, slight inhibition [22]) [22] sulfhydryl reagents ( strongly [24]) [24] Additional information ( conversion of labelled steroid to labelled aldosterone is inhibited by the addition of an excess of the same unlabelled steroid [16]; no inhibition with Harman, tetrahydronorharman and tetrahydroharman [26]) [16, 26]
"%  NADH ( less effective than NADPH [8]) [8, 15] NADP+ ( plus malate supports all 4 conversions, increase of aldosterone synthetase activity [15]) [15] NADPH ( no reaction with NADH or NADP+ [2]; adrenodoxin and NADPH-linked adrenodoxin reductase [11,13,17-20]; requirement during inactivation [18]; adrenodoxin and adrenodoxin reductase required in cell line homogenates, in living cells in absence of cofactors [22]; NADPH-linked cytochrome P-450 reductase [24]) [1-15, 17-22, 24] ascorbate ( plus NADH very low conversion and no aldosterone production, in synergism with NADH, malate and NADP+ increase of aldosterone synthetase activity in zona glomerulosa, not zona fasciculate [15]) [15] &

  2-oxoglutarate ( most potent activator in intact mitochondria [2]) [2] KCN ( slight [3]) [3] 32

1.14.15.4

Steroid 11b-monooxygenase

MnCl2 ( 1 mM, reactivation of dialysed enzyme [2]; causes most of activity to disappear in crude extracts [3]) [2, 3] NAD(P)H ( enhance activity of crude extracts that show hydroxylation without any cofactor [3]) [3] fumarate (increase of activity) [2] lipid extract ( from adrenocortical mitochondria, increase of aldosterone production from corticosterone [17]) [17] malate ( plus NADP+ and in synergism with ascorbate plus NADH increase of aldosterone synthetase activity in zona glomerulosa, no stimulation of 11b-/18-hydroxylation [15]) [15] phosphatidylcholine ( saturated acyl chains such as dipalmitoyl/dimyristoyl phosphatidylcholine [13]) [13] ) &  * +, 2.268 (corticosterone, +/-0.2268, calculatetd from Kcat /Km [17]) [17] 8 (11-deoxycorticosterone, dipalmitoyl phosphatidylcholine vesicles + diphosphatidyl glycerol [13]) [13] 18 (11-deoxycorticosterone, 18-hydroxylation [6]) [6] 35 (11-deoxycorticosterone, dipalmitoyl phosphatidylcholine vesicles [13]) [13] 39.6 (11-deoxycorticosterone, +/-2.112, calculatetd from Kcat /Km [17]) [17] 110 (11-deoxycorticosterone, 11b-hydroxylation [6]) [6] 207 (11-deoxycortisol, 11b-hydroxylation [24]) [24] " & *-% , 0.00000314 ( aldosterone production, mitochondria from zona glomerulosa [12]) [12] 0.0000236 ( 18-hydroxycorticosterone production, mitochondria from zona fasciculata [12]) [12] 0.00003 ( 18-hydroxylation) [10] 0.000119 ( 18-hydroxycorticosterone production, mitochondria from zona glomerulosa [12]) [12] 0.000171 ( 18-hydroxylation activity, 4% O2, 96% N2 [5]) [5] 0.000211 ( 18-hydroxylation activity, 21% O2, 79% N2 [5]) [5] 0.001061 ( deoxycorticosterone, 18-hydroxylation activity in [4]) [4] 0.00205 ( 11b-hydroxylation activity, 4% O2, 96% N2 [5]) [5] 0.00206 ( 18-hydroxydeoxycorticosterone production, mitochondria from zona glomerulosa [12]) [12] 0.00278 ( 11b-hydroxylation activity, 21% O2, 79% N2 [5]) [5] 0.0053 ( 11b-hydroxylation) [10] 0.00599 ( 18-hydroxydeoxycorticosterone production, mitochondria from zona fasciculata [12]) [12] 0.00691 ( corticosterone production, mitochondria from zona glomerulosa [12]) [12] 0.00951 ( corticosterone production, mitochondria from zona fasciculata [12]) [12]

33

Steroid 11b-monooxygenase

1.14.15.4

0.00968 ( +/-0.00126, 40.8% inhibition by 0.4 mM metyrapol, adrenal tissue [21]) [21] 0.00986 ( +/-0.00044, 39.7% inhibition by 0.4 mM metyrapone, adrenal tissue [21]) [21] 0.01011 ( +/-0.00126, 38.1% inhibition by 0.4 mM (+)-metyrapol, adrenal tissue [21]) [21] 0.01081 ( +/-0.00269, 33.8% inhibition by 0.4 mM (-)-metyrapol, adrenal tissue [21]) [21] 0.01193 ( deoxycorticosterone, 11b-hydroxylation activity [4]) [4] 0.01633 ( +/-0.00237, no inhibitor, adrenal tissue [21]) [21] 1.35-1.36 ( corticosterone, enzyme incorporated into liposomes [9]) [9] 1.38 ( corticosterone, soluble form of enzyme [9]) [9] Additional information ( activities of sonicated mitochondria and cholate extract of hydroxylation activities with deoxycotricosterone and 4-androstene-3,17-dione as substrates [6]; several activities of 49.5 and 51.5 kDa proteins for multihydroxylation steps [7]; conversion of 11-deoxycorticosterone [12]; dependent on DLPC concentration, with 0.05 mM maximum activity, 2fold increase with addition of 15% glycerol, 0.6 mM GSH, 0.01 mM FAD and 0.01 mM FMN [24]) [6, 7, 12, 24] . / *', 0.0008 (11-deoxycorticosterone, presence of methyltrienolone [20]) [20] 0.00087 (11-deoxycorticosterone, without phospholipids [17]) [17] 0.0011 (11-deoxycorticosterone, with phospholipids [17]) [17] 0.0015 (11-deoxycorticosterone, +/-0.00008 [17]) [17] 0.00197 (11-deoxycorticosterone, +/-0.00026 [19]) [19] 0.002 (11-deoxycorticosterone, absence of methyltrienolone [20]) [20] 0.005 (11-deoxycorticosterone, dipalmitoyl-phosphatidylcholine vesicles [13]) [13] 0.0059 (corticosterone, +/-0.00018 [19]) [19] 0.006 (deoxycorticosterone, identical for both enzyme activities [6]) [6] 0.0084 (corticosterone, without phospholipids [17]) [17] 0.009 (corticosterone, +/-0.0009 [17]) [17] 0.01 (11-deoxycorticosterone, dipalmitoyl/diphytanoyl-phosphatidylcholine vesicles [13]) [13] 0.013 (4-androstene-3,17-dione, identical for both enzyme activities [6]) [6] 0.02 (deoxycorticosterone, identical for both enzymes activities [4]) [4] 0.033 (11-deoxycorticosterone, dipalmitoyl phosphatidylcholine/diphosphatidyl glycerol vesicles [13]) [13] 0.06-0.09 (corticosterone, presence of methyltrienolone [20]) [20] 0.077 (11-deoxycortisol, 11b-hydroxylation [24]) [24]

34

1.14.15.4

Steroid 11b-monooxygenase

0.087 (corticosterone, with phospholipids [17]) [17] Additional information ( kinetic parameters only slightly modified by presence of phospholipids, 0.2% soybean lecithin, [17]; lipids such as DLPC required [24]) [17, 24] 0  7 ( assay at [4]) [4] 7.4 ( assay at [1,2,6,7,9-12,15,17-22]; 30 mM sodium phosphate buffer [24]) [1, 2, 6, 7, 9-12, 15, 17-22, 24] 8 [3] 0 

 7-7.2 )  *
, 20 ( assay at [3]) [3, 5] 30 ( assay at [4,10,15,17-19]) [4, 10, 15, 17-19] 35 ( 30 mM sodium phosphate buffer [24]) [24] 37 ( assay at [1,2,6,7,9,11,12,20-22]) [1, 2, 6, 7, 9, 11, 12, 20-22] ) 

 *
, 2-15 ( 2fold decrease of activity with increase of temperature, temperatures above 25 C unfavourable [3]) [3] 2-50 ( at 50 C almost no activity [5]) [5]

  #   ' 1  185000 ( exclusion chromatography [4]) [4] 1000000 ( glycerol density gradient [4]) [4]   ? ( x * 47500, SDS-PAGE [4]; x * 49500 of capsular portion and x * 51500 of capsular and decapsular portion, 11b-hydroxylase activities, SDS-PAGE [7]; x * 48000, SDS-PAGE [9]; x * 47500 [10]; x * 46000, two-dimensional gel [10]; x * 48500-51500, SDS-PAGE after Sepharose, x * 48400/49500, SDS-PAGE after HPLC [11]; x * 49000 and x * 51000, SDS-PAGE [12]; x * 46000 [14]; x * 48500 major form, x * 49500 isoform, SDS-PAGE [17]; x * 55555, calculated from full-length cDNA of 1.5 kb [22]; x * 51000, SDS-PAGE, enzyme expressed from mitochondria of interregnal tissue [23]; x * 60000, SDS-PAGE [24]) [4, 7, 9-12, 14, 17, 22, 23, 24] tetramer ( 4 * 47500, SDS-PAGE [4]) [4]

35

Steroid 11b-monooxygenase

1.14.15.4

 2 %$ %' %

% adrenal cortex ( capsular portion in zona glomerulosa cells, decapsular in zonae fasciculata and reticularis [7,12]) [2, 4-20, 25, 26] adrenal gland [1, 21, 22] brain [25] interrenal cell ( corresponding to mammalian adrenal gland, P450(11b,aldo) and adrenodoxin reductase exist more in outer regions of frogs during the spring and the summer [23]) [23] liver [1, 21] mycelium [24] plasma ( pharmacokinetics of metyrapone and metyrapol [21]) [21] testis [26] 3#

microsome ( CYP17 [26]) [24, 26] mitochondrion ( cytochrome P-45011b in inner mitochondrial membrane, adrenodoxin reductase and adrenodoxin are loosely associated, inclusion of purified P-45011b into phospholipid vesicles mimics the situation in vivo [9]; from zona glomerulosa [10,13,15,16]; from zona fasciculata [10,13,15,16]; from zona reticularis [13,16]; inner membrane, all 3 zones [11]; reconstitution of cytochrome P45011b, an intrinsic protein of inner membrane, into phospholipid vesicles via detergent dialysis procedure [13]; from zona glomerulosa and fasciculata [15]; CYP11B1 in zona fasciculata, CYP11B2 in zona glomerulosa, new CYP11B3 [25]; CYP11 [26]) [1, 2, 4-15, 17-20, 22, 23, 25, 26] $"

(copurification of 11b-hydroxylase with 18-hydroxylase, ammonium sulfate, DEAE-chromatography [4]; copurification of 11b-/18-hydroxylase with 11b-/19-hydroxylase, cholate extraction [6]; sonication, acetone and butanol extraction, Triton extraction [8]; ammonium sulfate precipitation, octyl-Sepharose adsorption with cholate and phosphotidylcholine extraction [9]; copurification of 11b-/18-hydroxylase, cholate extraction, ammonium sulfate precipitation, Tween extraction, octymine-Sepharose [10]; Na-cholate extraction, ammonium sulfate precipitation, aniline-Sepharose chromatography, hydroxylapatite HPLC [11]; without ionic detergents, ammonium sulfate fractionation, hexyl agarose [13]; ultracentrifugation, ammonium sulfate precipitation, w-aminooctyl-Sepharose, adrenodoxin-Sepharose, no glycerol used [14]; copurification of P-450 11b-2 and 11b-3, ammonium sulfate precipitation, octyl-Sepharose, separation of 2 isoforms by hydroxyapatite column connected to an FPLC system [17-19]; RP HPLC for detection of photolabeled peptide after digest with TPCK-treated trypsin of purified cytochrome P45011b, which is photolabeled with radioactive methyltrienolone [20]) [4, 6, 9-11, 13, 14, 17-20]

36

1.14.15.4

Steroid 11b-monooxygenase

(cholate extraction, hydroxyapatite HPLC [7]; octyl-Sepharose chromatography, cholate extraction [12]) [7, 12] (ammonium sulfate fractionation, ion-exchange chromatography [3]; Triton X-100, sodium cholate, column chromatographies [24]) [3, 24]
 

( 2 P-45011b cDNAs, pc P-450 11b-3 and -2, cloned into and expressed in COS-7 cells [17]) [17] ( CYP11B3 enzyme cDNA cloned into and expressed in COS-7 cells [25]) [25] ( cDNA transfected into COS-7 cells simultaneously with bovine adrenodoxin cDNA expression plasmid [16]) [16] ( full-length P45011B1 cDNA cloned into and expressed in lung fibroblast derived V79 Chinese hamster cells [22]) [22] ( P-450(11b,aldo) expressed in COS-7 cells [23]) [23] 

 

G59S ( mutation in CYP11B3, 5-6fold reduction of activity [25]) [25] Additional information ( all enzymes with aldosterone synthesis activity have a glycine residue at position 288, in nonaldosterone synthesising P45011b it is valine or serine [16]) [16] 

medicine ( inhibitors for P-45011b, against aldosterone overproduction which leads to oedematous diseases and hypertension [17-19]) [17-19]

4  )  2 ( 14% residual activity of 11b- and 18-hydroxylation [5]) [5] 2 ( crude extract, 7 mg/ml, 24 h, loss of half the activity, DEAE fraction unstable [3]) [3] 30 ( after preincubation for 20 min, 27% residual activity, after 60 min 5.7% [5]) [5] 30 ( in presence of phospholipids, catalytic activity stable to extended incubation and increased resistance of hemoprotein to denaturation [17]) [17] 30 ( 30 min, 30 mM sodium phosphate buffer, pH 8.0, decrease to 30%, improved to 45-70% by addition of 1 mM GSH, 0.2 mM 11-deoxycortisol, 0.5 mM PMSF or 20% glycerol [24]) [24] 50 ( complete inactivation [5]) [5] 5    "

, deoxycorticosterone stabilizes [4, 5] , deoxycorticosterone, protection of enzyme from inactivation by 18ethynylprogesterone [18] , inclusion into phospholipid vesicles stabilizes [9, 13]

37

Steroid 11b-monooxygenase

1.14.15.4

, phospholipid vesicles, 0.2% soybean lecithin, added to purified P45011b for storage and during incubation [17, 18] , 11-deoxycortisol, partially stabilizes [24] , EDTA, stabilizes during purification [3] , GSH, partially stabilizes [24] , GSH, stabilizes during purification, as well as other sulfhydryl compounds [3] , PMSF, partially stabilizes [24] , Tween 80, positive influence on activity conservation during extraction and fractionation [3] , a-lipoic acid, during DEAE chromatography stabilizes [3] , glycerol, partially stabilizes [24]    , -20 C, 50% glycerol, 1 week, without significant loss of activity [4] , -80 C, 20 mM potassium phosphate buffer, pH 7.4, 0.01 mM 11-deoxycorticosterone, 0.1 mM EDTA, 0.1 mM dithiothreitol, 1%, w/v, sodium cholate [20] , -80 C, 59 mM potassium phosphate buffer, pH 7.4, 0.1 mM EDTA, 0.1 mM dithiothreitol, 0.01 mM deoxycorticosterone, 0.3% cholate, 0.3% Tween 20 [6] , -80 C, after ammonium sulfate precipitation, 50 mM potassium phosphate, pH 7.4, 0.1 mM EDTA, 0.1 mM dithiothreitol, 0.5% sodium cholate, 0.5% soybean lecithin, several months, without loss of activity [17] , -80 C, after hydroxyapatite column/FPLC system, 50 mM potassium phosphate, pH 7.4, 0.01% sodium cholate, 0.05% soybean lecithin, 0.01 mM 11-deoxycorticosterone, 0.1 mM dithiothreitol, 0.1 mM EDTA, without loss of activity [17] , -80 C, after octyl-Sepharose, deoxycorticosterone-bound, 50 mM potassium phosphate, pH 7.4, 0.5% sodium cholate, 0.2% Tween 20, 0.01 mM 11deoxycorticosterone, 0.1 mM dithiothreitol, 0.1 mM EDTA [17, 18] , 4 C, 1 week, without significant loss of activity [4] , 4 C, 11 days, both activities decrease considerably in a similar way, deoxycorticosterone delays decline [5] , 4 C, enzyme incorporated into liposomes, stable during 2 weeks [9] , 4 C, soluble form, loses part of activity in few days, after 2 weeks 1/4 of original activity observed [9] , 4 C, sonicated mitochondria, stable [4] , blood samples, stable, 4 C [21] , -30 C, extract precipitate stable, solution less stable, DEAE fraction unstable [3] , -30 C, preserved by Tween 80, conservation of activity [3] , -80 C, purified, 200 mM sodium phosphate buffer, pH 8.0, 0.2 mM 11deoxycortisol, 20% glycerol, 0.1 mM GSH, 0.1% sodium cholate, several days without loss of activity [24] , 0 C, preserved by Tween 80, conservation of activity [3] , 2 C, crude extract loses 50% activity in 24 h [3] , purified or partially purified enzyme unstable during storage [3]

38

1.14.15.4

Steroid 11b-monooxygenase

"  [1] Tomkins, G.M.; Michael, P.J.; Curran, J.F.: Studies on the nature of steroid 11-b hydroxylation. Biochim. Biophys. Acta, 23, 655-656 (1957) [2] Grant, J.K.; Brownie, A.C.: The role of fumarate and TPN in steroid enzymic 11b-hydroxylation. Biochim. Biophys. Acta, 18, 433-434 (1955) [3] Zuidweg, M.H.J.: Hydroxylation of Reichstein's compound S with cell-free preparations from Curvularia lunata. Biochim. Biophys. Acta, 152, 144-158 (1968) [4] Watanuki, M.; Tilley, B.E.; Hall, P.F.: Purification and properties of cytochrome p-450 (11b- and 18-hydroxylase) from bovine adrenocortical mitochondria. Biochim. Biophys. Acta, 483, 236-247 (1977) [5] Watanuki, M.; Tilley, B.E.; Hall, P.F.: Cytochrome P-450 for 11b- and 18hydroxylase activities of bovine adrenocortical mitochondria: one enzyme or two?. Biochemistry, 17, 127-130 (1978) [6] Sato, H.; Ashida, N.; Suhara, K.; Itagaki, E.; Takemori, S.; Katagiri, M.: Properties of an adrenal cytochrome P-450 (P-45011b) for the hydroxylations of corticosteroids. Arch. Biochem. Biophys., 190, 307-314 (1978) [7] Ogishima, T.; Mitani, F.; Ishimura, Y.: Isolation of aldosterone synthase cytochrome P-450 from zona glomerulosa mitochondria of rat adrenal cortex. J. Biol. Chem., 264, 10935-10938 (1989) [8] Cooper, D.Y.; Schleyer, H.; Levin, S.S.; Rosenthal, O.: Studies on the partially purified heme protein P-450 from the adrenal cortex. Ann. N.Y. Acad. Sci., 212, 227-242 (1973) [9] Lombardo, A.; Defaye, G.; Guidicelli, C.; Monnier, N.; Chambaz, E.M.: Integration of purified adrenocortical cytochrome P-45011b into phospholipid vesicles. Biochem. Biophys. Res. Commun., 104, 1638-1645 (1982) [10] Yanigabashi, K.; Haniu, M.; Shively, J.E.; Shen, W.H.; Hall, P.: The synthesis of aldosterone by the adrenal cortex. Two zones (fasciculata and glomerulosa) possess one enzyme for 11b-, 18-hydroxylation, and aldehyde synthesis. J. Biol. Chem., 261, 3556-3562 (1986) [11] Ogishima, T.; Mitani, F.; Ishimura, Y: Isolation of two distinct cytochromes P-45011b with aldosterone synthase activity from bovine adrenocortical mitochondria. J. Biochem., 105, 497-499 (1989) [12] Lauber, M.; Muller, J.: Purification and characterization of two distinct forms of rat adrenal cytochrome P450(11) b: functional and structural aspects. Arch. Biochem. Biophys., 274, 109-119 (1989) [13] Seybert, D.: Lipid regulation of bovine cytochrome P45011b activity. Arch. Biochem. Biophys., 279, 188-194 (1990) [14] Tsubaki, M.; Ichikawa, Y.; Fujimoto, Y.; Yu, N.T.; Hori, H.: Active site of bovine adrenocortical cytochrome P-45011b studied by resonance Raman and electron paramagnetic resonance spectroscopies: distinction from cytochrome P-450scc . Biochemistry, 29, 8805-8812 (1990) [15] Yanigabashi, K.; Kobayashi, Y.; Hall, P.F.: Ascorbate as a source of reducing equivalents for the synthesis of aldosterone. Biochem. Biophys. Res. Commun., 170, 1256-1262 (1990)

39

Steroid 11b-monooxygenase

1.14.15.4

[16] Boon, W.C.; Roche, P.J.; Butkus, A.; McDougall, J.G.; Jeyaseelan, K.; Coghlan, J.P.: Functional and expression analysis of ovine steroid 11b-hydroxylase (cytochrome P 45011b). Endocr. Res., 23, 325-347 (1997) [17] Delorme, C.; Piffeteau, A.; Viger, A.; Marquet, A.: Inhibition of bovine cytochrome P-45011b by 18-unsaturated progesterone derivatives. Eur. J. Biochem., 232, 247-256 (1995) [18] Delorme, C.; Piffeteau, A.; Sobrio, F.; Marquet, A.: Mechanism-based inactivation of bovine cytochrome P-45011b by 18-unsaturated progesterone derivatives. Eur. J. Biochem., 248, 252-260 (1997) [19] Davioud, E.; Piffeteau, A.; Delorme, C.; Coustal, S.; Marquet, A.: 18-Vinyldeoxycorticosterone: a potent inhibitor of the bovine cytochrome P45011b. Bioorg. Med. Chem., 6, 1781-1788 (1998) [20] Ohnishi, T.; Miura, S.; Ichikawa, Y.: Photoaffinity labeling of cytochrome P45011b with methyltrienolone as a probe for the substrate binding region. Biochim. Biophys. Acta, 1161, 257-264 (1993) [21] Nagamine, S.; Horisaka, E.; Fukuyama, Y.; Maetani, K.; Matsuzawa, R.; Iwakawa, S.; Asada, S.: Stereoselective reductive metabolism of metyrapone and inhibitory activity of metyrapone metabolites, metyrapol enantiomers, on steroid 11b-hydroxylase in the rat. Biol. Pharm. Bull., 20, 188-192. (1997) [22] Denner, K.; Vogel, R.; Schmalix, W.; Doehmer, J.; Bernhardt, R.: Cloning and stable expression of the human mitochondrial cytochrome P45011B1 cDNA in V79 Chinese hamster cells and their application for testing of potential inhibitors. Pharmacogenetics, 5, 89-96 (1995) [23] Nonaka, Y.; Takemori, H.; Halder, S.K.; Sun, T.; Ohta, M.; Hatano, O.; Takakusu, A.; Okamoto, M.: Frog cytochrome P-450 (11b,aldo), a single enzyme involved in the final steps of glucocorticoid and mineralocorticoid biosynthesis. Eur. J. Biochem., 229, 249-256 (1995) [24] Suzuki, K.; Sanga, K.i.; Chikaoka, Y.; Itagaki, E.: Purification and properties of cytochrome P-450 (P-450lun) catalyzing steroid 11b-hydroxylation in Curvularia lunata. Biochim. Biophys. Acta, 1203, 215-223 (1993) [25] Zhou, M.Y.; Gomez-Sanchez, E.P.; Foecking, M.F.; Gomez-Sanchez, C.E.: Cloning and expression of the rat adrenal cytochrome P-450 11B3 (CYP11B3) enzyme cDNA: preferential 18-hydroxylation over 11b-hydroxylation of DOC. Mol. Cell. Endocrinol., 114, 137-145 (1995) [26] Kuhn-Velten, W.N.: Norharman (b-carboline) as a potent inhibitory ligand for steroidogenic cytochromes P450 (CYP11 and CYP17). Eur. J. Pharmacol., 250, R1-3 (1993)

40


  7    



   
 1.14.15.5    corticosterone,reduced-adrenal-ferredoxin:oxygen oxidoreductase (18-hydroxylating)    corticosterone 18-monooxygenase    corticosterone 18-hydroxylase corticosterone methyl oxidase oxygenase, corticosterone 18-mono
    37256-75-0

    



Bos taurus [1] Cavia porcellus [1] Ovis aries [1] Homo sapiens [2]

!    "
 # 

corticosterone + reduced adrenal ferredoxin + O2 = 18-hydroxycorticosterone + oxidized adrenal ferredoxin + H2 O    oxidation redox reaction reduction       corticosterone + reduced adrenal ferredoxin + O2 ( reaction in the biosynthesis of aldosterone [1]) (Reversibility: ? [1, 2]) [1, 2] $ 18-hydroxycorticosterone + oxidized adrenal ferredoxin + H2 O 41

Corticosterone 18-monooxygenase

1.14.15.5

     corticosterone + reduced adrenal ferredoxin + O2 (Reversibility: ? [1, 2]) [1, 2] $ 18-hydroxycorticosterone + oxidized adrenal ferredoxin + H2 O 2  1,2-bis(3-pyridyl)-2-methyl-1-propanone (SU 4885) [1] 18-hydroxycorticosterone ( product inhibition [1]) [1] 3-(1,2, 3,4-tetrahydr-1-oxo-2-naphthyl)pyridine (SU 9055) [1] Co2+ [1] Cu2+ [1] Fe3+ [1] Hg2+ [1] Mn2+ [1] NEM ( higher concentrations [1]) [1] SU 10603 ( less effective [1]) [1] Zn2+ [1] diethyldithiocarbamate [1] formamidine acetate [1] p-chloromercuribenzoate [1]
"%  NADPH ( activation, no activation by NADH, FAD, FMN, cytochrome c, GSH, dehydroascorbate, in vitro best stimulation by NADPH-regenerating system instead of exogenous NADPH [1]) [1] reduced adrenal ferredoxin [1] '(   Ca2+ ( increase of activity, Na+ , K+ alone or together are ineffective [1]) [1] 0  7.3 ( assay at [1]) [1] )  *
, 37 ( assay at [1]) [1]

 2 %$ %' %

% adrenal cortex [1] blood [2] 3#

mitochondrial crista [1, 2] mitochondrial membrane [1, 2]
 

(expression in COS7 cells [2]) [2]

42

1.14.15.5

Corticosterone 18-monooxygenase

4  5    "

, lyophilization, mitochondria lose 90% activity [1]

"  [1] Raman, P.B.; Sharma, D.C.; Dorfman, R.I.: Studies on aldosterone biosynthesis in vitro. Biochemistry, 5, 1795-1804 (1966) [2] Nomoto, S.; Massa, G.; Mitani, F.; Ishimura, Y.; Miyahara, K.; Toda, K.; Nagano, I; Yamashiro, T.; Ogoshi, S.; Fukata, J.I.; Onishi, S.; Hashimoto, K.; Doi, Y.; Imura, H.; Shizuta; Y.: CMO I deficiency caused by a point mutation in exon 8 of the human CYP11B2 gene encoding steroid 18-hydroxylase (P450C18). Biochem. Biophys. Res. Commun., 234, 382-385 (1997)

43


     *  &

,

4

   
 1.14.15.6    cholesterol,reduced-adrenal-ferredoxin:oxygen oxidoreductase (side-chaincleaving)    cholesterol monooxygenase (side-chain-cleaving)    C27-side chain cleavage enzyme CYPXIA1 P450(scc) cholesterol 20-22-desmolase cholesterol C20 22 desmolase cholesterol C20 -C22 lyase cholesterol desmolase cholesterol side-chain cleavage enzyme cholesterol side-chain-cleaving enzyme cytochrome P-450scc desmolase, steroid 20-22 endoenzymes, cholesterol side-chain-cleaving enzymes, cholesterol side-chain-cleaving steroid 20-22 desmolase steroid 20-22-lyase
    37292-81-2

     Bos taurus [1-22]

44

1.14.15.6

Cholesterol monooxygenase (side-chain-cleaving)

!    "
 # 

cholesterol + reduced adrenal ferredoxin + O2 = pregnenolone + 4-methylpentanal + oxidized adrenal ferredoxin + H2 O (a heme-thiolate protein. The reaction proceeds in three stages, with hydroxylation an C-20 and C-22 preceding scission of the side-chain at C-20)    oxidation redox reaction reduction       cholesterol + reduced adrenal ferredoxin + O2 ( first step in biosynthesis of all steroid hormones [1, 4, 6, 7, 9-12, 15, 17]) (Reversibility: ? [1, 4, 6, 7, 9-12, 15, 17-22]) [1, 4, 6, 7, 9-12, 14, 15, 17-22] $ pregnenolone + oxidized adrenal ferredoxin + H2 O ( isocaproic aldehyde additional product [15]; isocapraldehyde additional product [17, 20]; precursor of all steroid hormones [18, 19, 21, 22]) [1, 4, 6, 7, 9-12, 14, 15, 17-22]      (20S)-22-thiacholesterol + reduced adrenodoxin + O2 (Reversibility: ? [17]) [17] $ (20S,22R)-22-thiacholesterol S-oxide + (20S,22S)-22-thiacholesterol Soxide ( (22R)-sulfoxide preferentially formed by a factor of 4.2 to 1 over (22S)-sulfoxide [17]) [17]  20a-hydroxycholesterol + reduced adrenodoxin + O2 (Reversibility: ? [7]) [7] $ pregnenolone + oxidized adrenodoxin + H2 O [7]  cholesterol + reduced adrenodoxin + O2 ( 20- or 22-hydroxycholesterol and 20,22-dehydroxycholesterol are putative intermediates [5]; stable intermediates: 22-hydroxycholesterol and 20,22-dihydroxycholesterol [6]; without demonstrable accumulation of putative intermediates 20a-hydroxycholesterol and 20a,22-dehydroxycholesterol [7]; intermediates 22R-hydroxycholesterol and 20a,22R-dihydroxycholesterol from hydroxylation [10]; intermediates (22R)-22-hydroxycholesterol and (20R,22R)-20,22-dihydroxycholesterol from hydroxylation [10,14]; regioselective and stereospecific transformation [10]; hydroxylation of cholesterol at C-22, then C-20, followed by oxidative scission of the glycol to get pregnenolone [14]) (Reversibility: ? [1-22]) [1-22] $ pregnenolone + oxidized adrenodoxin + H2 O ( isocaproaldehyde produced besides [11]) [1-22]  cholesterol sulfate + reduced adrenodoxin + O2 (Reversibility: ? [15]) [15]

45

Cholesterol monooxygenase (side-chain-cleaving)

1.14.15.6

$ pregnenolone sulfate + 17-hydroxy-pregnenolone + dehydroisoandrosterone sulfate + oxidized adrenodoxin + H2 O [15]  Additional information ( suggested shuttle mechanism in which adrenodoxin transfers electrons between adrenodoxin reductase and cytochrome P-450. No ternary adrenodoxin-adrenodoxin reductase-P-450 complex [1-3, 10]; reduction of adrenodoxin must be rate-limiting step [1]; dissociation of oxidized adrenodoxin from P-450scc must be potential rate-limiting factor [2, 3]; cholesterol side chain cleavage activity is dependent on free reduced adrenodoxin [3]; one-enzymethree-step hypothesis [5]; transfer of cholesterol to cytochrome P450scc is rate-limiting [9]; electron shuttle between flavoprotein and cytochrome, biphasic reduction kinetics, rapid association of cytochrome and adrenodoxin, slower intracomplex electron transfer from iron-sulfur center of adrenodoxin to heme of cytochrome P-450scc [10]; only a single active site of enzyme [14]; 3 high regioselective and stereospecific oxidation steps at the same heme active site [17]; 21 amino-acid residues from Pro-8 to Arg-28 in the amino-terminus are located in or near the cholesterol-binding site, this site is in a quite different region from the adreno-ferredoxin binding site Pro374 to Ile389 [21]; 2 interaction processes with 2 different sensitivities to ionic strength [22]) [13, 5, 8, 10, 14, 17, 21, 22] $ ? 2  (20R,S)-20-amino-5-pregnen-3b-ol ( 20-amine derivative, amine is attached closer to the d-ring than in the 22-amine, very weak inhibitor, 0.1 mM causes less than 20% inhibition [13]) [13] (20S)-22-thiacholesterol ( competitive inhibitor, is converted enzymatically to a more potent inhibitor, the (22S) and (22R) sulfoxides, inhibition by approximately 75% at 0.001 mM, no inactivation in absence of NADPH and O2 [17]) [17] (20S,22R)-22-thiacholesterol S-oxide ( competitive versus cholesterol, binds 10 times more tightly than (22S) diastereomer, 25 and 44% inhibition at 0.00005 and 0.0001 mM, respectively, complete inhibition at 0.001 mM, no substrate for P-450 [17]) [17] (20S,22S)-22-thiacholesterol S-oxide ( competitive versus cholesterol, no substrate for P-450 [17]) [17] (22R)-22-aminocholesterol ( completely inhibited by 0.001 mM, affinity toward the P-450scc almost 3fold greater than that for the (22S)-form, competitive versus cholesterol, no substrate for P-450 [11]) [11] (22S)-22-aminocholesterol ( not inhibited below 0.001 mM, competitive versus cholesterol, no substrate for P-450 [11]) [11] 17b-amino-5-androsten-3b-ol ( 17-amine derivative, amine is attached closer to the d-ring than in the 22-amine, very weak inhibitor, 0.1 mM causes less than 20% inhibition [13]) [13] 22-amino-23,24-bisnor-5-cholen-3b-ol ( 22-amine derivative, same steroid ring structure as cholesterol, competitive inhibitor with respect to

46

1.14.15.6

Cholesterol monooxygenase (side-chain-cleaving)

cholesterol, 50% reversible inhibition at 0.0001 mM, reversible cooperative binding [13, 14]; dual interaction: binding of steroid ring to cholesterol site and binding of the amine to the heme iron. Need not be metabolically activated in order to inhibit the enzyme, not metobolized to pregnenolone. 60% competitive inhibition at 0.00015 mM [14]) [13, 14] 22-amino-23,24-bisnor-5a-cholen-3b-ol ( 50% inhibition at 0.003 mM [13, 14]) [13, 14] 23,24-bisnor-5-cholene-3b,22-diol ( competitive inhibitor, 40% inhibition at 0.01 mM, 50% at 0.015 mM, resembles the intermediate 22-hydroxycholesterol but acts as an inhibitor rather than serving as a substrate [14]) [14] 23-amino-24-nor-5-cholen-3b-ol ( 23-amine derivative, same steroid ring structure as cholesterol, competitive inhibitor with respect to cholesterol, 50% inhibition at 0.0001 mM, reversible cooperative binding [13]) [13] 24-amino-5-cholen-3b-ol ( 24-amine derivative, amine attached in greater distance from steroid ring, same steroid ring structure as cholesterol, causes a progressive decrease in inhibitory potency, 50% inhibition at 0.0023 mM, reversible noncooperative binding [13]) [13] 25-amino-26,27-bisnor-5-cholesten-3b-ol ( 25-amine derivative, amine attached in greater distance from steroid ring, causes a progressive decrease in inhibitory potency, 50% inhibition at more than 0.1 mM [13]) [13] Ca2+ ( inhibits side chain cleavage activity optimally activated by 100 mM NaCl [2]; CaCl2 inhibits side chain cleavage activity at 100 mM NaCl, 50% inhibition with 0.07 mM in Tween 20 and 0.03 mM for vesicle-incorporated, Ca2+ does not affect binding of either cholesterol of oxidized adrenodoxin to P-450scc [3]) [2, 3] adrenodoxin ( oxidized form, high affinity to P-450scc , inhibits side chain cleavage by competition with reduced form [1, 3]) [1, 3] cholesterol ( inhibition above 0.003 mM, mitochondrial [15]) [15] cholesterol sulfate ( inhibition above 0.005 mM, mitochondrial [15]) [15] methoxychlor ( pesticide of DDT, suicide inhibitor, competitive to cholesterol, substantial irreversible loss of activity, 5% inhibition within 5 min at 0.2 mM, decrease is suppressed by the presence of cholesterol [21]) [21] phosphatidyl choline [4] phosphatidyl ethanolamine [4] Additional information ( cholesterol analogues have shortened side chain and primary amine group, tested in presence of 0.07 mM cholesterol [13,14]; steroid ring is suggested to bind to the substrate site on the enzyme and the amine is coordinated to the heme iron [13]; amine binding to heme is important for tight inhibitor potency rather than the 5-androstene ring, 23,24-bisnor-5-cholen-3b-ol-22-carboxamide is ineffective as inhibitor, side chain carbons 23-27 may play some role in positioning the substrate for hydroxylation [14]; no substrate inhibition with cytosolic enzyme, inhibitor of cholesterol-side-chain cleavage isolated from the cytosol of bovine 47

Cholesterol monooxygenase (side-chain-cleaving)

1.14.15.6

adrenal cortex [15]; mechanism-based inhibition, no inhibition with (20R)-22-thiacholesterol at 0.001 mM, inhibition by occupying the cholesterol binding pocket [17]; inhibitors bind to substrate-heme complex, longer chain compounds possess inhibitory activity, phenyl ethyl compounds not [20]; inactivation different in absence and presence of cholesterol, protective effect of cholesterol as well as 20a-hydroxycholesterol and deoxycorticosterone against inactivation by methoxychlor [21]) [13-15, 17, 20, 21]
"%  NADPH ( NADPH-linked adrenodoxin reductase [1-3, 6, 8, 10, 12, 15, 17-19, 21]) [1-22] adrenodoxin ( transports electrons from adrenodoxin reductase to cytochrome P-450 by shuttling [1-3]; required [9]; promotes cholesterol binding [14]; electron shuttle [22]) [1-10, 12-22] heme ( low heme content [4]; haemoprotein [6]; 8 heme groups per molecule P-450 [7]; 0.78 heme group/unit of 48000 Da P-450, 1 mol of iron/44000 g [12]; 3 mixed function oxidation cycles at the single heme center [14]; 8 molecules of meme per molecule of enzyme [16]; content of 18.9 nmol/mg protein [21]; hemeprotein [21]) [2, 4, 6, 7, 10-12, 14, 16-21] &

  1,2-di-(2'-hexyl-decanoyl)-sn-glycero-3-phosphocholine ( abranched phophatidylcholine, inclusion in vesicle-reconstituted system, partially in connection with the nonactivator lipids dimyristoyl-/dioleoyl-phosphatidylcholine, efficiency close to cardiolipin [18]) [18] 1,2-di-(2'-octyl-dodecanoyl)-sn-glycero-phosphocholine ( abranched phophatidylcholine, inclusion in vesicle-reconstituted system, partially in connection with the nonactivator lipids dimyristoyl-/dioleoyl-phosphatidylcholine, efficiency close to cardiolipin [18]) [18] Brij ( 56, 76 and 96 [5]) [5] Emulgen ( 911 and 913 [5]) [5] Triton X-100 ( effect onto P-450 itself suggested [5]) [5] Tween 20 ( small stabilizing and binding activating effect on ternary complex of adrenodoxin-cholesterol-P-450scc [2]) [2, 5] cardiolipin ( affinity of P-450scc for cholesterol is increased [9]; most potent and effective activator lipid, binds to enzyme and enhances the binding of cholesterol [18]) [9, 18] fatty acid ( C18 , natural detergents in DMPC vesicles, stimulation similar to octyl glucoside [9]) [9] glycerol ( 5-20% concentration, 20-50% increase of enzyme activity [7]) [7] lipid ( from adrenal mitochondria accelerates activity [7]) [7] octyl glucoside ( high concentrations of this detergent cause 50% stimulation of cholesterol side-chain-cleavage in large unilamellar vesicles at low cholesterol concentration, 0.01 mM increase the proportion of P-450 bound by cholesterol [9]) [9]

48

1.14.15.6

Cholesterol monooxygenase (side-chain-cleaving)

phospholipid ( cholesterol gains access to the cytochrome within micelles [2]; inclusion of P-450 into vesicles [3]; P-450scc reconstituted in vesicles, cholate-solubilized for small unilamellar dioleoylphosphatidylcholine vesicles, non-ionic detergent n-octyl glucoside used for large ones [9]) [2, 3, 9] Additional information ( activator of cholesterol side-chain cleavage isolated from the cytosol of bovine adrenal cortex [15]; enzymatic activity is greatest at low ionic strength with a number of different ions [16]; head and fatty acid groups have large effects on stimulation of activity, head group appears as major determinant of the lipid interaction with the P-450scc , 1-2 and 3-4 molecules of cardiolipin and the branched phophatidylcholines, respectively, bind highly specific to P-450scc at an effector site distant from cholesterol binding site with a resultant stabilization of an optimal cholesterol-binding conformation of P-450scc [18]) [15, 16, 18] '(   Ca2+ ( very little [2]; particulary but also other metal ions modulate adrenodoxin binding to adrenodoxin reductase and P-450, therefore activation of cholesterol side chain cleavage and adrenodoxin reduction [3]; ineffective activator [3]; increased rate of association of cholesterol to partially purified cytochrome P-450scc [6]) [2, 3, 6] Mg2+ ( increased MgCl2 concentrations continue to increase side chain cleavage activity even after all adrenodoxin is reduced [3]) [2, 3] Additional information ( Kd for adrenodoxin increases with increase of ions from NaCl, binding of adrenodoxin to P-450scc is more sensitive to changes in metal ion concentration than cholesterol binding, high salt concentrations cause fall-off in activity [2]; increase of side chain cleavage activity from 50 to 100 mM NaCl, higher concentrations cause a decrease, total loss at 300 mM [3]) [2, 3] Additional information ( several cations and anions promote the dissociation of adrenodoxin-cytochrome P-450scc complex, decreasing order: K+ , Cs+ , Rb+, Na+ , Li+ and SCN- , Br-, Cl- , respectively. The order for dissotiation of adrenodoxin-adrenodoxin reductase complex: Rb+, Cs+ , K+ , Li+ , Na+ [10]) [10] ) &  * +, 3 (cholesterol) [11, 17] 25-30 (cholesterol, by large unilamellar vesicles [9]) [9] Additional information ( for small unilamellar vesicles 5fold lower [9]) [9] . / *', 0.0003 (cholesterol sulfate, mitochondrial [15]) [15] 0.0004 (adrenodoxin, free [1]) [1] 0.0005 (cholesterol, mitochondrial [15]) [15] 0.00056 (adrenodoxin, +/-0.00004, complex between adrenodoxin and adrenodoxin reductase [1]) [1] 0.0012 (adrenodoxin) [2]

49

Cholesterol monooxygenase (side-chain-cleaving)

1.14.15.6

0.002 (cholesterol, low concentration, both vesicle types [9]) [9] 0.012 (20a-hydroxycholesterol) [7] 0.0165 (cholesterol, cytosolic [15]) [15] 0.0232 (cholesterol sulfate, cytosolic [15]) [15] 0.164 (cholesterol) [21] 0.19 (cholesterol) [7] 0.3 (cholesterol, large unilamellar vesicles, second type of reaction [9]) [9] 0.39 (cholesterol) [2] Additional information ( Km for reduced adrenodoxin is approximately equal to the Ki for oxidized adrenodoxin at 80 mM NaCl [3]) [3] 0  6.8 ( for cholesterol and 20a-hydroxycholesterol, 75 mM potassium phosphate buffer [7]) [7] 7 ( assay at [5, 7, 10]; reduction of P-450scc -cholesterol complex by reduced adrenodoxin [10]) [5, 7, 10] 7.2 ( assay at [1, 3, 9, 11, 13, 14, 17, 21, 22]; maximal adrenodoxin-induced absorbance with 0.03 mM cholesterol [10]) [1, 3, 9-11, 13, 14, 17, 21, 22] 7.4 ( assay at [4, 6, 12]) [4, 6, 12] 0 

 6-7.5 ( less than 50% of maximal activity below and above range [7]) [7] )  *
, 37 ( assay at [1-4, 6, 7, 9, 11, 12, 14, 17, 18, 20, 21]) [1-4, 6, 7, 9, 1114, 17, 18, 20, 21] ) 

 *
, 15-42 ( tested in different dimyristoyl-phosphatidylcholine vesicles, breaks in activity at 27-30 C [18]) [18]

  #   ' 1  97000 ( sedimentation equilibrium [12]) [12] 100000 ( native-PAGE, incomplete enzyme or molecular environmental conditions not optimal [12]) [12] 200000 ( sedimentation equilibrium analysis [8]; native-PAGE, lowest single unit with high enzymatic activity [12]; sedimentation equilibrium performed in 100 mM potassium phosphate buffer, pH 7.6 [16]) [8, 12, 16] 220000 ( corpus luteum [12]) [12] 225000 ( sedimentation equilibrium [12]) [12] 400000 ( native-PAGE [12]) [12] 415000 ( sedimentation equilibrium [12]) [12] 50

1.14.15.6

Cholesterol monooxygenase (side-chain-cleaving)

470000 ( sedimentation equilibrium performed in 100 mM potassium phosphate buffer, pH 7.6 [16]) [16] 800000 ( exclusion chromatography [7]) [7] 850000 ( sedimentation equilibrium analysis [7]; adrenal mitochondria [12]) [7, 12, 16]   ? ( x * 60000, SDS-PAGE, molecular weights of bovine P-450 reported: 1 * 46000 and x * 53000 [4]; 2,4,6,8 * 48000, SDS-PAGE, corpus luteum. x * 46000, x * 60000, x * 53000, x * 52000-53000 adrenal mitochondria [12]; single band, SDS-PAGE [14]) [4, 12, 14] hexadecamer ( 16 * 53000, sedimentation equilibrium performed in 6 M guanidine after heating, 16 * 52000, SDS-PAGE. Can also exist in forms of 4 and 8 subunits after treatment with 100 mM potassium phosphate buffer, pH 7.6, with molecular weights of 200000 and 470000, respectively [16]) [16] tetramer ( 4 * 46000, sedimentation equilibrium analysis after guanidine treatment and SDS-PAGE [8]) [8]

 2 %$ %' %

% adrenal cortex [1-10, 13-22] adrenal gland [12] corpus luteum [12] 3#

cytosol [15] mitochondrial inner membrane ( integral to membrane [1, 3, 10]; adrenodoxin reductase and adrenodoxin are peripheral proteins on matrix side of inner membrane, P-450scc is integral membrane protein [2,3]; electron carriers and P-450 liquid-bound within mitochondrion [7]; matrix side [9, 18]; inner membrane [15, 18, 19, 22]) [1-22] $"

(affinity chromatography [1]; ammonium sulfate fractioning, aniline-Sepharose, adrenodoxin-Separose [2]; DEAE-cellulose, adrenodoxin-Sepharose [3, 9]; cholate extraction, diphosphate treatment, affinity chromatography [4,5]; iso-octane and ammonium sulfate fractionating, gel filtration [6]; cholic acid extraction, ammonium sulfate precipitation, DEAE-cellulose, hydroxylapatite and gel filtration [7, 16]; ammonium sulfate precipitation, affinity chromatography [8, 9]; several ion exchange and heptyl-Sepharose hydrophobic chromatography [12]; column chromatography, gel filtration [15]; precipitation with polyethylene glycol, affinity chromatography [19, 22]) [119, 22]
 

(mutants and wild-type expressed in Escherichia coli [19, 22]) [19, 22]

51

Cholesterol monooxygenase (side-chain-cleaving)

1.14.15.6



 

K103Q ( decrease of stability [19]; expression similar to wildtype, decreased stability or an altered heme or substrate pocket, B'-C loop, no change in interaction of P-450scc and adrenodoxin [22]) [19, 22] K104Q ( dramatic decrease in expression level [19]; decreased expression, B'-C loop, no change in interaction of P-450scc and adrenodoxin [22]) [19, 22] K109Q ( dramatic changes in folding and heme insertion [19]; change in folding, thus, an inability of heme to be retained, helix C, no change in interaction of P-450scc and adrenodoxin [22]) [19, 22] K110Q ( folding and heme insertion not affected [19]; expression similar to wild-type, helix C, does not appear to play a role in adrenodoxin binding because it shows no change in interaction of P-450scc and adrenodoxin [22]) [19, 22] K145Q ( expression similar to wild-type, helix D [22]) [19, 22] K148Q ( expression level is not affected, extreme instable and rapid denaturation [19]; extremely unstable, helix D [22]) [19, 22] K267Q ( participation in electrostatic interaction of P-450scc with adrenodoxin [21]) [21] K338Q ( removed from heme group but still very important for interaction with adrenodoxin, K helix [22]) [22] K342Q ( removed from heme group but still very important for interaction with adrenodoxin, K helix [22]) [22] K394Q ( functional parameters decreased because substitution is close to 405 position, but to much lower extend [19]; expression similar to wild-type, ªmeanderª, involved in the interaction of P-450scc with its electron-transfer partners [22]) [19, 22] K403Q ( functional parameters decreased because substitution is close to 405 position, but to much lower extend, shows importance of this residue for electrostatic interaction with negatively charged residues of ferredoxin [19]; participation in electrostatic interaction of P-450scc with adrenodoxin [21]; expression similar to wild-type, located between the meander and the heme-binding region, important role in electrostatic interactions with adrenodoxin, ability to bind adrenodoxin affected to lower extent than K405Q [22]) [19, 21, 22] K405Q ( dramatic loss of activity, shows importance of this residue for electrostatic interaction with negatively charged residues of ferredoxin [19]; participation in electrostatic interaction of P-450scc with adrenodoxin [21]; expression similar to wild-type, 4fold decrease in efficiency of enzymatic reduction by adrenodoxin and adrenodoxin reductase, and a 3.3fold decrease of cholesterol side chain cleavage activity, located between the meander and the heme-binding region, important role in electrostatic interactions with adrenodoxin [22]) [19, 21, 22] R425Q ( most harmful substitution, L-helix, heme-binding region [22]) [22] R425Q/R426C ( double mutant, most harmful substitution [22]) [22] R425Q/R426Q ( double mutant, most harmful substitution [22]) [22] 52

1.14.15.6

Cholesterol monooxygenase (side-chain-cleaving)

R426Q ( participation in electrostatic interaction of P-450scc with adrenodoxin [21]; expression similar to wild-type, serious changes in proteine folding and ability to insert and bind heme correctly, unable to catalyze cholesterol side chain cleavage reaction, although it is able to bind cholesterol, L-helix, heme-binding region, important role in electrostatic interactions with adrenodoxin [22]) [21, 22] Additional information ( site-directed mutagenesis, no evident changes in functional properties for KQ mutants, 103, 110, 145, 394 and 403 with activities between 69 and 86% of wild-type [19]; site directed mutagenesis. Complex stabilizing salt bridges: K403 of P-450scc with D76 of adrenodoxin, K405 with D72, R426 with E73 and K267 with E47, multiple electrostatic interactions between the negatively charged residues of adrenodoxin and positively charged groups of P-450scc [22]) [19, 22] 

medicine ( treatment of certain hormone-related pathologies [17]) [17]

4  )  Additional information ( increase of complex formation of adrenodoxin and cytochrome P-450scc in absence of cholesterol with decrease of temperature to 6 C, elevated temperature decreases the affinity of P-450scc for cholesterol [2]) [2] 5    "

, catalase, protects P-450scc against activity loss [11] , glycerol, 50%, stabilizes [7]    , -20 C, 10 mM sodium phosphate buffer, pH 7.4, 0.1 mM EDTA, 20% glycerol, stable for months [4] , -20 C, 50 mM potassium phosphate buffer, pH 7.0, 50% glycerol, 3 months [7] , -20 C, 50% glycerol, for months [5] , -20 C, mitochondrial precipitate, 10 mM sodium phosphate buffer, pH 7.4, 0.1 mM EDTA [4] , -20 C, purified, 10 mM sodium phosphate buffer, pH 7.4, 0.1 mM EDTA, 20% glycerol, for months without loss of P-450 activity [4] , -70 C, mitochondrial pellet, 30-40 mg/ml, 100 mM potassium phosphate, pH 7.3, 0.2 mM EDTA [2] , -80 C, 50 mM potassium phosphate buffer, pH 7.0, 0.1 mM EDTA, 0.1 mM dithiothreitol, 0.01% cholate, a few days without substantial loss of activity [8]

53

Cholesterol monooxygenase (side-chain-cleaving)

1.14.15.6

, 0 C on ice, purified, 20 mM potassium phosphate buffer, pH 7.4, 20% glycerol, v/v, 0.1 mM EDTA, 0.1 mM dithioerythritol, 0.01% Emulgen 911, 3 weeks, without substantial loss of activity [12] , 4 C, purified, 50 mM potassium phosphate, pH 7.3, 0.1 mM dithiothreitol, 10% glycerol, stable a few weeks [2] , 5 C, 50 mM potassium phosphate buffer, pH 7.0, without glycerol, 2143% decrease of activity within 30 days, completely lost in 4 months [7] , purified, 25 mM potassium phosphate, pH 7.3, 0.05 mM EDTA, 0.05 mM dithiothreitol, 0.025 mM deoxycorticosterone, 0.25% Tween 20, 0.25% sodium cholate [2]

"  [1] Hanukoglu, I.; Jefcoate, C.R.: Mitochondrial cytochrome P-450scc . Mechanism of electron transport by adrenodoxin. J. Biol. Chem., 255, 3057-3061 (1980) [2] Hanukoglu, I.; Spitsberg, V.; Bumpus, J.A.; Dus, K.M.; Jefcoate, C.R.: Adrenal mitochondrial cytochrome P-450scc . Cholesterol and adrenodoxin interactions at equilibrium and during turnover. J. Biol. Chem., 256, 4321-4328 (1981) [3] Hanukoglu, I.; Privalle, C.T.; Jefcoate, C.R.: Mechanisms of ionic activation of adrenal mitochondrial cytochromes P-450scc and P-45011b. J. Biol. Chem., 256, 4329-4335 (1981) [4] Wang, H.P.; Kimura, T.: Purification and characterization of adrenal cortex mitochondrial cytochrome P-450 specific for cholesterol side chain cleavage activity. J. Biol. Chem., 251, 6068-6074 (1976) [5] Nakajin, S.; Ishii, Y.; Shinoda, M.: Binding of Triton X-100 to purified cytochrome P-450scc and enhancement of the cholesterol side chain cleavage activity. Biochem. Biophys. Res. Commun., 87, 524-531 (1979) [6] Hume, R.; Kelly, R.W.; Taylor, P.L.; Boyd, G.S.: The catalytic cycle of cytochrome P-450scc and intermediates in the conversion of cholesterol to pregnenolone. Eur. J. Biochem., 140, 583-591 (1984) [7] Shikita, M.; Hall, P.F.: Cytochrome P-450 from bovine adrenocortical mitochondria: an enzyme for the side chain cleavage of cholesterol. I. Purification and properties. J. Biol. Chem., 248, 5598-5604 (1973) [8] Takemori, S.; Sukara, K.; Hashimoto, K.; Hashimoto, M.; Sato, H.; Gomi, T.; Katagiri, M.: Purification of cytochrome P-450 from bovine adrenocortical mitochondria by an aniline-Sepharose and the properties. Biochem. Biophys. Res. Commun., 63, 588-593 (1975) [9] Dhariwal, M.S.; Jefcoate, C.R.: Cholesterol metabolism by purified cytochrome P-450scc is highly stimulated by octyl glucoside and stearic acid exclusively in large unilamellar phospholipid vesicles. Biochemistry, 28, 8397-8402 (1989) [10] Lambeth, J.D.; Kriengsiri, S.: Cytochrome P-450scc -adrenodoxin interactions. Ionic effects on binding, and regulation of cytochrome reduction by bound steroid substrates. J. Biol. Chem., 260, 8810-8816 (1985) 54

1.14.15.6

Cholesterol monooxygenase (side-chain-cleaving)

[11] Nagahisa, A.; Foo, T.; Gut, M.; Orme-Johnson, W.H.: Competitive inhibition of cytochrome P-450scc by (22R)- and (22S)-22-aminocholesterol. Sidechain stereochemical requirements for C-22 amine coordination to the active-site heme. J. Biol. Chem., 260, 846-851 (1985) [12] Kashiwagi, K.; Dafeldecker, W.P.; Salhanick, H.A.: Purification and characterization of mitochondrial cytochrome P-450 associated with cholesterol side chain cleavage from bovine corpus luteum. J. Biol. Chem., 255, 26062611 (1980) [13] Sheets, J.J.; Vickery, L.E.: Active site-directed inhibitors of cytochrome P450scc . Structural and mechanistic implications of a side chain-substituted series of amino-steroids. J. Biol. Chem., 258, 11446-11452 (1983) [14] Sheets, J.J.; Vickery, L.E.: C-22-Substituted steroid derivatives as substrate analogues and inhibitors of cytochrome P-450scc . J. Biol. Chem., 258, 17201725 (1983) [15] Warne, P.A.; Greenfield, N.J.; Lieberman, S.: Modulation of the kinetics of cholesterol side-chain cleavage by an activator and by an inhibitor isolated from the cytosol of the cortex of bovine adrenals. Proc. Natl. Acad. Sci. USA, 80, 1877-1881 (1983) [16] Shikita, M.; Hall, P.F.: Cytochrome P-450 from bovine adrenocortical mitochondria: an enzyme for the side chain cleavage of cholesterol. II. Subunit structure. J. Biol. Chem., 248, 5605-5609 (1973) [17] Miao, E.; Joardar, S.; Zuo, C.; Cloutier, N.J.; Nagahisa, A.; Byon, C.; Wilson, S.R.; Orme-Johnson, W.H.: Cytochrome P-450scc -mediated oxidation of (20S)-22-thiacholesterol: Characterization of mechanism-based inhibition. Biochemistry, 34, 8415-8421 (1995) [18] Schwarz, D.; Kisselev, P.; Wessel, R.; Jueptner, O.; Schmid, R.D.: a-Branched 1,2-diacyl phosphatidylcholines as effectors of activity of cytochrome P450scc (CYP11A1). Modeling the structure of the fatty acyl chain region of cardiolipin. J. Biol. Chem., 271, 12840-12846 (1996) [19] Lepesheva, G.I.; Azeva, T.N.; Strushkevich, N.V.; Gilep, A.A.; Usanov, S.A.: Site-directed mutagenesis of cytochrome P450scc (CYP11A1). Effect of lysine residue substitution on its structural and functional properties. Biochemistry (Moscow), 65, 1409-1418 (2000) [20] Ahmed, S.: The use of the novel substrate-heme complex approach in the derivation of a representation of the active site of the enzyme cholesterol side chain cleavage. Biochem. Biophys. Res. Commun., 274, 821-824 (2000) [21] Tsujita, M.; Ichikawa, Y.: Substrate-binding region of cytochrome P-450scc (P-450 XIA1). Identification and primary structure of the cholesterol binding region in cytochrome P-450scc . Biochim. Biophys. Acta, 1161, 124-130 (1993) [22] Usanov, S.A.; Graham, S.E.; Lepesheva, G.I.; Azeva, T.N.; Strushkevich, N.V.; Gilep, A.A.; Estabrook, R.W.; Peterson, J.: Probing the interaction of bovine cytochrome P450scc (CYP11A1) with adrenodoxin: evaluating site-directed mutations by molecular modeling. Biochemistry, 41, 8310-8320 (2002)

55


     

8

   
 1.14.15.7    choline,reduced-ferredoxin:oxygen oxidoreductase    choline monooxygenase    CMO EC 1.14.14.4 (formerly) choline oxygenase
    118390-76-4

     Spinacia oleracea (spinach, cv. Savoy Hybrid 612 [1-4]) [1-4] Beta vulgaris (sugar beet, cv. Great Western D-2 [1]) [1, 5] Amaranthus caudatus (amaranth, cv. RRC 1036 [5]) [5]

!    "
 # 

choline + O2 + 2 reduced ferredoxin 2 H+ = betaine aldehyde hydrate + H2 O + 2 oxidized ferredoxin    oxidation redox reaction reduction      choline + O2 + reduced ferredoxin (Reversibility: ? [1]) [13] $ betaine aldehyde hydrate + oxidized ferredoxin

56

1.14.15.7

Choline monooxygenase

2  (NH4 )2 SO4 ( more than 50% inhibition at 25 mM [3]) [3] N,N-dimethyldiethanolamine ( 32% inhibition at 5 mM [3]) [3] Zn2+ ( 10 mM inhibits activity [2]) [2] betaine ( 300 mM betaine give 50% inhibition [2]) [2] chlorocholine ( 98% inhibition at 2.5 mM [3]) [3] diethylcholine ( 58% inhibition at 5 mM [3]) [3] ethylcholine ( 88% inhibition at 5 mM [3]) [3] sulfocholine ( 65% inhibition at 2.5 mM [3]) [3] triethylcholine ( 43% inhibition at 5 mM [3]) [3] trimethylaminobutanol ( 15% inhibition at 2.5 mM [3]) [3] trimethylaminopropanol ( 27% inhibition at 2.5 mM [3]) [3]
"%  O2 ( half-maximal rate with a O2 partial pressure of 2 kPa, corresponding to an concentration of 0.025 mM [2]) [2] ferredoxin ( ferredoxin is reduced via NADPH and ferredoxinNADP+ reductase [2]) [2] &

  catalase ( stimulation of activity by countering negative effects of H2 O2 produced by ferredoxin autoxidation [2]) [2] '(   Ca2+ ( 10 mM Ca2+ can replace Mg2+ [2]) [2] Mg2+ ( maximum activity with more than 10 mM [2]) [2] Additional information ( Mn2+ , Cu2+ or Co2+ have no effect on activity [2]) [2] " & *-% , 0.00003 ( in leaves of untreated plants [5]) [5] 0.000036 ( in leaves of untreated plants [5]) [5] 0.0001 ( in leaves of plants after salinization with 300 mM NaCl for three days [5]) [5] 0.00012 ( in roots of untreated plants and after salinization with 100 mM NaCl for three days [5]) [5] 0.00018 ( in leaves of plants after salinization with 100 mM NaCl for three days [5]) [5] 0.00024 ( in leaves of plants after salinization with 400 mM NaCl for three days [5]) [5] 0.00025 ( activity varies with leaf age [2]) [2] 0.00048 ( in roots of plants after salinization with 400 mM NaCl for three days [5]) [5] 0.00058 ( after salinization of plants with 200 mM NaCl for two weeks [2]) [2] . / *', 0.1 (choline, reduced ferredoxin provided by NADPH/ferredoxinNADP+ reductase or reconstituted chloroplasts [2]) [2]

57

Choline monooxygenase

1.14.15.7

0  7.7 ( reduced ferredoxin provided by NADPH/ferredoxin-NADP+ reductase or reconstituted chloroplasts [2]) [2]

  #   ' 1  42860 ( MALDI-MS after fractionation with HPLC [4]) [4] 42880 ( prediction from cloned polypeptide [4]) [4] 45000 ( SDS-PAGE [2]) [2] 98000 ( gel filtration with Ultrogel AcA 34 [2]) [2] 133000 ( gel filtration with tandem Superose 12-Superdex 200 [4]) [4] 135000 ( native electrophoresis [4]) [4]

 2 %$ %' %

3#

chloroplast ( in stromal fraction of chloroplasts [2]) [1, 2] $"

[3]
 

[4]

4     , -20 C, 50% glycerol, 1 month, without loss of activity [3]

"  [1] Lerma, C.; Hanson, A.D.; Rhodes, D.: Oxygen-18 and deuterium labeling studies of choline oxidation by spinach and sugar beet. Plant Physiol., 88, 695702 (1988) [2] Brouquisse, R.; Weigel, P.; Rhodes, D.; Yocum, C.F.; Hanson, A.D.: Evidence for a ferredoxin-dependent choline monooxygenase from spinach chloroplast stroma. Plant Physiol., 90, 322-329 (1989) [3] Burnet, M.; Lafontaine, P.J.; Hanson, A.D.: Assay, purification, and partial characterization of choline monooxygenase from spinach. Plant Physiol., 108, 581-588 (1995) [4] Rathinasabapathi, B.; Burnet, M.; Russell, B.L.; Gage, D.A.; Liao, P.C.; Nye, G.J.; Scott, P.; Golbeck, J.H.; Hanson, A.D.: Choline monooxygenase, an unu58

1.14.15.7

Choline monooxygenase

sual iron-sulfur enzyme catalyzing the first step of glycine betaine synthesis in plants: prosthetic group characterization and cDNA cloning. Proc. Natl. Acad. Sci. USA, 94, 3454-3458 (1997) [5] Russell, B.L.; Rathinasabapathi, B.; Hanson, A.D.: Osmotic stress induces expression of choline monooxygenase in sugar beet and amaranth. Plant Physiol., 116, 859-865 (1998)

59

$        

4

   
 1.14.16.1    l-phenylalanine,tetrahydrobiopterin:oxygen oxidoreductase (4-hydroxylating)    phenylalanine 4-monooxygenase    EC 1.14.3.1 (formerly) EC 1.99.1.2 (formerly) PAH oxygenase, phenylalanine 4-monophenylalaninase phenylalanine 4-hydroxylase phenylalanine hydroxylase
    9029-73-6

     Rattus norvegicus [1-4, 9-11, 13, 14, 17, 19, 23, 24, 25, 27, 28, 30, 37, 42] Macaca irus [16] Chromobacterium violaceum (most probably 2 phenylalanine hydroxylases encoded by different genes [34]) [6, 7, 18, 20, 28, 34, 44] Homo sapiens [1, 4, 12, 15, 22, 26, 29, 30, 31, 32, 33, 35, 38, 40, 41, 42, 43, 44] Bos taurus [5, 13] Pseudomonas sp. (ATCC 11299a [8]) [8, 21] Homo sapiens [36] Callorhinus ursinus (Schreber, bank vole [39]) [39] Apodemus sylvaticus (field mouse [39]) [39]

60

1.14.16.1

Phenylalanine 4-monooxygenase

!    "
 # 

l-phenylalanine + tetrahydrobiopterin + O2 = l-tyrosine + 4a-hydroxytetrahydrobiopterin ( enzyme requires an initial reduction of Cu2+ to Cu+ for activation [18]; proposed catalytic cycle [43])    oxidation redox reaction reduction       l-phenylalanine + (6R)-tetrahydrobiopterin + O2 ( in mammals rate-limiting step in complete catabolism of phenylalanine to CO2 and water [2]) (Reversibility: ? [2]) [2] $ l-tyrosine + (6R)-dihydrobiopterin + H2 O [2]      2-fluorophenylalanine + tetrahydrobiopterin + O2 (Reversibility: ? [2]) [2] $ ?  3-fluorophenylalanine + tetrahydrobiopterin + O2 (Reversibility: ? [2]) [2] $ ?  3-phenylserine + tetrahydrobiopterin + O2 (Reversibility: ? [2, 6]) [2, 6] $ ?  4-chlorophenylalanine + tetrahydrobiopterin + O2 (Reversibility: ? [2]) [2] $ ?  4-fluorophenylalanine + tetrahydrobiopterin + O2 (Reversibility: ? [2]) [2] $ ?  4-methylphenylalanine + 6,7-dimetyl-tetrahydropterin + O2 (Reversibility: ? [28]) [28] $ 4-(hydroxymethyl)phenylalanine + 3-methyltyrosine + H2 O + 6,7-dimethyl-dihydropterin ( 74% methyl-hydroxylation, 26% parahydroxylation, shift of para-substituent by NIH shift mechanism [28]; 79% methyl-hydroxylation, 21% para-hydroxylation, shift of parasubstituent by NIH shift mechanism [28]) [28]  l-cyclohexylalanine + 6,7-dimetyl-tetrahydropterin + O2 ( 4times slower reaction than with l-phenylalanine [28]; 50% less activity than the enzyme from Chromobacterium violaceum [28]) (Reversibility: ? [28]) [28] $ 4-hydroxy-l-cyclohexylalanine + H2 O + 6,7-dimethyl-dihydropterin [28]

61

Phenylalanine 4-monooxygenase

1.14.16.1

 l-methionine + tetrahydrobiopterin + O2 ( lysolecithin activated enzyme [2]) (Reversibility: ? [2]) [2] $ ?  l-norleucine + tetrahydrobiopterin + O2 ( lysolecithin activated enzyme [2]) (Reversibility: ? [2]) [2] $ ?  l-phenylalanine + 6-methyltetrahydropterin + O2 (Reversibility: ? [34]) [34] $ l-tyrosine + 6-methyldihydropterin + H2 O ( in the presence of FeSO4 and dithiothreitol [34]) [34]  l-phenylalanine + 6-methyltetrahydropterin + O2 ( copper-depleted enzyme, in the absence of Fe2+ , 6-methyltetrahydropterin oxidation can be uncoupled from substrate hydroxylation by the exclusion of iron [34]) (Reversibility: ? [34]) [34] $ l-phenylalanine + 6-methyldihydropterin + H2 O2 [34]  l-phenylalanine + tetrahydrobiopterin + O2 ( low activity with tetrahydrobiopterin can be selectively increased by a wide variety of reversible and irreversible modificators of the enzyme, e.g. interaction with phospholipids [1-4, 19]; relatively low activity with tetrahydrobiopterin can be selectively increased by limited proteolysis [1,4, 19]; relatively low activity with tetrahydrobiopterin can be selectively increased by limited proteolysis, alkylation of sulfhydryl groups with Nethylmaleimide or phosphorylation by cAMP-dependent protein kinase [1]; non activated enzyme has much greater activity with 6-methyltetrahydropterin and dimethyltetrahydropterin than with tetrahydrobiopterin [1]; additional electron donors: 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine, 2-amino-4-hydroxy-6-methyltetrahydropteridine, tetrahydrofolate, and 6-methyl-5-deazatetrahydropterin [6, 7, 20]; additional electron donors: 6-methyltetrahydropterin, 7-methylpterin, and 2,4,5-triamino-6-hydroxypyrimidine [2, 3]; additional electron donors: 6-methylpterin, 6,7-dimethyltetrahydropterin [2, 3, 5, 7, 20]; specificity is quantitatively altered when the enzyme is activated by lysolecithin [2]) (Reversibility: ir [2, 3]; ? [1, 4-22]) [122] $ l-tyrosine + dihydrobiopterin + H2 O ( 4-a-carbinolamine is the first free pterin product formed [2]) [1-22]  l-tryptophan + tetrahydrobiopterin + O2 ( 0.4% of activity with l-phenylalanine [6, 7]; truncated enzyme containing C-terminal 334 amino acids [33]) (Reversibility: ? [2, 6, 7, 33]) [2, 6, 7, 33] $ ?  S-methyl-l-cysteine + tetrahydrobiopterin + O2 ( lysolecithin activated enzyme [2]) (Reversibility: ? [2]) [2] $ ?  b-2-thienylalanine + tetrahydrobiopterin + O2 (Reversibility: ? [2]) [2] $ ?  m-tyrosine + tetrahydrobiopterin + O2 (Reversibility: ? [2]) [2] 62

1.14.16.1

Phenylalanine 4-monooxygenase

$ ?  p-methylphenylalanine + tetrahydrobiopterin + O2 (Reversibility: ? [2]) [2] $ ? 2  (6R)-l-erythro-tetrahydrobiopterin ( inhibits phenylalanine and lysolecithin activation [3]; modulator that inhibits activation [24]) [3, 24] (7R)-5,6,7,8-tetrahydropterin ( 0.001 mM, 50% inhibition at 0.5 mM phenylalanine, 0.004 mM, 50% inhibition at 0.1 mM phenylalanine, recombinant enzyme [30]) [30] 2,2'-dipyridine [2] 2,3-dihydroxynaphthalene ( binds to Fe3+ on enzyme that is oxidized during catalysis [23]) [23] 2-mercaptoethanol ( 2 mM, 80% inhibition [14]) [14] 3,4-dihydroxyphenylpropane ( 0.0016 mM, 50% inhibition [11]) [11] 3,4-dihydroxyphenylpropionic acid ( 0.24 mM, 50% inhibition [11]) [11] 3,4-dihydroxystyrene ( 0.0005-0.005 mM, 50% inhibition, noncompetitive vs. 6,7-dimethyltetrahydropterin and l-phenylalanine [11]) [11] 4-chloromercuribenzoate ( 1 mM, complete inhibition after 10 min [22]) [22] 4-chlorophenylalanine ( recombinant enzyme, 3 mM, almost complete inhibition [15]; competitive vs. phenylalanine [22]) [15, 22] 4-fluorophenylalanine ( above 1 mM [22]) [22] 4-hydroxyphenylpyruvic acid ( above 0.4 mM iron, activation below [21]) [21] 8-hydroxyquinoline [2] dl-DOPA ( 0.1 mM, approx. 60% inhibition [21]) [21] dl-m-tyrosine ( 0.4 mM, approx. 80% inhibition [21]) [21] dl-a-tocopherol ( strong inhibition [22]) [22] DOPA [2] EDTA [7] H2 O2 ( inactivates the reduced form of the enzyme [3]) [3] l-3,4-dihydroxyphenylalanine [21] l-DOPA ( 0.3 mM, approx. 40% inhibition [21]) [21] l-cysteine ( 2 mM, 42% inhibition [14]) [14] S-methyl-l-cysteine ( 6 mM, 55% inhibition [2]) [2] Tris [3, 13] Tween 80 [3] acetohydroxamate ( competitive vs. tetrahydrobiopterin, most probably due to chelation of enzyme's iron [23]) [23] ascorbate ( 2 mM, 78% inhibition [14]) [14] bathocuproine [7] bathophenanthroline ( competitive vs. 6-methyl-5,6,7,8-tetrahydropterin and tetrahydrobiopterin, most probably due to chelation of enzyme's iron [23]) [23]

63

Phenylalanine 4-monooxygenase

1.14.16.1

bathophenanthroline disulfonate ( 0.025 mM, 50% inhibition [5]) [5] benzohydroxamate ( competitive vs. tetrahydrobiopterin, most probably due to chelation of enzyme's iron [23]) [23] catechol ( 0.033 mM, 50% inhibition [11]) [2, 3, 11] citrate [7] copper-chelating agents [22] deaza-6-methyltetrahydropterin ( competitive vs. 6-methyltetrahydropterin [24]) [24] diethyldithiocarbamate ( 1 mM, 58% inhibition [22]) [2, 7, 22] dithiothreitol ( 2 mM, 94% inhibition, dithiothreitol induces the dissociation of iron from the enzyme, catalase and phenylalanine partially protect from inhibition [14]) [13, 14] dopamine ( 0.025 mM, 50% inhibition [11]; 0.5 mM, approx. 40% inhibition [21]) [2, 7, 11, 21] epinephrine [17] glycerol [3] halogenated phenylalanine ( moderate [2]) [2] iron-chelating agents [22] norepinephrine [2] o-phenanthroline ( 1 mM, removes Fe2+ from the reduced enzyme [23]) [2, 7, 23] phenylalanine [21] phosphate [7] thiol-binding reagents [22] tryptophan ( recombinant enzyme, 3 mM, approx. 75% inhibition [15]) [15] tyrosine ( recombinant enzyme, 3 mM, approx. 50% inhibition [15]) [15, 21] Additional information ( not inhibited by EDTA [2]; not inhibited by bathocuproine [5]) [2, 5]
"%  l-threo-neopterin [8] tetrahydrobiopterin [1-22] tetrahydrofolate ( 10% of activity with 6,7-dimethyltetrahydropterin [20]) [6, 20] Additional information ( artificial cofactors: 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine, 6-methyl-5-deazatetrahydropterin and 2amino-4-hydroxy-6-methyltetrahydropteridine can replace tetrahydrobiopterin [6, 7]; artificial cofactors: 6-methyltetrahydropterin, 7-methylpterin [2, 3, 22]; artificial cofactors: 6-methyltetrahydropterin, 6,7-dimethyltetrahydropterin [2, 3, 5, 7, 20]; artificial cofactor: 2,4,5-triamino-6-hydroxypyrimidine [2]) [2, 3, 5, 6, 7, 20, 22] &

  4-hydroxyphenylacetic acid ( 0.4 mM, approx. 30% activation [21]) [21]

64

1.14.16.1

Phenylalanine 4-monooxygenase

N-ethylmaleimide ( activation by alkylation of sulfhydryl groups [1]) [1] a-chymotrypsin ( limited proteolysis of purified liver enzyme, 2030fold increase in activity, cofactor tetrahydrobiopterin [19]) [19] cAMP-dependent protein kinase ( 2-4fold increase in activity in the presence of tetrahydrobiopterin [1]; 1.6fold increase in activity, cofactor tetrahydrobiopterin [29]; 1.5fold stimulation of recombinant enzyme [30]) [1, 4, 29, 30] dithiothreitol [7] liver lysosomal proteases ( limited proteolysis of liver enzyme [19]) [19] lysolecithin ( 1 mM, approx. 7.5fold stimulation of phosphorylated recombinant wild-type enzyme, S16E and S16D mutant enzymes, approx. 24fold stimulation of non-phosphorylated recombinant wild-type enzyme, S16A, S16Q, S16N and S16K mutant enzymes [25]; 2fold activation of recombinant enzyme, cofactor tetrahydrobiopterin [30]) [2-4, 19, 25, 30] phenylalanine ( major regulator of liver enzyme, binds at an effector site converting the inactive to an active enzyme [3]; binds to activation site distinct from catalytic site with a stoichiometry of 1/enzyme subunit, binding induces a conformation change converting the enzyme tetramer from an inactive to an active form, activation site binds phenylalanine in a cooperative manner, regulation of activation [24]; homotropic allosteric activator of both hepatic and recombinant enzymes [27]; approx. 4.5fold increase in activity of non-phosphorylated and phosphorylated enzyme, cofactor tetrahydrobiopterin [29]; recombinant enzyme, 2fold increase in activity, cofactor tetrahydrobiopterin [30]; after activation with phenylalanine the dimer/tetramer equilibrium is shifted towards the tetrameric form [42]; wild-type tetramer reveals a kinetic positive coorperativity of lphenylalanine binding leading to a 5-6fold activation of wild-type tetramer, dimeric form shows a hyperbolic rate vs. substrate concentration, 1.6fold activation by phenylalanine, tetrameric T427P mutant enzyme shows no cooperativity, dimeric forms of wild-type and T427P mutant have similar kinetic properties [42]) [3, 24, 27, 29, 30, 42] phospholipids ( activate [1-4, 19]; increase in activity in the presence of tetrahydrobiopterin but not in the presence of synthetic pterin cofactors [19]) [1-4, 19] trypsin ( limited proteolysis of purified liver enzyme [19]) [19] Additional information ( relatively low activity with tetrahydrobiopterin can be selectively increased by limited proteolysis [1,4,19]; relatively low activity with tetrahydrobiopterin can be selectively increased by phosphorylation [1]; non activated enzyme has much greater activity with 6-methyltetrahydropterin and dimethyltetrahydropterin than with tetrahydrobiopterin [1]; enzyme activity is not stimulated by phosphorylation [4]; activity of recombinant enzyme is activated 1.5fold by exposure to pH 8.5-9.0 [32]) [1-4, 19, 32]

65

Phenylalanine 4-monooxygenase

1.14.16.1

'(   Ca2+ ( substoichiometric amounts after removal of copper with dithiothreitol [34]) [34] Zn2+ ( substoichiometric amounts after removal of copper with dithiothreitol [34]) [34] copper ( less than 0.01 copper atoms per 50000 Da subunit [5]; 1 mol of Cu2+ per mol of enzyme [7,18]; electron paramagnetic resonance spectroscopy indicates a type II copper-containing enzyme [18]; copper does not support enzyme activity and can be removed by dithiothreitol [34]) [5, 7, 18, 34] iron ( iron-protein [2, 5, 9, 13, 17]; 1.5-3.0 iron atoms per 50000 Da subunit [5]; 0.6 mol of iron per mol of 51000 Da subunit [9]; electron paramagnetic resonance spectrum [13, 17]; onedimensional 1H-NMR spectroscopy of iron [13]; 1 iron atom per subunit, fully active enzyme has non-heme high-spin ferric iron coordinated at the active site [14]; iron can be removed by treatment with o-phenanthroline, reconstitution of apoenzyme with iron restores 90% of the initial activity [17]; kinetic data suggest, that enzyme's iron is solvent-accessible and resides in a hydrophobic pocket of the enzyme [23]; maximal 1 iron per subunit, hepatic and recombinant enzyme [27]; copper depleted enzyme can be reconstituted with approx. 1 molecule of iron per enzyme molecule, iron reconstituted enzyme hydroxylates phenylalanine [34]; conformation and distances to the catalytic iron of both l-phenylalanine and the cofactor analog l-erythro-7,8-dihydrobiopterin simultaneously bound to the recombinant enzyme estimated by 1H-NMR [35]; enzyme is active only in the presence of Fe2+ [44]) [2, 5, 9, 13, 14, 17, 23, 27, 35, 44] Additional information ( enzyme does not contain iron [7]) [7] ) &  * +, 1.1 (phenylalanine, Ser2-Gln428 deletion mutant, tetrameric form [31]) [31] 8.1 (phenylalanine, Ser2-Gln428 deletion mutant, l-phenylalanine activated, dimeric form [31]) [31] 9.9 (phenylalanine, Ser2-Gln428 deletion mutant, lysophosphatidylcholine activated, dimeric form [31]) [31] 14 (phenylalanine, Ser2-Gln428 deletion mutant, lysophosphatidylcholine and phenylalanine activated, dimeric form [31]) [31] 21.8 (phenylalanine, recombinant enzyme, tetrameric form [31]) [31] 27 (phenylalanine, Asp112-Lys452 deletion mutant, lysophosphatidylcholine activated, tetrameric form [31]) [31] 30 (phenylalanine, Asp112-Lys452 deletion mutant, tetrameric form [31]) [31] 38 (phenylalanine, V379D mutant enzyme [37]) [37] 40 (phenylalanine, Gly103-Gln428 deletion mutant, lysophosphatidylcholine activated, dimeric form [31]) [31]

66

1.14.16.1

Phenylalanine 4-monooxygenase

52.6 (phenylalanine, recombinant enzyme, lysophosphatidylcholine activated, tetrameric form [31]) [31] 57.1 (phenylalanine, Gly103-Gln428 deletion mutant, tetrameric form [31]) [31] 62.8 (phenylalanine, Asp112-Lys452 deletion mutant, l-phenylalanine activated, tetrameric form [31]) [31] 62.9 (phenylalanine, Asp112-Lys452 deletion mutant, lysophosphatidylcholine and phenylalanine activated, tetrameric form [31]) [31] 74.2 (phenylalanine, Gly103-Gln428 deletion mutant, lysophosphatidylcholine and phenylalanine activated, dimeric form [31]) [31] 78.1 (phenylalanine, recombinant enzyme, l-phenylalanine activated, tetrameric form [31]) [31] 80 (phenylalanine, Gly103-Gln428 deletion mutant, l-phenylalanine activated, dimeric form [31]) [31] 95.7 (phenylalanine, recombinant enzyme, lysophosphatidylcholine and phenylalanine activated, tetrameric form [31]) [31] 125 (phenylalanine, H264Q/Y277H/V379D mutant enzyme [37]) [37] 281 (phenylalanine, H264Q/V379D mutant enzyme [37]) [37] 392 (phenylalanine, truncated enzyme containing C-terminal 334 amino acids [33]) [33] 395 (phenylalanine, recombinant wild-type enzyme [33]) [33] 545 (phenylalanine, cofactor 6,7-dimethyl-5,6,7,8-tetrahydropterin [9]) [9] 604 (phenylalanine, Y277H mutant enzyme [37]) [37] 840 (phenylalanine, H264Q mutant enzyme [37]) [37] 960 (phenylalanine, recombinant wild-type enzyme [37]) [37] " & *-% , 0.00009 ( activity in liver of males living in an area with high emissions of SO2 and nitrogen oxides, cofactor tetrahydrobiopterin [39]) [39] 0.00033 ( activity in liver of males, cofactor tetrahydrobiopterin [39]) [39] 0.00097 ( activity in liver of males, cofactor tetrahydrobiopterin [39]) [39] 0.006 ( activity in liver of males, cofactor 6,7-dimethyltetrahydropterin [39]) [39] 0.055 ( I65T mutant enzyme, cofactor 6-methyltetrahydropterin [38]) [38] 0.09 ( cofactor 6,7-dimethyltetrahydropterin [5]) [5] 0.099 ( R270K mutant enzyme, expression in the absence of glycerol in the growth medium, cofactor 6-methyltetrahydropterin [41]) [41] 0.105 ( activity in liver of males living in an area with high emissions of SO2 and nitrogen oxides, cofactor 6,7-dimethyltetrahydropterin [39]) [39] 0.106 [4] 0.17 ( cofactor 6-methyltetrahydropterin [5]) [5]

67

Phenylalanine 4-monooxygenase

1.14.16.1

0.225 ( liver enzyme [22]) [22] 0.23 ( R270K mutant enzyme, expression in the presence of glycerol in the growth medium, cofactor 6-methyltetrahydropterin [41]) [41] 0.23 ( cofactor tetrahydrobiopterin [5]) [5] 0.247 ( activity in liver of males, cofactor 6,7-dimethyltetrahydropterin [39]) [39] 0.408 ( V388M mutant enzyme, cofactor tetrahydrobiopterin [38]) [38] 0.424 ( maltose-binding-protein phenylalanine hydroxylase fusion protein, dimeric form [26]) [26] 0.44 ( liver enzyme [16]) [16] 0.536 ( R261Q mutant enzyme, cofactor tetrahydrobiopterin [38]) [38] 0.59 [2, 9] 0.745 ( V388M mutant enzyme, cofactor 6-methyltetrahydropterin [38]) [38] 0.78 ( R261Q mutant enzyme, cofactor 6-methyltetrahydropterin [38]) [38] 1.283 ( maltose-binding-protein phenylalanine hydroxylase fusion protein, tetrameric form [26]) [26] 1.41 [10] 1.46 [9] 1.46 ( enzyme form II [9]) [9] 1.6 ( fetal liver enzyme [12]) [12] 1.742 ( recombinant wild-type enzyme, cofactor tetrahydrobiopterin [38]) [38] 1.76 ( adult liver enzyme [12]) [12] 1.773 ( V388M mutant enzyme, expression in the absence of glycerol in the growth medium, cofactor 6-methyltetrahydropterin [41]) [41] 1.8 ( enzyme form I [9]) [9] 2.49 ( recombinant wild-type enzyme, cofactor 6-methyltetrahydropterin [38]) [38] 2.6 ( recombinant enzyme [30]) [30] 2.91 ( V388M mutant enzyme, expression in the presence of glycerol in the growth medium, cofactor 6-methyltetrahydropterin [41]) [41] 3.9 [21] 4.9 ( recombinant wild-type enzyme [25]) [25] 5 ( phosphorylated recombinant wild-type enzyme, S16N and S16D mutant enzyme [25]) [25] 5.1 ( S16A and S16K mutant enzymes [25]) [25] 5.2 ( S16E and S16Q mutant enzymes [25]) [25] 6.8 ( recombinant enzyme [27]) [27] 12.5-13 [3] 12.9 [7] 13.2 [6, 20] 14 ( truncated enzyme containing C-terminal 334 amino acids [33]) [33] 68

1.14.16.1

Phenylalanine 4-monooxygenase

. / *', 0.002 (tetrahydrobiopterin, native liver enzyme [19]) [19] 0.002-0.004 (tetrahydrobiopterin) [2] 0.0024 (l-cyclohexylalanine) [28] 0.0025 (tetrahydrobiopterin, S16A mutant enzyme [25]) [25] 0.0026 (tetrahydrobiopterin) [4] 0.0027 (tetrahydrobiopterin, S16K mutant enzyme [25]) [25] 0.0028 (tetrahydrobiopterin, S16D mutant enzyme [25]) [25] 0.003 ((6R)-5,6,7,8-tetrahydrobiopterin, recombinant enzyme [30]) [30] 0.0034 (tetrahydrobiopterin, S16E mutant enzyme [25]) [25] 0.0036 (tetrahydrobiopterin, S16N mutant enzyme [25]) [25] 0.0044 (tetrahydrobiopterin, S16Q mutant enzyme [25]) [25] 0.0046 (tetrahydrobiopterin, phosphorylated recombinant wild-type enzyme [25]) [25] 0.01-0.015 (6-methyltetrahydrobiopterin) [15] 0.01-0.015 (tetrahydrobiopterin, substrate phenylalanine [3]) [3] 0.012 (tetrahydrobiopterin, chymotrypsin activated liver enzyme [19]) [19] 0.016 (tetrahydrobiopterin, recombinant enzyme [15]) [15] 0.021 (tetrahydrobiopterin, recombinant wild-type enzyme, dimeric form [31]) [31] 0.022 (tetrahydrobiopterin) [38] 0.023 (tetrahydrobiopterin, native liver enzyme in crude extract [15]) [15] 0.024 (tryptophan, truncated enzyme containing C-terminal 334 amino acids, pH 8.0 [33]) [33] 0.025 ((6R)-5,6,7,8-tetrahydrobiopterin, recombinant enzyme, phenylalanine-activated [30]) [30] 0.025 (tetrahydrobiopterin, recombinant wild-type enzyme [25]) [25] 0.025 (tetrahydrobiopterin, cleaved maltose-binding-protein phenylalanine fusion protein [26]) [26] 0.025 (tetrahydrobiopterin, recombinant wild-type enzyme, tetrameric form [31]) [31] 0.029 (tetrahydrobiopterin, recombinant enzyme [26]) [26] 0.03-0.04 (tetrahydrobiopterin, recombinant wild-type, C237S and C237D mutant enzyme [32]) [32] 0.031 (tetrahydrobiopterin, Gly103-Gln428 deletion mutant, dimeric form [31]) [31] 0.031 (tetrahydrobiopterin, maltose-binding-protein phenylalanine fusion protein [26]) [26] 0.032 (tetrahydrobiopterin, Ser2-Gln428 deletion mutant, dimeric form [31]) [31] 0.033 (6,7-dimethyltetrahydropterin) [4] 0.034 (tetrahydrobiopterin, Asp112-Lys452 deletion mutant, tetrameric form [31]) [31] 69

Phenylalanine 4-monooxygenase

1.14.16.1

0.0344 (6,7-dimethyl-5,6,7,8-tetrahydropterin, enzyme form II, substrate l-phenylalanine [9]) [9] 0.037 (6-methyl-5,6,7,8-tetrahydropterin, enzyme form I, substrate l-phenylalanine [9]) [9] 0.041 (tetrahydrobiopterin, truncated enzyme containing C-terminal 334 amino acids [33]) [33] 0.043 (phenylalanine, recombinant enzyme, cofactor (6R)-methyltetrahydropterin [30]) [30] 0.0444 (6,7-dimethyl-5,6,7,8-tetrahydropterin, enzyme form I, substrate l-phenylalanine [9]) [9] 0.045 (6-methyltetrahydrobiopterin, native liver enzyme [19]) [19] 0.045 (6-methyltetrahydropterin) [27] 0.045 (6-methyltetrahydropterin, substrate phenylalanine [3]) [3] 0.045 (6-methyltetrahydropterin, recombinant enzyme [30]) [30] 0.0455 (6-methyl-5,6,7,8-tetrahydropterin, enzyme form II, substrate l-phenylalanine [9]) [9] 0.049 (phenylalanine, phenylalanine-activated recombinant enzyme, cofactor tetrahydrobiopterin [30]) [30] 0.05 (phenylalanine, cofactor tetrahydrobiopterin [4]) [4] 0.05 (phenylalanine, recombinant enzyme, cofactor tetrahydrobiopterin [30]) [30] 0.05-0.06 (6,7-dimethyltetrahydropterin) [2] 0.05-0.075 (l-phenylalanine, recombinant enzyme, cofactor tetrahydrobiopterin [15]) [15] 0.053 (tetrahydrobiopterin, recombinant enzyme [33]) [33] 0.054 (2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine) [6, 20] 0.055 (phenylalanine, recombinant enzyme, cofactor (7R)-5,6,7,8tetrahydrobiopterin [30]) [30] 0.061 (6-methyltetrahydropterin, recombinant enzyme [27]) [27] 0.065 (6,7-dimethyltetrahydrobiopterin, native liver enzyme [19]) [19] 0.073 (6-methyltetrahydropterin, maltose-binding-protein phenylalanine fusion protein [26]) [26] 0.082 (tetrahydrobiopterin, V388M mutant enzyme [38]) [38] 0.085 (6-methyltetrahydropterin, truncated enzyme containing Cterminal 334 amino acids [33]) [33] 0.087 (6-methyltetrahydrobiopterin, chymotrypsin activated liver enzyme [19]) [19] 0.088 (6-methyltetrahydropterin, recombinant enzyme [26]) [26] 0.09 (6-methyltetrahydropterin) [38] 0.09 (l-phenylalanine, native liver enzyme in crude extract, cofactor 6-methyltetrahydrobiopterin [15]) [15] 0.096 (tryptophan, truncated enzyme containing C-terminal 334 amino acids, pH 7.0 [33]) [33] 0.1 (6-methyltetrahydropterin, recombinant enzyme [33]) [33] 0.105 (6,7-dimethyltetrahydrobiopterin, chymotrypsin activated liver enzyme [19]) [19] 70

1.14.16.1

Phenylalanine 4-monooxygenase

0.138 (l-phenylalanine, cofactor tetrahydrobiopterin [38]) [38] 0.14 (l-phenylalanine) [6, 20] 0.145 (l-phenylalanine, truncated enzyme containing C-terminal 334 amino acids [33]) [33] 0.17 (phenylalanine, cofactor 6-methyltetrahydropterin, recombinant enzyme [27]) [27] 0.175 (l-phenylalanine, cofactor tetrahydrobiopterin, recombinant enzyme [26]) [26] 0.18 (phenylalanine, cofactor 6-methyltetrahydropterin [3]) [3, 27] 0.183 (phenylalanine, S16E mutant enzyme [25]) [25] 0.187 (phenylalanine, phosphorylated recombinant wild-type enzyme [25]) [25] 0.194 (l-phenylalanine, cofactor tetrahydrobiopterin, maltose-binding-protein phenylalanine fusion protein [26]) [26] 0.2 ((7R)-5,6,7,8-tetrahydrobiopterin, recombinant enzyme [30]) [30] 0.2 (phenylalanine, cofactor tetrahydrobiopterin [2,3]) [2, 3] 0.2 (phenylalanine, recombinant wild-type enzyme [25]) [25] 0.217 (phenylalanine, S16Q mutant enzyme [25]) [25] 0.236 (l-phenylalanine, cofactor tetrahydrobiopterin, cleaved maltose-binding-protein phenylalanine fusion protein [26]) [26] 0.254 (phenylalanine, S16K mutant enzyme [25]) [25] 0.266 (phenylalanine, S16D mutant enzyme [25]) [25] 0.287 (phenylalanine, S16N mutant enzyme [25]) [25] 0.288 (phenylalanine, S16A mutant enzyme [25]) [25] 0.3-0.4 (l-phenylalanine, native and recombinant enzyme, cofactor 6-methyltetrahydrobiopterin [15]) [15] 0.318 (l-phenylalanine) [22] 0.329 (l-phenylalanine, recombinant enzyme [33]) [33] 0.382 (l-phenylalanine, cofactor 6-methyltetrahydropterin, recombinant enzyme [26]) [26] 0.393 (l-phenylalanine, cofactor 6-methyltetrahydropterin [38]) [38] 0.454 (l-phenylalanine, cofactor 6-methyl-5,6,7,8-tetrahydropterin, enzyme form II [9]) [9] 0.47 (thienylalanine, native enzyme, cofactor tetrahydrobiopterin [2]) [2] 0.504 (l-phenylalanine, cofactor 6-methyltetrahydropterin, maltosebinding-protein phenylalanine fusion protein [26]) [26] 0.55 (phenylalanine, cofactor dimethyltetrahydropterin [4]) [4] 0.72 (l-phenylalanine, cofactor 6-methyl-5,6,7,8-tetrahydropterin, enzyme form I [9]) [9] 1 (4-fluorophenylalanine, approx. value [22]) [22] 1.3 (phenylalanine, cofactor 6,7-dimethyltetrahydrobiopterin [2]) [2] 1.43 (l-phenylalanine, cofactor 6,7-dimethyl-5,6,7,8-tetrahydropterin, enzyme form II [9]) [9] 71

Phenylalanine 4-monooxygenase

1.14.16.1

1.7 (thienylalanine, cofactor tetrahydrobiopterin, lysolecithin activated enzyme [2]) [2] 1.92 (l-phenylalanine, cofactor 6,7-dimethyl-5,6,7,8-tetrahydropterin, enzyme form I [9]) [9] 4.9 (l-tryptophan, cofactor 2-amino-4-hydroxy-6-methyltetrahydropteridine [9]) [9] 8.5 (l-tryptophan, cofactor 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine, enzyme form I [9]) [9] . / *', 1.5e-006 (bathophenanthroline, vs. tetrahydrobiopterin [23]) [23] 1.8e-006 (bathophenanthroline, vs. 6-methyl-5,6,7,8-tetrahydropterin [23]) [23] 0.00027 (3,4-dihydroxystyrene, with 15 min preincubation [11]) [11] 0.0015 ((7R)-5,6,7,8-tetrahydrobiopterin) [30] 0.0032 (3,4-dihydroxystyrene, without preincubation [11]) [11] 0.01 (catechol) [2] 0.026 (deaza-6-methyltetrahydropterin) [24] 0.07 (benzohydroxymate) [23] 0.11 (phenylalanine, competitive vs. iron [21]) [21] 0.3 (l-tryptophan, competitive vs. iron [21]) [21] 0.3 (tyrosine, competitive vs. iron [21]) [21] 1.1 (4-chlorophenylalanine) [22] 1.7 (acethydroxamate) [23] 0 

 5-9.5 ( recombinant enzyme, 25% loss of activity at pH 5.5, 15% loss of activity at pH 7.0-8.0 [32]) [32] )  *
, 25 ( assay at [3]) [3] 27 ( assay at [10]) [10] 30 ( native and recombinant enzyme [15]) [15] ) 

 *
, 15-42 ( native and recombinant enzyme [15]) [15]

  #   ' 1  25000-27000 ( sucrose density gradient centrifugation, gel filtration [21]) [21] 31000-32400 ( gel filtration, SDS-PAGE, sedimentation studies [20]) [6, 20] 51000-55000 ( monomeric form, enzyme exists as monomer, dimer and tetramer [2]) [2]

72

1.14.16.1

Phenylalanine 4-monooxygenase

100000-110000 ( dimeric form, enzyme exists as monomer, dimer and tetramer [2]) [2, 1] 107000 ( fetal liver enzyme, sucrose density gradient centrifugation [4]) [4] 110000 ( fetal liver enzyme, gel filtration [22]) [22] 150000 ( adult liver enzyme, gel filtration [12]) [12] 160000 ( fetal liver enzyme, gel filtration [12]) [12] 165000 ( gel filtration [4]) [4] 200000-210000 ( tetrameric species accounts for 78% of the total enzyme, enzyme exists as monomer, dimer and tetramer [2]) [1, 2] 240000 ( sedimentation equilibrium [9]) [9] 275000 [4]   ? ( x * 51000, SDS-PAGE [5]; x * 51000-55000, SDS-PAGE [10]; x * 46200, equilibrium sedimentation [10]; x * 49000, adult liver enzyme, SDS-PAGE [12]; x * 52000 + x * 49000, fetal liver enzyme, SDS-PAGE [12]) [5, 10, 12] dimer ( 2 * 54000, fetal liver enzyme, SDS-PAGE [4, 22]; enzyme exists as monomer, dimer and tetramer [2]) [2, 4, 22] monomer ( 1 * 33000, SDS-PAGE [6]; 1 * 28000, SDS-PAGE [21]; enzyme exists as monomer, dimer and tetramer [2]; 1 * 33613, deduced from nucleotide sequence [34]; 1 * 33483-33488, recombinant enzyme, mass spectrometry [34]) [2, 6, 21, 34] tetramer ( 4 * 51000, enzyme form I and II, SDS-PAGE [9]; enzyme exists as monomer, dimer and tetramer [2]; tetrameric species accounts for 78% of the total enzyme [1]) [1, 2, 9] Additional information ( some authors report that the enzyme exists as a mixture of dimers and tetramers, others report that it exists solely as tetramer or as dimer, percentage of dimers increases on frozen storage, preincubation of the enzyme with phenylalanine leads to even higher than 200000 Da molecular weight forms [1]; rat liver enzyme and recombinant human enzyme are in a tetramer/dimer equilibrium [42]) [1, 42] $   "

side-chain modification ( liver enzyme in untreated animals is a mixture of phosphorylated and nonphosphorylated forms, enzyme may be phosphorylated in vivo by cAMP-dependent protein kinase [1]; enzyme contains 0.3 mol of phosphate per mol of enzyme subunit [4]; enzyme form I contains 0.3 mol phosphate per mol subunit, enzyme form II contains 0.4 mol [9]; activity of liver enzyme might be regulated by phosphorylation-dephosphorylation [1]; phosporylated enzyme shows a 3.3fold increase in Vmax [25]; recombinant enzyme incorporates 0.97 phosphate per subunit [27]; recombinant enzyme incorporates 1 mol phosphate per mol of enzyme subunit when phosphorylated in vitro by cyclic AMP-dependent protein kinase, Ser-16 seems to be the phosphorylation site [29]; incorporation of 0.6 mol phosphate per enzyme subunit, recombinant enzyme [30]) [1, 3, 4, 9, 25, 27, 29] 73

Phenylalanine 4-monooxygenase

1.14.16.1

Additional information ( enzyme is phosphorylated in vitro by the catalytic subunit of cAMP-dependent protein kinase [5]; hepatic enzyme consists of a mixture of phosphorylated and nonphosphorylated forms [1]) [1, 5]

 2 %$ %' %

% liver [1-5, 9-14, 19, 22] 3#

cytoplasm [3] cytosol [2] $"

(distinct forms I and II differing in phosphate content and isoelectric point [9]; affinity chromatography on 6,7-dimethyl-5,6,7,8-tetrahydropterinSepharose 4B [10]; recombinant wild-type S16E, S16Q, S16N, S16D, S16A, and S16K mutant enzyme [25]; recombinant enzyme [27]) [2, 3, 9, 10, 13, 17, 25, 27] (pteridine affinity resin, 2 non-interconvertible forms [16]) [16] (protamine, DEAE-Sephadex, acid, DEAE-cellulose, Ultrogel, hydroxylapatite, Blue dextran-phenyl-butylamine-Sepharose [6]; metal-free enzyme by extraction of copper with dithiothreitol [28]) [6, 7, 20, 28] (adult and fetal liver enzyme, monoclonal antibody affinity chromatography [12]; fetal, newborn and adult enzyme are probably identical [22]; Phenyl-Sepharose, DEAE-Sepharose [4]; recombinant enzyme, partially purified [15]; recombinant maltose-binding-protein phenylalanine hydroxylase fusion protein [26]; ammonium sulfate, Phenyl-Sepharose, DEAE-Sepharose, recombinant enzyme [30]; recombinant wild-type enzyme and deletion mutants [31]; truncated enzyme containing the C-terminal 343 amino acids [33]; recombinant His-tagged wild-type, I65T, R261Q and V388M mutant enzymes, affinity chromatography [38]; recombinant wild-type, R270K and V388M mutant enzymes expressed in the presence and absence of glycerol [41]; double truncated mutant enzyme DN1 102 /DC428 452 [43]) [4, 12, 15, 22, 26, 30, 31, 33, 38, 41, 43] (polyethylene glycol, Phenyl-Sepharose, DEAE-Sepharose [5]) [5, 13] [21] (recombinant wild-type, L348V and V388M mutant enzyme, affinity chromatography [36]) [36]
 #

(vapour diffusion, 1 mg enzyme dissolved in 0.1 ml 35% ammonium sulfate, 50 mM acetate, pH 6.0, 1 mM dithiothreitol, reservoir contains 60% ammonium sulfate, crystals appear after 3-4 d at 4 C [20]; vapor-diffusion hanging drop method at 4 C, reservoir solution contains 1 ml of 1.65-1.9 M ammonium sulfate, 40-100 mM NaCl and 20 mM HEPES pH 7.5, hanging

74

1.14.16.1

Phenylalanine 4-monooxygenase

drops are made using equal volumes of enzyme, 20 mg/ml, and reservoir solution, crystals grow in about one week, crystal structures of Fe-free apoenzyme, Fe3+ -bound enzyme and Fe3+ plus 7,8-dihydro-l-biopterin-bound enzyme at 1.7 A, 2.0 A and 1.4 A resolution respectively [44]) [20, 44] (crystal structure of the catalytic domain in its catalytic active Fe2+ form and as binary complex with tetrahydrobiopterin, 1.7 and 1.5 A resolution [40]; crystal structure of ternary complex of catalytic domain, Fe2+ form, with tetrahydrobiopterin and 3-(2-thienyl)-l-alanine [43]) [40, 43]
 

(expression of wild-type and S16E, S16Q, S16N, S16D, S16A, and S16K mutant enzymes in Escherichia coli [25]; expression of truncated enzyme containing the catalytic domain and various mutants in Escherichia coli [37]) [25, 27, 37] (expression in Escherichia coli [34]) [34, 44] (expression in Escherichia coli [15]; expression as maltose-binding-protein fusion protein in Escherichia coli circumvents proteolytic degradation by the host cell [26]; expression of wild-type enzyme and Asp112-Lys452, Ser2Gln428 and Gly103-Gln428 deletion mutants in Escherichia coli [31]; truncated enzyme containing the C-terminal 336 amino acids bearing the catalytic domain [33]; expression of His-tagged wild-type, I65T, R261Q and V388M mutant enzymes in Escherichia coli [38]; expression of wild-type, R270K and V388M mutant enzymes in the presence of the chemical chaperone glycerol [41]; expression of wild-type and T427P mutant enzyme in Escherichia coli [42]; expression of double truncated mutant enzyme DN1 102 /DC428 452 in Escherichia coli [43]) [15, 26, 29, 30, 31, 32, 33, 38, 41, 42, 43] (expression of wild-type, L348V, L349L and V388M mutant enzyme maltose-binding-protein fusions in Escherichia coli and COS cells [36]) [36] 

 

A322S/V379D ( truncated enzyme containing the catalytic domain, mutant shows tyrosine hydroxylation activity [37]) [37] C237D ( approx. 3fold higher activity than wild-type [32]) [32] C237S ( approx. 2fold higher activity than wild-type [32]) [32] H264Q ( mutant of full length enzyme, no tyrosine hydroxylation activity [37]) [37] H264Q/V379D ( double mutant of full length enzyme, shows significant tyrosine hydroxylation activity [37]) [37] H264Q/V379D ( truncated enzyme containing the catalytic domain, mutant shows tyrosine hydroxylation activity [37]) [37] H264Q/Y277H/V379D ( triple mutant of full length enzyme, shows significant tyrosine hydroxylation activity [37]) [37] H264Q/Y277H/V379D ( truncated enzyme containing the catalytic domain, mutant shows tyrosine hydroxylation activity [37]) [37] I65T ( 22% of wild-type phenylalanine hydroxylase activity [38]) [38] L293M ( truncated enzyme containing the catalytic domain, mutant shows tyrosine hydroxylation activity [37]) [37] 75

Phenylalanine 4-monooxygenase

1.14.16.1

L348V ( 25% activity after expression in Escherichia coli in the absence of GroESL, 55% in the presence of GroESL, 77% activity after expression in COS cells at 27 C [36]) [36] R261Q ( 31% of wild-type phenylalanine hydroxylase activity [38]) [38] R270K ( expression in the presence of the chemical chaperone glycerol enhances activity after purification [41]) [41] S16A ( similar Km for tetrahydrobiopterin and activity as wild-type [25]) [25] S16D ( similar Km for tetrahydrobiopterin and activity as wild-type [25]) [25] S16E ( slightly higher Km for tetrahydrobiopterin than wild-type, approx. 3fold higher Vmax with phenylalanine [25]) [25] S16K ( similar Km for tetrahydrobiopterin and activity as wild-type [25]) [25] S16N ( slightly higher Km for tetrahydrobiopterin than wild-type, approx. 3fold higher Vmax with phenylalanine [25]) [25] S16Q ( slightly higher Km for tetrahydrobiopterin than wild-type, similar Vmax with phenylalanine [25]) [25] S251A ( truncated enzyme containing the catalytic domain, no tyrosine hydroxylation activity [37]) [37] S251A/H264Q ( truncated enzyme containing the catalytic domain, no tyrosine hydroxylation activity [37]) [37] S251A/H264Q/V379D ( truncated enzyme containing the catalytic domain, mutant shows tyrosine hydroxylation activity [37]) [37] S251A/H264Q/Y277H ( truncated enzyme containing the catalytic domain, no tyrosine hydroxylation activity [37]) [37] S251A/H264Q/Y277H/A322S ( truncated enzyme containing the catalytic domain, no tyrosine hydroxylation activity [37]) [37] S251A/H264Q/Y277H/A322S/V379D ( truncated enzyme containing the catalytic domain, mutant shows tyrosine hydroxylation activity [37]) [37] S251A/H264Q/Y277H/A322S/V379D/Y356H ( truncated enzyme containing the catalytic domain, mutant shows tyrosine hydroxylation activity [37]) [37] S251A/H264Q/Y277H/A322S/V379D/Y356H/L293M ( truncated enzyme containing the catalytic domain, mutant shows tyrosine hydroxylation activity [37]) [37] S251A/H264Q/Y277H/V379D ( truncated enzyme containing the catalytic domain, mutant shows tyrosine hydroxylation activity [37]) [37] S251A/V379D ( truncated enzyme containing the catalytic domain, mutant shows tyrosine hydroxylation activity [37]) [37] S348L ( instable enzyme forming aggregates after expression in Escherichia coli in the presence of GroESL [36]) [36] T427P ( increase in the amount of oligomeric forms higher than tetramers after preincubation of a mixture of dimeric and tetrameric forms with phenylalanine, tetrameric form exhibits approx. 50% of wild-type tetramer phenylalanine hydroxylase activity [42]) [42] 76

1.14.16.1

Phenylalanine 4-monooxygenase

V379D ( truncated enzyme containing the catalytic domain, mutant shows tyrosine hydroxylation activity [37]) [37] V388M ( 30% of wild-type phenylalanine hydroxylase activity [38]) [38] V388M ( expression in the presence of the chemical chaperone glycerol enhances activity after purification [41]) [41] V388M ( 40% activity after expression in Escherichia coli in the absence of GroESL, 82% in the presence of GroESL, 78% activity after expression in COS cells at 27 C [36]) [36] Y277H ( mutant of full length enzyme, no tyrosine hydroxylation activity [37]) [37] Y277H/V379D ( truncated enzyme containing the catalytic domain, mutant shows tyrosine hydroxylation activity [37]) [37]

4  )  50 ( V388M mutant enzyme, 50% activity after 10 min [36]) [36] 51 ( L348V mutant enzyme, 50% activity after 10 min [36]) [36] 59 ( recombinant wild-type enzyme, 50% activity after 10 min [36]) [36] 5    "

, NaCl or KCl above 200 mM stabilize [3] , frozen and thawed enzyme is 10-20% less active than before freezing [3] , glycerol + EDTA stabilize [3] , nonionic detergents e.g. Triton X-100 and Tween 80, 0.03-0.1%, stabilize [3] , phenylalanine stabilizes [22]    , -70 C, 2 years, no loss of activity [3] , -80 C, 1 month, 30% loss of activity [9] , -80 C, 1 year, 30% loss of activity [2] , -20 C, 50 mM acetate, pH 6.0, 3 months, 20% loss of activity [6] , -80 C, enzyme concentration 1 mg/ml, several months, no loss of activity [4]

"  [1] Kaufman, S.: Aromatic amino acid hydroxylases. The Enzymes, 3rd Ed. (Boyer, P.D., Krebs, E.G., eds.), 18, 217-282 (1987) [2] Kaufman, S.: Phenylalanine 4-monooxygenase from rat liver. Methods Enzymol., 142, 3-17 (1987) [3] Shiman, R.: Purification and assay of rat liver phenylalanine 4-monooxygenase. Methods Enzymol., 142, 17-27 (1987) 77

Phenylalanine 4-monooxygenase

1.14.16.1

[4] Abita, J.P.; Blandin-Savoja, F.; Rey, F.: Phenylalanine 4-monooxygenase from human liver. Methods Enzymol., 142, 27-35 (1987) [5] Doskeland, A.P.; Doskeland, S.O.; Flatmark, T.: Phenylalanine 4-monooxygenase from bovine liver. Methods Enzymol., 142, 35-44 (1987) [6] Fujisawa, H.; Nakata, H.: Phenylalanine 4-monooxygenase from Chromobacterium violaceum. Methods Enzymol., 142, 44-49 (1987) [7] Pember, S.O.; Villafranca, J.J.; Benkovic, S.J.: Chromobacterium violaceum phenylalanine 4-monooxygenase. Methods Enzymol., 142, 50-56 (1987) [8] Guroff, G.; Rhoads, C.A.: Phenylalanine hydroxylation by Pseudomonas species (ATCC 11299a). Nature of the cofactor. J. Biol. Chem., 244, 142-146 (1969) [9] Nakata, H.; Fujisawa, H.: Purification and characterization of phenylalanine 4-monooxygenase from rat liver. Biochim. Biophys. Acta, 614, 313-327 (1980) [10] Al-Janabi, J.M.: Purification of rat liver phenylalanine hydroxylase by affinity chromatography. Arch. Biochem. Biophys., 200, 603-608 (1980) [11] Koizumi, S.; Matsushima, Y.; Nagatsu, T.; Linuma, H.; Takeuchi, T.; Umezawa, H.: 3,4-Dihydroxystyrene, a novel microbial inhibitor for phenylalanine hydroxylase and other pteridine-dependent monooxygenases. Biochim. Biophys. Acta, 789, 111-118 (1984) [12] Yamashita, M.; Minato, S.; Arai, M.; Kishida, Y.; Nagatsu, T.; Umezawa, H.: Purification of phenylalanine hydroxylase from human adult and foetal livers with a monoclonal antibody. Biochem. Biophys. Res. Commun., 133, 202-207 (1985) [13] Martinez, A.; Andersson, K.K.; Haavik, J.; Flatmark, T.: EPR and 1H-NMR spectroscopic studies on the paramagnetic iron at the active site of phenylalanine hydroxylase and its interaction with substrates and inhibitors. Eur. J. Biochem., 198, 675-682 (1991) [14] Martinez, A.; Olafsdottir, S.; Haavik, J.; Flatmark, T.: Inactivation of purified phenylalanine hydroxylase by dithiothreitol. Biochem. Biophys. Res. Commun., 182, 92-98 (1992) [15] Ledley, F.D.; Grenett, H.E.; Woo, S.L.C.: Biochemical characterization of recombinant human phenylalanine hydroxylase produced in Escherichia coli. J. Biol. Chem., 262, 2228-2233 (1987) [16] Cotton, R.G.H.; Grattan, P.J.: Phenylalanine hydroxylase of Macaca irus A simple purification by affinity chromatography. Eur. J. Biochem., 60, 427430 (1975) [17] Bloom, L.M.; Benkovic, S.J.; Gaffney, B.J.: Characterization of phenylalanine hydroxylase. Biochemistry, 25, 4204-4210 (1986) [18] Pember, S.O.; Villafranca, J.J.; Benkovic, S.J.: Phenylalanine hydroxylase from Chromobacterium violaceum is a copper-containing monooxygenase. Kinetics of the reductive activation of the enzyme. Biochemistry, 25, 66116619 (1986) [19] Abita, J.P.; Parniak, M.; Kaufman, S.: The activation of rat liver phenylalanine hydroxylase by limited proteolysis, lysolecithin, and tocopherol phosphate. Changes in conformation and catalytic properties. J. Biol. Chem., 259, 14560-14566 (1984) 78

1.14.16.1

Phenylalanine 4-monooxygenase

[20] Nakata, H.; Yamauchi, T.; Fujisawa, H.: Phenylalanine hydroxylase from Chromobacterium violaceum. Purification and characterization. J. Biol. Chem., 254, 1829-1833 (1979) [21] Letendre, C.H.; Dickens, G.; Guroff, G.: Phenylalanine hydroxylase from Pseudomonas sp. (ATCC 11299a). Purification, molecular weight, and influence of tyrosine metabolites on activation and hydroxylation. J. Biol. Chem., 250, 6672-6678 (1975) [22] Woo, S.L.C.; Gillam, S.S.; Woolf, L.I.: The isolation and properties of phenylalanine hydroxylase from human liver. Biochem. J., 139, 741-749 (1974) [23] Shiman, R.; Gray, D.W.; Hill, M.A.: Regulation of rat liver phenylalanine hydroxylase. I. Kinetic properties of the enzyme's iron and enzyme reduction site. J. Biol. Chem., 269, 24637-24646 (1994) [24] Shiman, R.; Xia, T.; Hill, M.A.; Gray, D.W.: Regulation of rat liver phenylalanine hydroxylase. II. Substrate binding and the role of activation in the control of enzymic activity. J. Biol. Chem., 269, 24647-24656 (1994) [25] Kowlessur, D.; Yang, X.J.; Kaufman, S.: Further studies of the role of Ser-16 in the regulation of the activity of phenylalanine hydroxylase. Proc. Natl. Acad. Sci. USA, 92, 4743-4747 (1995) [26] Martinez, A.; Knappskog, P.M.; Olafsdottir, S.; Doskeland, A.P.; Eiken, H.G.; Svebak, R.M.; Bozzini, M.; Apold, J.; Flatmark, T.: Expression of recombinant human phenylalanine hydroxylase as fusion protein in Escherichia coli circumvents proteolytic degradation by host cell proteases. Isolation and characterization of the wild-type enzyme. Biochem. J., 306, 589-597 (1995) [27] Kappock, T.J.; Harkins, P.C.; Friedenberg, S.; Caradonna, J.P.: Spectroscopic and kinetic properties of unphosphorylated rat hepatic phenylalanine hydroxylase expressed in Escherichia coli. Comparison of resting and activated states. J. Biol. Chem., 270, 30532-30544 (1995) [28] Carr, R.T.; Balasubramanian, S.; Hawkins, P.C.D.; Benkovic, S.J.: Mechanism of metal-independent hydroxylation by Chromobacterium violaceum phenylalanine hydroxylase. Biochemistry, 34, 7525-7532 (1995) [29] Doeskeland, A.P.; Martinez, A.; Knappskog, P.M.; Flatmark, T.: Phosphorylation of recombinant human phenylalanine hydroxylase: effect on catalytic activity, substrate activation and protection against non-specific cleavage of the fusion protein by restriction protease. Biochem. J., 313, 409-414 (1996) [30] Kowlessur, D.; Citron, B.A.; Kaufman, S.: Recombinant human phenylalanine hydroxylase: novel regulatory and structural properties. Arch. Biochem. Biophys., 333, 85-95 (1996) [31] Knappskog, P.M.; Flatmark, T.; Aarden, J.M.; Haavik, J.; Martinez, A.: Structure/function relationships in human phenylalanine hydroxylase. Effect of terminal deletions on the oligomerization, activation and cooperativity of substrate binding to the enzyme. Eur. J. Biochem., 242, 813-821 (1996) [32] Knappskog, P.M.; Martinez, A.: Effect of mutations at Cys237 on the activation state and activity of human phenylalanine hydroxylase. FEBS Lett., 409, 7-11 (1997) [33] Daubner, S.C.; Hillas, P.J.; Fitzpatrick, P.F.: Expression and characterization of the catalytic domain of human phenylalanine hydroxylase. Arch. Biochem. Biophys., 348, 295-302 (1997) 79

Phenylalanine 4-monooxygenase

1.14.16.1

[34] Chen, D.; Frey, P.A.: Phenylalanine hydroxylase from Chromobacterium violaceum. Uncoupled oxidation of tetrahydropterin and the role of iron in hydroxylation. J. Biol. Chem., 273, 25594-25601 (1998) [35] Teigen, K.; Froystein, N.A.; Martinez, A.: The structural basis of the recognition of phenylalanine and pterin cofactors by phenylalanine hydroxylase: implications for the catalytic mechanism. J. Mol. Biol., 294, 807-823 (1999) [36] Gamez, A.; Perez, B.; Ugarte, M.; Desviat, L.R.: Expression analysis of phenylketonuria mutations: effect on folding and stability of the phenylalanine hydroxylase protein. J. Biol. Chem., 275, 29737-29742 (2000) [37] Daubner, S.C.; Melendez, J.; Fitzpatrick, P.F.: Reversing the substrate specificities of phenylalanine and tyrosine hydroxylase: aspartate 425 of tyrosine hydroxylase is essential for l-DOPA formation. Biochemistry, 39, 9652-9661 (2000) [38] Leandro, P.; Rivera, I.; Lechner, M.C.; de Almeida, I.T.; Konecki, D.: The V388M mutation results in a kinetic variant form of phenylalanine hydroxylase. Mol. Genet. Metab., 69, 204-212 (2000) [39] Donlon, J.; Fallon, B.; Barrett, P.; Carroll, O.; Henderson, P.; Fairley, J.S.: Hepatic phenylalanine hydroxylase of Clethrionomys glareolus Schreber as a bioindicator of pollution. Biochem. Soc. Trans., 26, S64 (1998) [40] Andersen, O.A.; Flatmark, T.; Hough, E.: High resolution crystal structures of the catalytic domain of human phenylalanine hydroxylase in its catalytically active Fe(II) form and binary complex with tetrahydrobiopterin. J. Mol. Biol., 314, 279-291 (2001) [41] Leandro, P.; Lechner, M.C.; Tavares de Almeida, I.; Konecki, D.: Glycerol increases the yield and activity of human phenylalanine hydroxylase mutant enzymes produced in a prokaryotic expression system. Mol. Genet. Metab., 73, 173-178 (2001) [42] Bjorgo, E.; De Carvalho, R.M.N.; Flatmark, T.: A comparison of kinetic and regulatory properties of the tetrameric and dimeric forms of wild-type and Thr427!Pro mutant human phenylalanine hydroxylase. Contribution of the flexible hinge region Asp425-Gln429 to the tetramerization and cooperative substrate binding. Eur. J. Biochem., 268, 997-1005 (2001) [43] Andreas Andersen, O.; Flatmark, T.; Hough, E.: Crystal structure of the ternary complex of the catalytic domain of human phenylalanine hydroxylase with tetrahydrobiopterin and 3-(2-thienyl)-l-alanine, and its implications for the mechanism of catalysis and substrate activation. J. Mol. Biol., 320, 1095-1108 (2002) [44] Erlandsen, H.; Kim, J.Y.; Patch, M.G.; Han, A.; Volner, A.; Abu-Omar, M.M.; Stevens, R.C.: Structural comparison of bacterial and human iron-dependent phenylalanine hydroxylases: similar fold, different stability and reaction rates. J. Mol. Biol., 320, 645-661 (2002)

80

)   !    

4

   
 1.14.16.2    l-tyrosine,tetrahydrobiopterin:oxygen oxidoreductase (3-hydroxylating)    tyrosine 3-monooxygenase    l-tyrosine hydroxylase TH [22, 23, 25-31] monophenol monooxygenase [19] oxygenase, tyrosine 3-monotyrosine 3-hydroxylase tyrosine hydroxylase tyrosine-3-mono-oxygenase [24, 25] tyrosine-3-monooxygenase [26]
    9036-22-0

     Bos taurus [1, 3, 4, 6, 7, 11, 13-15, 17, 26] Rattus norvegicus (PC12 cells [22]) [1-3, 8-10, 12, 16, 18, 20-22, 24, 26, 29, 31] Oryctolagus cuniculus [5] Canis familiaris [6] Cavia porcellus [6, 11] Rana esculenta (ridibunda, frog [19]; tyrosinase activity is modified to tyrosine hydroxylase activity via immobilization [19]) [19] Portulaca grandiflora [32] Homo sapiens (isoform 1 hTH1 [28,30]; 4 isoforms hTH1, hTH2, hTH3, hTH4 [23, 25, 26]) [23, 25, 26, 28, 30] Schistosoma mansoni [27]

81

Tyrosine 3-monooxygenase

1.14.16.2

!    "
 # 

l-tyrosine + tetrahydrobiopterin + O2 = 3,4-dihydroxy-l-phenylalanine + 4ahydroxytetrahydrobiopterin (requires Fe2+ , activated by phosphorylation, catalysed by EC 2.7.1.128 [acetyl-CoA carboxylase] kinase; mechanism [6, 21, 31]; stereochemical analysis of ligand binding [20]; active binding site [20]; H-NMR analysis of conformation of the complex between phenylalanine, 6-methyltetrahydropterin and isoform hTH1 [23]; secondary structure and conformation analysis of phosphorylated and unphosphorylated isoform hTH1 [28]; phosphorylation site of hTH1 is Ser-40 [28]; ligand binding model, hTH1 [30])    oxidation redox reaction reduction       l-tyrosine + (6R)-l-erythro-1',2'-dihydroxypropyltetrahydropterin + O2 ( first step in biosynthesis of catecholamines such as norepinephrine, epinephrine and dopamine [1, 8, 10, 14]) (Reversibility: ? [1, 8, 10, 14]) [1, 8, 10, 14] $ ?  l-tyrosine + (6R)-l-erythro-tetrahydrobiopterin + O2 ( probable regulatory role of cosubstrate for all 4 isoforms [25]) (Reversibility: ? [25]) [25] $ ?  l-tyrosine + tetrahydropterin + O2 ( reaction is coupled with ascorbate oxidation [19]; rate-limiting step in catecholamine biosynthesis [3, 22, 24, 28]; first step in biosynthesis of catecholamines such as norepinephrine, epinephrine and dopamine [1, 8, 10, 11, 14, 19, 24, 28]) (Reversibility: ? [1, 3, 8, 10, 11, 14, 19, 22-24, 28]) [1, 3, 8, 10, 11, 14, 19, 22-24, 28] $ 3,4-dihydroxy-l-phenylalanine + dihydrobiopterin + H2 O      3,4-dihydroxy-l-phenylalanine + 6,7-dimethyltetrahydropteridine + O2 ( i.e. l-dopa [26]; l-dopa-oxidase activity [26]) (Reversibility: ? [26]) [26] $ ?  3,4-dihydroxy-l-phenylalanine + tetrahydropterin + O2 ( i.e. ldopa [26]; 4 isoforms, thiols required, stimulation by Fe2+ and tetrahydropterin [26]) (Reversibility: ? [26]) [26] $ ?  4-bromo-l-phenylalanine + 6-methyltetrahydropterin + O2 ( low activity [21]) (Reversibility: ? [21]) [21] $ ?

82

1.14.16.2

Tyrosine 3-monooxygenase

 4-chloro-l-phenylalanine + 6-methyltetrahydropterin + O2 (Reversibility: ? [21]) [21] $ ?  4-fluoro-l-phenylalanine + 6-methyltetrahydropterin + O2 (Reversibility: ? [21]) [21] $ ?  4-methoxy-l-phenylalanine + 6-methyltetrahydropteridine + O2 (Reversibility: ? [21]) [21] $ ?  4-methyl-l-phenylalanine + 6-methyltetrahydropterin + O2 (Reversibility: ? [21]) [21] $ ?  l-phenylalanine + tetrahydropteridine + O2 ( recombinant wild-type and mutant [31]) (Reversibility: ? [6, 23, 31]) [6, 23, 31] $ l-tyrosine + dihydropteridine + H2 O [6, 23, 31]  l-phenylalanine + tetrahydropteridine + O2 ( wild-type and mutants [29]) (Reversibility: ? [6, 21, 23, 29]) [6, 21, 23, 29] $ 3,4-dihydroxy-l-phenylalanine + dihydropteridine + H2 O [6, 21, 23, 29]  l-tyrosine + (6R)-l-erythro-tetrahydrobiopterin + O2 ( recombinant hTH1 [30]; cosubstrate has a regulatory role for all 4 isoforms [25]; tyrosine at 0.1 mM and O2 at 4.8% inhibit with (6R)-lerythro-tetrahydrobiopterin as electron donor, depending on O2 and cofactor concentration [14]; preferred electron donor [7]) (Reversibility: ? [7, 14, 25, 30]) [7, 14, 25, 30] $ ?  l-tyrosine + (6RS)-l-erythro-tetrahydrobiopterin + O2 (Reversibility: ? [25]) [25] $ ?  l-tyrosine + (6S)-l-erythro-tetrahydrobiopterin + O2 (Reversibility: ? [25]) [25] $ ?  l-tyrosine + 2-amino-4-hydroxy-6,7-dimethyltetrahydropterin + O2 ( synthetic pterin as electron donor [8,10,32]) (Reversibility: ? [7, 8, 10, 11, 13, 32]) [7, 8, 10, 11, 13, 32] $ ?  l-tyrosine + 2-amino-4-hydroxy-6-methyltetrahydropterin + O2 ( synthetic pterin as electron donor [8,10,32]) (Reversibility: ? [7, 8, 10, 13, 18, 32]) [7, 8, 10, 13, 18, 32] $ ?  l-tyrosine + 2-methyl-4-oxo-5,6,7,8-tetrahydropterin + O2 ( recombinant hTH1 [30]) (Reversibility: ? [30]) [30] $ ?  l-tyrosine + 6-methyltetrahydropterin + O2 ( higher activity than with tetrahydropterin [23]; artificial cosubstrate [7]) (Rever83

Tyrosine 3-monooxygenase

$  $ 

$  $ 

$

1.14.16.2

sibility: ? [3, 7, 10, 13, 15, 16, 20-23, 25]) [3, 7, 10, 13, 15, 16, 2023, 25] ? l-tyrosine + ascorbate + O2 (Reversibility: ? [19]) [19] 3,4-dihydroxy-l-phenylalanine + dihydroascorbate + H2 O [19] l-tyrosine + tetrahydrobiopterin + O2 ( recombinant wild-type and mutant [31]; recombinant hTH1 [30]; tetrahydropterin absolutely required [27]; substrate inhibition occurs at 0.1 mM tyrosine or O2 with (6R)- and (6RS)-tetrahydrobiopterin [7]) (Reversibility: ? [1-32]) [1-32] 3,4-dihydroxy-l-phenylalanine + dihydropteridine + H2 O ( 3,4-dihydroxy-l-phenylalanine is identical with dopa [1-32]) [1-32] l-tyrosine + tetrahydrofolic acid + O2 (Reversibility: ? [11]) [11] ? Additional information ( active with diverse tetrahydropterin analogues, substituted at C6, C7 or C3, overview [30]; under aerobic conditions can generate significant amounts of reactive oxygen species, including hydrogen peroxide and hydroxyl radicals [26]; 4-substituted substrate analogues get hydroxylated at position 4 or 3, the latter is preferred with big-sized substituents, multiply hydroxylated products occur as well [21]; tyrosinase activity is modified to tyrosine hydroxylase activity via immobilization [19]) [19, 21, 26, 30] ?

2  1,10-phenanthroline ( complete [26]) [4, 26] 2,2'-dipyridyl [11] 2,4-diamino-6-dihydroxypropyl-5,6,7,8-tetrahydropterin ( competitive against (6R)-l-erythro-tetrahydrobiopterin [30]) [30] 2-amino-4,6-dioxo-3,4,5,6,8,8a,9,10-octaahydrooxazolo[1,2-f]pteridine ( competitive against (6R)-l-erythro-tetrahydrobiopterin [30]) [30] 2-methyl-l-tyrosine [11] 3,4-dihydroxybenzoic acid [11] 3,4-dihydroxystyrene [2] 3-iodo-l-tyrosine ( complete [26]) [7, 13, 26] 5(N-phenylthiocarbamoyl)-5,6,7,8-tetrahydropterin [30] 5-[(3-azido-6-nitrobenzylidene)amino]-2,6-diamino-4-pyrimidinone

( competitive against tetrahydrobiopterin [20]) [20] 5-deaza-6-methyltetrahydropterin [13] 5-methyl-5,6,7,8-tetrahydropterin ( competitive against (6R)-l-erythro-tetrahydrobiopterin [30]) [30] 6-[2-(4-benzoylphenyl)propionyloxymethyl]-5,6,7,8-tetrahydropterin

( competitive against (6R)-l-erythro-tetrahydrobiopterin [30]) [30] 8-methyl-6,7-dimethyl-5,6,7,8-tetrahydropterin ( competitive against (6R)-l-erythro-tetrahydrobiopterin [30]) [30] Ba2+ ( weak inhibition [4]) [4] Co2+ ( competitive against Fe2+ [23]) [4, 23]

84

1.14.16.2

Tyrosine 3-monooxygenase

Fe3+ [4] l-dopa ( above 0.15 mM inhibiting l-dopa-oxidase activity in presence of tetrahydropterin, but not with 6,7-dimethyltetrahydropterin [26]; modest feedback inhibition [22]) [1, 11, 22, 26] l-erythro-7,8-dihydrobiopterin ( competitive against tetrahydropterin [23]) [23] Ni2+ ( competitive against Fe2+ [23]) [4, 23] O2 ( above 4.8% [14]) [8, 14] RNA ( above 0.015 mg/ml [3]) [3] Zn2+ ( competitive against Fe2+ [23]) [4, 23] a-propyldihydroxyphenylacetamide [11] bathocuproine sulfonate ( slightly [26]) [26] bathophenanthroline sulfonate [4, 26] catecholamines ( feedback inhibition, reversible by phosphorylation [28]) [8, 27, 28] dihydrobiopterin ( l-dopa-oxidase activity [26]) [26] dopamine [8, 11] dopamine quinone ( covalent modification and inactivation [22]) [22] epinephrine [8] methylcatechol [13] noradrenaline [26] norepinephrine [8, 11] phenylalanine ( l-isomer, not d-isomer [11]) [6, 11] tetrahydrobiopterin ( substrate inhibition at tyrosine and O2 concentrations higher than 0.1 mM and 2.2 mM, respectively [7]) [7] tyrosine ( at concentrations above 0.03 mM [27]; substrate inhibition, with (6R)-l-erythro-tetrahydrobiopterin [25]; 0.1 mM [14]) [14, 25, 27] Additional information ( inhibited by metal chelating agents [32]; pH-dependence of inhibitor binding [13]) [13, 32] &

  RNA ( below 0.015 mg/ml activation, rat brain enzyme contains RNA, about 10% of the total mass [3]) [3] SDS ( 0.01%, activates [10]) [10] bathocuproine sulfate ( slight activation [4]) [4] dextran sulfate ( activates the enzyme in the crude extract, less effective with the purified enzyme [18]) [18] heparin ( 0.1 mg/ml, activates [10,16]; activates the enzyme in the crude extract, less effective with the purified enzyme [18]) [10, 16, 18] phosphatidyl-l-serine ( activates [17]; no stimulation [18]) [17] phosphatidylinositol ( no stimulation [18]; 0.1 mg/ml activates [10,16]) [10, 16] phospholipid ( activates [17]; no stimulation [18]) [17] thiols ( activate the l-dopa activity [26]) [26]

85

Tyrosine 3-monooxygenase

1.14.16.2

Additional information ( inhibitory effect of several compounds on l-dopa-oxidase activity [26]; activation by phosphorylation [10, 16, 17, 26, 28]; long lasting induction of expression in anterior and posterior locus coelereus after injection of vindeburnol, i.e. RU24722, can be reversed by housing of the animals at 28 C [24]) [10, 16, 17, 24, 26, 28] '(   Cu2+ ( slightly activating [26]) [26] Mn2+ ( activates [32]) [32] NaCl ( activates [16]) [16] Zn2+ ( 0.13 mol per mol of subunit [4]) [4] iron ( required [25, 26, 31]; incorporation of stoichiometric amounts of Fe2+ leads to 40fold increase in activity [23]; 0.1 atom per subunit [12]; 0.5-0.75 mol of iron per mol of enzyme [17]; 0.66 mol per mol of subunit [4]; high spin Fe(III) in an environment of nearly axial symmetry [4]; Fe2+ activates [11, 26, 32]) [4, 11, 12, 17, 23, 25, 26, 30-32] phosphate ( 1 covalently bound residue per subunit [12]; activated enzyme contains: 0.75 mol per mol of subunit [3]; 0.62 mol per mol of subunit [4]; 1 mol per mol of subunit [16]) [3, 4, 12, 16] " & *-% , 0.00003 ( crude extract [27]) [27] 0.03-0.1 ( recombinant enzyme, cell lysate [12]) [12] 0.04 ( purified enzyme [5]) [5] 0.0916 ( purified enzyme from brain [3]) [3] 0.16 ( purified recombinant isoform hTH3 [26]) [26] 0.18 ( purified enzyme from adrenal medulla [3]) [3] 0.2 ( purified enzyme [26]; purified recombinant isoform hTH4 [26]) [26] 0.21 ( purified enzyme [14]) [14] 0.29 ( purified recombinant isoform hTH2 [26]) [26] 0.33 ( purified recombinant isoform hTH1, tyrosine concentration is 0.2 mM [26]) [26] 0.36 ( purified enzyme [17]) [17] 0.41 ( purified enzyme [15]) [15] 0.425 ( purified enzyme [4]) [4] 0.78 ( purified enzyme [27]) [27] 0.79 ( purified recombinant isoform hTH1, tyrosine concentration is 0.05 mM [26]) [26] 1 ( about, purified recombinant enzyme [30]) [30] 1.6 ( purified enzyme [16,18]) [16, 18] 1.7 ( purified, recombinant enzyme [12]) [12] 1.88 ( purified enzyme [7]) [7] 2.5 ( purified enzyme, tyrosine concentration is 0.2 mM [26]) [26] 3.46 ( purified enzyme, immobilized [19]) [19] 20.7 ( purified enzyme, tyrosine concentration is 0.05 mM [26]) [26] 330 ( purified enzyme [10]) [10] 86

1.14.16.2

Tyrosine 3-monooxygenase

1604 ( purified enzyme [8]) [8] Additional information ( dopa-oxidase activity of the 4 isoforms [26]; activity of immobilized enzyme under different conditions in bioreactors [19]) [19, 26] . / *', 0.00125 (phenylalanine, recombinant mutant D425V [29]) [29] 0.006 (O2, below, recombinant enzyme [12]) [12] 0.0061 (tyrosine) [3] 0.0077 (phenylalanine, recombinant mutant H323Y [29]) [29] 0.0088 (phenylalanine, recombinant mutant Q310H [29]) [29] 0.0094 (tyrosine, recombinant enzyme, pH-independent [12]) [12] 0.0095 (tyrosine) [27] 0.01 (phenylalanine, recombinant mutant Y371F [31]) [31] 0.01 (tetrahydropterin, recombinant phosphorylated hTH1, l-dopaoxidase activity [26]) [26] 0.011 (tyrosine) [23] 0.016 (tyrosine, recombinant wild-type [29]) [29] 0.021 (tetrahydropterin, recombinant enzyme, value is pH-dependent [12]) [12] 0.027 (tetrahydropterin, recombinant wild-type, pH 7.1 [31]) [31] 0.032 (tyrosine, recombinant wild-type, pH 6.0 [31]) [31] 0.033 (6-methyl-5,6,7,8-tetrahydropterin, recombinant wild-type, pH 7.1 [31]) [31] 0.045 (tetrahydropterin) [27] 0.045 (tyrosine, recombinant mutant D425V [29]) [29] 0.05 (tyrosine, bovine adrenal gland [6]) [6] 0.051 (tyrosine, recombinant wild-type, pH 7.1 [31]) [31] 0.053 (tetrahydropterin, recombinant mutant Y371F, pH 7.1 [31]) [31] 0.054 (tyrosine, recombinant mutant Q310H [29]) [29] 0.055 (6-methyl-5,6,7,8-tetrahydropterin, recombinant wild-type, pH 6.0 [31]) [31] 0.055 (tyrosine, recombinant isoform hTH2, with 6-methyltetrahydropterin [25]) [25] 0.056 (l-dopa, recombinant hTH1, dopa-oxidase activity with tetrahydropterin [26]) [26] 0.058 (2-amino-4-hydroxy-6-methyl-5,6,7,8-tetrahydropteridine) [8, 18] 0.059 (6-methyl-5,6,7,8-tetrahydropterin, recombinant mutant Y371F, pH 7.1 [31]) [31] 0.06-0.1 (6-methyl-5,6,7,8-tetrahydropterin) [23] 0.065 (6-methyl-5,6,7,8-tetrahydropterin, recombinant mutant Y371F, pH 6.0 [31]) [31] 0.065 (tyrosine, recombinant mutant Y371F, pH 7.1 [31]) [31] 0.066 (tyrosine, recombinant isoform hTH4, with 6-methyltetrahydropterin [25]) [25] 0.071 (tyrosine, recombinant mutant Y371F, pH 6.0 [31]) [31]

87

Tyrosine 3-monooxygenase

1.14.16.2

0.074 (tyrosine, recombinant isoform hTH3, with 6-methyltetrahydropterin [25]) [25] 0.075 (tyrosine, with 6-methyltetrahydropterin [10]) [10] 0.08 (tetrahydropterin, low Km form [3]) [3] 0.085 (phenylalanine) [23] 0.092 (tyrosine, recombinant mutant H323Y [29]) [29] 0.1 (phenylalanine, recombinant wild-type [29]) [29] 0.11 (phenylalanine, recombinant wild-type [31]) [31] 0.146 (l-dopa, recombinant, dopa-oxidase activity with 6,7-dimethyltetrahydropterin [26]) [26] 0.15 (6-methyl-5,6,7,8-tetrahydropterin) [32] 0.166 (tyrosine, recombinant isoform hTH1, with 6-methyltetrahydropterin [25]) [25] 0.3 (phenylalanine, bovine adrenal [6]) [6] 0.5 (tyrosine) [32] 0.63 (tetrahydropterin, high Km form [3]) [3] 0.95 (6-methyl-5,6,7,8-tetrahydropterin) [3] Additional information ( Km -values of diverse tetrahydropterin analogues, substituted at C6 , C7 or C3 [30]; Km -values for tyrosine of mutant phenylalanine hydroxylase with tyrosine hydroxylation activity [29]; Km dependent on cosubstrate type and concentration, overview for 4 isoforms [25]; Km is dependent on substrate and cofactor concentration when (6R)-l-erythro-tetrahydrobiopterin is used due to inhibitory effects [14]; Km dependence on substrate concentrations [7, 15]; effect of RNA on Km [3]; rat brain: 2 kinetically distinguishable forms: low Km form, high Km form [3, 10]; phosphorylation converts the enzyme from a form possessing a high Km for pterin cofactor to a form with a low Km for pterin cofactor [10]) [1, 3, 7, 10, 14-18, 25, 29, 30] . / *', 0.001 (6-[2-(4-benzoylphenyl)propionyloxymethyl]-5,6,7,8-tetrahydropterin) [30] 0.011 (3-iodo-l-tyrosine, versus tetrahydrobiopterin [13]) [13] 0.016 (2,4-diamino-6-dihydroxypropyl-5,6,7,8-tetrahydropterin) [30] 0.024 (3-iodo-l-tyrosine, versus tyrosine, in presence of tetrahydrobiopterin [13]) [13] 0.063 (5-methyl-5,6,7,8-tetrahydropterin) [30] 0.07 (l-erythro-7,8-dihydrobiopterin) [23] 0.138 (2-amino-4,6-dioxo-3,4,5,6,8,8a,9,10-octaahydrooxazolo[1,2-f]pteridine) [30] 0.617 (8-methyl-6,7-dimethyl-5,6,7,8-tetrahydropterin) [30] 2.83 (5(N-phenylthiocarbamoyl)-5,6,7,8-tetrahydropterin) [30] Additional information ( value dependent on electron donor, overview [13]) [13] 0  5.4-5.5 ( crude extract [8]) [8] 5.9 ( nonphosphorylated enzyme [16]) [16] 88

1.14.16.2

Tyrosine 3-monooxygenase

6 ( assay at [22]) [22] 6-7 ( 4 isoforms [25]) [25] 6-7.5 ( purified enzyme [8]) [8, 18] 6.2 [10, 11, 16] 7 ( recombinant hTH1, assay at [30]; recombinant enzyme isoforms, l-dopa-oxidase activity [26]; broad optimum, recombinant enzyme [12]) [12, 26, 30] 7.1 ( assay at [21]) [12, 21] Additional information ( pH profile is affected by a variety of conditions: enzymatic phosphorylation by cAMP-dependent protein kinase, calmodulin-dependent protein kinase II and presence of polyanions [8]) [8] 0 

 5-8.5 ( 4 isoforms [25]) [25] 5.9-7.4 ( activity declines precipitously below pH 5.9 and above pH 7.4 [16]) [16] )  *
, 25 ( recombinant hTH1, assay at [30]; assay at [19, 21]) [19, 21, 30] 30 ( assay at [8]) [8] 37 ( assay at [5, 10, 14, 16, 22]) [5, 10, 14, 16, 22]

  #   ' 1  24000 ( 4 isoforms, gel filtration [25]; sucrose density gradient centrifugation [16]) [16, 25] 210000-211000 ( adrenal, gel filtration [4]; gel filtration [3]) [3, 4] 225000 ( pheochromocytoma tumors, asymmetric protein, native PAGE, gel filtration and sucrose density gradient centrifugation [10]) [10] 239000 ( brain, gel filtration [3]) [3] 250000 ( gel filtration [12]) [12] 260000 ( from adrenal medulla [1]; gel filtration [8,18]) [1, 8, 18] 280000 ( form I [15]; gel filtration [7, 14, 15]) [1, 7, 14, 15] 310000 ( from caudate nucleus, gel filtration [15]) [15] 390000 ( from adrenal medulla, form II, gel filtration [15]) [15] Additional information ( high molecular weight of brain enzyme is partly due to association with RNA, this makes it difficult to decide whether tyrosine hydroxylase molecules of different structure are present in the various regions of the brain, cell bodies of noradrenergic neurons: MW 200000, substancia nigra and caudate nucleus, dopaminergic neurons: MW 65000 [1]; peripheral noradrenergic neurons in superior cervical ganglion: MW 130000 [1]) [1, 18]

89

Tyrosine 3-monooxygenase

1.14.16.2

  tetramer ( 2 * 61100 + 2 * 62400, SDS-PAGE [3]; 4 * 55000, recombinant enzyme, SDS-PAGE [12]; 4 * 59000, SDS-PAGE [1, 8, 18]; 4 * 60000, 4 isoforms, SDS-PAGE [25]; 4 * 60000, SDS-PAGE [1, 7, 15, 16]; 4 * 56000, about, pheochromocytoma tumor, SDS-PAGE and amino acid sequence analysis [10]; 4 * 62000 [1]; 4 * 63300, SDSPAGE [3]) [1, 3, 7, 8, 10, 12, 15, 16, 18, 25] $   "

no glycoprotein [12, 17]

 2 %$ %' %

% PC-12 cell [22] adrenal gland [2, 5, 6, 13, 17, 18, 26] adrenal medulla [1, 3, 4, 7, 8, 11, 14, 15] brain [1, 3, 11] callus (cell culture) [32] caudate nucleus [6, 15] epidermis [19] heart [6] locus ceruleus ( anterior and posterior [24]) [24] nervous system ( sympathetic, innervated [11]) [11] pheochromocytoma cell ( tumor [10]) [10, 22, 26] striatum [16] 3#

cytosol [4, 10, 11, 14, 22] membrane ( particle-bound [7]) [7] $"

(2 forms with different MW in adrenal medulla [15]; from adrenal medulla [3, 4, 7, 14, 17]; large scale [4]; from caudate nuclei [15]) [3, 4, 7, 11, 14, 15, 17] (recombinant wild-type and mutants from Escherichia coli [29]; recombinant [21]; recombinant from Escherichia coli [20]; 2 kinetically distinguishable forms of the enzyme: low Km form and high Km form [3]; from brain [3]; recombinant enzyme from Spodoptera frugiperda [12]) [3, 8, 10, 12, 16, 18, 20, 21, 29] [5] (immobilization on polyacrylamide-based support [19]) [19] [32] (4 isoforms, recombinant from Escherichia coli [25]) [23, 25] (recombinant from Escherichia coli [27]) [27]

90

1.14.16.2

Tyrosine 3-monooxygenase


 #

(crystals of the binary complex with iron and 7,8-dihydrobiopterin obtained by equilibrium dialysis, solutions degassed by helium and crystal growth in nitrogen atmosphere at 4 C [20]; 3D-structure model, ligand binding of pterin analogues [20]) [20] (crystal structure, analysis of tetrahydropterin analogues binding to isoform hTH1 [30]) [30]
 

(expression of wild-type and mutant enzymes in Escherichia coli [29,31]; expression in Escherichia coli [20]; expression in Spodoptera frugiperda cells via baculovirus expression system [12]) [9, 12, 20, 29, 31] (isoform 1 hTH1 and His-tagged truncated mutant, expression in Escherichia coli [30]; 4 isoforms, overexpression in Escherichia coli, phosphorylation-free [25,26]) [25, 26, 30] (expression as His-tagged protein in Escherichia coli, amino acid sequence analysis [27]) [27] 

 

D425V ( 335fold reduced tyrosine hydroxylation/dopa formation activity, 120fold reduced reaction velocity with tyrosine, 3fold enhanced phenylalanine hydroxylation activity, active site mutant [29]) [29] H323Y ( enhanced Km for tyrosine, 4.5fold enhanced phenylalanine hydroxylation activity, active site mutant [29]) [29] Q310H ( 4fold reduced tyrosine hydroxylation/dopa formation activity, slightly enhanced phenylalanine hydroxylation activity, active site mutant [29]) [29] Y371F ( increased Km for tyrosine and pterin cosubstrates, highly decreased Km for phenylalanine [31]) [31] Additional information ( truncated hTH1 isoform lacking the 150 N-terminal amino acids [30]; active site mutants of phenylalanine hydroxylase lead to highly increased tyrosine hydroxylation activity of the enzyme mutants [29]; investigation of the role of several amino acid residues in binding of substrate and ligands by site-specific mutagenesis [20]) [20, 29, 30] 

synthesis ( immobilization of tyrosinase on polyacrylamide-based support for production of l-dopa from l-tyrosine thereby modifying the enzyme activity to tyrosine hydroxylase [19]) [19]

4  0  7 ( preincubation at 37 C for 2 h below, enzyme isoforms are unstable, especially isoforms hTH1 and hTH3 [25]; preincubation at 37 C for 2 h above, isoforms are stable [25]) [25] 8 ( isoforms, optimal stability at [25]) [25]

91

Tyrosine 3-monooxygenase

1.14.16.2

)  4 ( 5 h, without stabilizing agent, complete loss of activity [18]) [18] 37 ( unstable in presence of phosphatidylinositol or NaCl at high concentration, but relatively stable in presence of heparin [16]) [16] 50 ( half-life of activated, phosphorylated enzyme: 5 min, halflife of nonphosphorylated enzyme: 15 min [1]) [1] Additional information ( thermal stability of hTH1 is increased by phosphorylation at Ser-40 [28]) [28] 5    "

, 0.1 M 2-mercaptoethanol is necessary for stabilization during purification [11] , EDTA, 0.1 mM stabilizes [8, 18] , Tween 80, 0.05% stabilizes [8, 18] , glycerol, 25% stabilizes [8, 18] , heparin stabilizes [16] , enzyme is stabilized by immobilization on polyacrylamide-based support [19] , phosphorylated enzyme is less stable than nonphosphorylated form [1]    , -70 C, in presence of diisopropylfluorophosphate, 2 months [3] , -80 C, highly purified enzyme, 2 months [15] , -80 C, protein concentration above 0.05 mg/ml, 2 months [7] , 4 C, in presence of diisopropylfluorophosphate, 1 week [3] , -80 C, 0.05% Tween 80, 0.1 mM EDTA, 25% glycerol, 5 mM sodium phosphate buffer, pH 7.5, 3 months without loss of activity [18] , -80 C, 0.05% Tween 80, 1 mM EDTA, 25% glycerol, 3 months [8] , 4 C, 24 h, 0.05% Tween 80, 1 mM EDTA, 25% glycerol, 68% loss of activity [8] , 4 C, 5 h, without stabilizing agents, almost complete loss of activity [8, 18] , -80 C, 20 mM Tris-HCl, pH 7.4, 8% sucrose, dithiothreitol, several months [5] , -30 C, immobilized and lyophilized enzyme, stored in dry environment, unaltered more than 150 days [19]

"  [1] Kaufman, S.: Aromatic amino acid hydroxylases. The Enzymes, 3rd Ed. (Boyer, P.D., Krebs, E.G., eds.), 18, 217-282 (1987) [2] Koizumi, S.; Matsushima, Y.; Nagatsu, T.; Linuma, H.; Takeuchi, T.; Umezawa, H.: 3,4-Dihydroxystyrene, a novel microbial inhibitor for phenylalanine hydroxylase and other pteridine-dependent monooxygenases. Biochim. Biophys. Acta, 789, 111-118 (1984)

92

1.14.16.2

Tyrosine 3-monooxygenase

[3] Nelson, T.J.; Kaufman, S.: Interaction of tyrosine hydroxylase with ribonucleic acid and purification with DNA-cellulose or poly(A)-sepharose affinity chromatography. Arch. Biochem. Biophys., 257, 69-84 (1987) [4] Haavik, J.; Andersson, K.K.; Petersson, L.; Flatmark, T.: Soluble tyrosine hydroxylase (tyrosine 3-monooxygenase) from bovine adrenal medulla: large-scale purification and physicochemical properties. Biochim. Biophys. Acta, 953, 142-156 (1988) [5] Lloyd, T.; Walega, M.A.: Purification of tyrosine hydroxylase by high-pressure liquid chromatography. Anal. Biochem., 116, 559-563 (1981) [6] Ikeda, M.; Levitt, M.; Udenfriend, S.: Phenylalanine as substrate and inhibitor of tyrosine hydroxylase. Arch. Biochem. Biophys., 120, 420-427 (1967) [7] Nagatsu, T.; Oka, K.: Tyrosine 3-monooxygenase from bovine adrenal medulla. Methods Enzymol., 142, 56-62 (1987) [8] Fujisawa, H.; Okuno, S.: Tyrosine 3-monooxygenase from rat adrenals. Methods Enzymol., 142, 63-71 (1987) [9] Grima, A.; Lamouroux, F.; Blanot, F.; Biguet, N.F.; Mallet, J.: Complete coding sequence of rat tyrosine hydroxylase mRNA. Proc. Natl. Acad. Sci. USA, 82, 617-621 (1985) [10] Tank, A. W.; Weiner, N.: Tyrosine 3-monooxygenase from rat pheochromocytoma. Methods Enzymol., 142, 71-82 (1987) [11] Nagatsu, T.; Levitt, M.; Udenfriend, S.: Tyrosine hydroxylase.The initial step in norepinephrine biosynthesis. J. Biol. Chem., 239, 2910-2917 (1964) [12] Fitzpatrick, P.F.; Chlumsky, L.J.; Dauber, S.C.; O' Malley, K.L.: Expression of rat tyrosine hydroxylase in insect tissue culture cells and purification and characterization of the cloned enzyme. J. Biol. Chem., 265, 2042-2047 (1990) [13] Fitzpatrick, P.F.: The pH dependence of binding of inhibitors to bovine adrenal tyrosine hydroxylase [published erratum appears in J Biol Chem 1989 Mar 25;264(9):5313]. J. Biol. Chem., 263, 16058-16062 (1988) [14] Oka, K.; Kato, T.; Sugimoto, T.; Matsuura, S.; Nagatsu, T.: Kinetic properties of tyrosine hydroxylase with natural tetrahydrobiopterin as cofactor. Biochim. Biophys. Acta, 661, 45-53 (1981) [15] Oka, K.; Ashiba, G.; Sugimoto, T.; Matsuura, S.; Nagatsu, T.: Kinetic properties of tyrosine hydroxylase purified from bovine adrenal medulla and bovine caudate nucleus. Biochim. Biophys. Acta, 706, 188-196 (1982) [16] Richtand, N.M.; Inagami, T.; Misono, K.; Kuczenski, R.: Purification and characterization of rat striatal tyrosine hydroxylase. Comparison of the activation by cyclic AMP-dependent phosphorylation and by other effectors. J. Biol. Chem., 260, 8465-8473 (1985) [17] Hoeldtke, R.; Kaufman, S.: Bovine adrenal tyrosine hydroxylase: purification and properties. J. Biol. Chem., 252, 3160-3169 (1977) [18] Okuno, S.; Fujisawa, H.: Purification and some properties of tyrosine 3monooxygenase from rat adrenal. Eur. J. Biochem., 122, 49-55 (1982) [19] Villanova, E.; Manjon, A.; Iborra, J.L.: Tyrosine hydroxylase activity of immobilized tyrosinase on Enzyacryl-AA and CPG-AA supports: stabilization and properties. Biotechnol. Bioeng., 26, 1306-1312 (1984)

93

Tyrosine 3-monooxygenase

1.14.16.2

[20] Goodwill, K.E.; Sabatier, C.; Stevens, R.C.: Crystal structure of tyrosine hydroxylase with bound cofactor analog and iron at 2.3. ANG. resolution: selfhydroxylation of Phe300 and the pterin-binding site. Biochemistry, 37, 13437-13445 (1998) [21] Hillas, P.J.; Fitzpatrick, P.F.: A mechanism for hydroxylation by tyrosine hydroxylase based on partitioning of substituted phenylalanines. Biochemistry, 35, 6969-6975 (1996) [22] Xu, Y.; Stokes, A.H.; Roskoski, R., Jr.; Vrana, K.E.: Dopamine, in the presence of tyrosinase, covalently modifies and inactivates tyrosine hydroxylase. J. Neurosci. Res., 54, 691-697 (1998) [23] Martinez, A.; Abeygunawardana, C.; Haavik, J.; Flatmark, T.; Mildvan, A.S.: Interaction of substrate and pterin cofactor with the metal of human tyrosine hydroxylase as determined by 1 H-NMR. Adv. Exp. Med. Biol., 338, 7780 (1993) [24] Schmitt, P.; Reny-Palasse, V.; Bourde, O.; Garcia, C.; Pujol, J.F.: Further characterization of the long-term effect of RU24722 on tyrosine hydroxylase in the rat locus coeruleus. J. Neurochem., 61, 1423-1429 (1993) [25] Nasrin, S.; Ichinose, H.; Hidaka, H.; Nagatsu, T.: Recombinant human tyrosine hydroxylase types 1-4 show regulatory kinetic properties for the natural (6R)-tetrahydrobiopterin cofactor. J. Biochem., 116, 393-398 (1994) [26] Haavik, J.: l-DOPA is a substrate for tyrosine hydroxylase. J. Neurochem., 69, 1720-1728 (1997) [27] Hamdan, F.F.; Ribeiro, P.: Cloning and characterization of a novel form of tyrosine hydroxylase from the human parasite, Schistosoma mansoni. J. Neurochem., 71, 1369-1380 (1998) [28] Martinez, A.; Haavik, J.; Flatmark, T.; Arrondo, J.L.; Muga, A.: Conformational properties and stability of tyrosine hydroxylase studied by infrared spectroscopy. Effect of iron/catecholamine binding and phosphorylation. J. Biol. Chem., 271, 19737-19742 (1996) [29] Daubner, S.C.; Melendez, J.; Fitzpatrick, P.F.: Reversing the substrate specificities of phenylalanine and tyrosine hydroxylase: aspartate 425 of tyrosine hydroxylase is essential for l-DOPA formation. Biochemistry, 39, 9652-9661 (2000) [30] Almas, B.; Toska, K.; Teigen, K.; Groehn, V.; Pfleiderer, W.; Martinez, A.; Flatmark, T.; Haavik, J.: A kinetic and conformational study on the interaction of tetrahydropteridines with tyrosine hydroxylase. Biochemistry, 39, 13676-13686 (2000) [31] Daubner, S.C.; Fitzpatrick, P.F.: Mutation to phenylalanine of tyrosine 371 in tyrosine hydroxylase increases the affinity for phenylalanine. Biochemistry, 37, 16440-16444 (1998) [32] Yamamoto, K.; Kobayashi, N.; Yoshitama, K.; Teramoto, S.; Komamine, A.: Isolation and purification of tyrosine hydroxylase from callus cultures of Portulaca grandiflora. Plant Cell Physiol., 42, 969-975 (2001)

94

   !    

4!

   
 1.14.16.3    anthranilate,tetrahydrobiopterin:oxygen oxidoreductase (3-hydroxylating)    anthranilate 3-monooxygenase    anthranilate 3-hydroxylase anthranilate hydroxylase anthranilic acid hydroxylase anthranilic hydroxylase oxygenase, anthranilate 3-mono
    37256-79-4

     Tecoma stans [1]

!    "
 # 

anthranilate + tetrahydrobiopterin + O2 = 3-hydroxanthranilate + dihydrobiopterin + H2 O    oxidation redox reaction reduction      anthranilate + tetrahydrofolic acid + O2 ( specific for anthranilate, the optimum concentration is 0.5 mM, tetrahydrofolic acid is the most active electron donor when tested alone [1]) (Reversibility: ? [1]) [1]

95

Anthranilate 3-monooxygenase

1.14.16.3

$ 3-hydroxyanthranilate + dihydrofolic acid + H2 O2  Additional information ( phenylalanine, benzoic acid, acetanilide, tryptophan, kynurenine and cinnamic acid do not serve as substrates [1]) [1] $ ? 2  1,10-phenanthroline ( 74.2% inhibition at 0.5 mM [1]) [1] 2,2'-dipyridyl ( 97.4% inhibition, reversed by Fe3+ [1]) [1] 2,3-dimercaptopropanol [1] Ag+ ( 100% inhibition at 0.5 mM [1]) [1] Co2+ ( 62.4% inhibition at 0.5 mM]) [1] Cu2+ ( 77% inhibition at 0.5 mM [1]) [1] EDTA ( 64.6% inhibition [1]) [1] Hg2+ ( 97% inhibition at 0.5 mM [1]) [1] aminopterin ( 100% inhibition at 0.5 mM, 78.4% inhibition at 0.2 mM [1]) [1] anthranilic acid ( above 1 mM [1]) [1] cysteine ( 91.9% inhibition at 0.5 mM [1]) [1] glutathione ( 86.8% inhibition at 0.5 mM [1]) [1] iodoacetate ( 57% inhibition at 0.5 mM [1]) [1] p-chloromercuribenzoate ( slight at 0.5 mM [1]) [1]
"%  tetrahydrofolic acid ( absolute requirement [1]) [1] &

  NADH ( increases activity [1]) [1] NADPH ( increases activity [1]) [1] '(   Fe3+ ( activator, required for the reaction [1]) [1] " & *-% , 0.549 [1] 0  5 [1] 0 

 3.6-6.2 ( about 50% of activity maximum at pH 3.6 and pH 6.2 [1]) [1] )  *
, 30 ( assay at [1]) [1]

 2 %$ %' %

% leaf [1] 96

1.14.16.3

Anthranilate 3-monooxygenase

$"

(using fractional precipitation with acetone and adsorption on DEAEcellulose [1]) [1]

4     , -20 C, 1 month stable [1]

"  [1] Nair, P.M.; Vaidyanathan, C.S.: Anthranilic acid hydroxylase from Tecoma stans. Biochim. Biophys. Acta, 110, 521-531 (1965)

97

)      

4

   
 1.14.16.4    l-tryptophan,tetrahydrobiopterin:oxygen oxidoreductase (5-hydroxylating)    tryptophan 5-monooxygenase    l-tryptophan hydroxylase indoleacetic acid-5-hydroxylase oxygenase, tryptophan 5-monotryptophan 5-hydroxylase tryptophan hydroxylase
    9037-21-2

    







Rattus norvegicus [1-3, 6, 9, 10, 14, 17] Canis familiaris [1] Oryctolagus cuniculus [1, 6, 12, 15, 19, 20, 21] Bos taurus [1, 7, 13] Mus musculus (mastocytoma cell line P-815 [16]; transgenic mice containing exon 1 of the human huntigton gene [25]) [4, 5, 8, 16, 17, 25] Homo sapiens (enzyme from a carcinoid tumor [11]) [11, 22, 23, 24, 26] Cavia porcellus [13] Thunnus albacares (sp. scombroidei, yellofin tuna [18]) [18] Schistosoma mansoni [20]

!    "
 # 

l-tryptophan + tetrahydrobiopterin + O2 = 5-hydroxy-l-tryptophan + 4ahydroxytetrahydrobiopterin ( phenylalanine hydroxylase, EC 1.14.16.1 also catalyzes this reaction [7]; hydroxylation regiospecificity [21])

98

1.14.16.4

Tryptophan 5-monooxygenase

   oxidation redox reaction reduction       l-tryptophan + tetrahydropteridine + O2 ( enzyme catalyzes the rate-limiting step in biosynthesis of putative neurotransmitter 5-hydroxytryptamine, serotonin [1, 3, 13, 14]; believed to be the ratelimeting enzyme in the biosynthesis of serotonin in the central nervous system [5]; initial and uncommited step in the biosynthesis of melatonin [24]) (Reversibility: ? [1, 3, 5, 13, 14]) [1, 3, 5, 13, 14, 24] $ 5-hydroxy-l-tryptophan + dihydropteridine + H2 O [1, 3, 5, 13, 14, 24]      2-azaisotryptophan + 6-methyltetrahydrobiopterin + O2 ( recombinant N- and C-terminal truncated enzyme [21]) (Reversibility: ? [21]) [21] $ 5-hydroxy-2-azaisotryptophan + 6-hydroxy-2-azaisotryptophan + 6methyldihydrobiopterin + H2 O [21]  4-azatryptophan + 6-methyltetrahydrobiopterin + O2 ( recombinant N- and C-terminal truncated enzyme [21]) (Reversibility: ? [21]) [21] $ 5-hydroxy-4-azatryptophan + 6-methyldihydrobiopterin + H2 O [21]  4-methyltryptophan + 6-methyltetrahydrobiopterin + O2 ( recombinant N- and C-terminal truncated enzyme [21]) (Reversibility: ? [21]) [21] $ 5-hydroxy-4-methyltryptophan + 6-methyldihydrobiopterin + H2 O [21]  5-methyltryptophan + 6-methyltetrahydrobiopterin + O2 ( recombinant N- and C-terminal truncated enzyme [21]) (Reversibility: ? [21]) [21] $ 5-hydroxymethyltryptophan + 5-hydroxy-4-methyltryptophan + 6methyldihydrobiopterin + H2 O ( more than 99% 5-hydroxymethyltryptophan [21]) [21]  6-azatryptophan + 6-methyltetrahydrobiopterin + O2 ( recombinant N- and C-terminal truncated enzyme [21]) (Reversibility: ? [21]) [21] $ 5-hydroxy-6-azatryptophan + 6-methyldihydrobiopterin + H2 O [21]  6-methyltryptophan + 6-methyltetrahydrobiopterin + O2 ( recombinant N- and C-terminal truncated enzyme [21]) (Reversibility: ? [21]) [21] $ 5-hydroxy-6-methyltryptophan + 6-methyldihydrobiopterin + H2 O [21]  7-azatryptophan + 6-methyltetrahydrobiopterin + O2 ( recombinant N- and C-terminal truncated enzyme [21]) (Reversibility: ? [21]) [21] 99

Tryptophan 5-monooxygenase

1.14.16.4

$ 5-hydroxy-7-azatryptophan + 6-methyldihydrobiopterin + H2 O [21]  7-methyltryptophan + 6-methyltetrahydrobiopterin + O2 ( recombinant N- and C-terminal truncated enzyme [21]) (Reversibility: ? [21]) [21] $ 5-hydroxy-7-methyltryptophan + 6-methyldihydrobiopterin + H2 O [21]  l-phenylalanine + tetrahydropteridine + O2 ( tryptophan hydroxylase form I, 39% of activity with l-tryptophan [3, 9]; at a comparable rate of l-tryptophan hydroxylation [5, 7]; 28.8% of the rate observed with l-tryptophan [15]) (Reversibility: ? [3, 5, 7, 9, 15]) [3, 5, 7, 9, 15, 19] $ tyrosine + dihydropteridine + H2 O [3, 5, 7, 9, 15, 19]  l-tryptophan + tetrahydropteridine + O2 ( cofactor 2-amino-4-hydroxy-6-methyltetrahydropteridine [3, 12, 15]; cofactor 2amino-4-hydroxy-6,7-dimethyltetrahydropteridine [5, 12, 15]; cofactor tetrahydrobiopterin [12, 15]) (Reversibility: ? [1-15, 18, 20]) [115, 18, 19, 20] $ 5-hydroxy-l-tryptophan + dihydropteridine + H2 O [1-15, 18, 19, 20]  l-tyrosine + tetrahydropteridine + O2 ( 1% of activity with ltryptophan [5]) (Reversibility: ? [5]) [5] $ ? 2  (7R)-5,6,7,8-tetrahydrobiopterin ( recombinant pinal enzyme, 2.1 mM, 50% inhibition [22]) [22] 1,10-phenanthroline ( 0.0024 mM, 50% inhibition, competitive vs. tryptophan [11]) [5, 8, 11] 2,2'-dipyridyl [5, 8] 3,4-dihydroxyphenylalanine [8] 3,4-dihydroxyphenylethylamine [8] 3,4-dihydroxystyrene [2] 3,4-methylenedioxymethamphetamine ( inactivation of the enzyme [24]) [24] 4-fluorotryptophan [5, 8] 5-fluorotryptophan [5, 8] 5-hydroxytryptophan ( 0.018 mM, 50% inhibition [20]) [8, 20] 6-fluorotryptophan ( 1 mM, 90% inhibition of pineal tryptophan hydroxylase [7]) [5, 7, 8] 8-hydroxyquinoline [5, 8] CoCl2 ( 1 mM, complete inhibition [18]) [18] dl-4-chlorophenylalanine ( 0.009 mM, 505 inhibition [20]) [18, 20] d-DOPA ( noncompetitive vs. l-tryptophan and (6R)-l-erythro5,6,7,8-tetrahydrobiopterin [16]) [16] H2 O2 ( complete dependence on catalase in the absence of a mercaptan [12]) [12] l-DOPA ( noncompetitive vs. l-tryptophan and (6R)-l-erythro5,6,7,8-tetrahydrobiopterin [16]) [16]

100

1.14.16.4

Tryptophan 5-monooxygenase

l-erythro-7,8-dihydrobiopterin ( recombinant N-terminal truncated enzyme, competitive vs. (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin [23]) [23] l-p-chlorophenylalanine [5, 8] l-phenylalanine ( competitive vs. tryptophan [8]; substrate inhibition with tetrahydrobiopterin as cofactor, above 0.5 mM [15]) [8, 15] l-tryptophan ( competitive to l-phenylalanine [8]; substrate inhibition with 2-amino-4-hydroxy-6-methyl-5,6,7,8-tetrahydropteridine, not with 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine, above 0.2 mM [15]) [8, 15] l-tyrosine ( noncompetitive vs. l-tryptophan and (6R)-l-erythro5,6,7,8-tetrahydrobiopterin [16]) [16] ZnSO4 ( 1 mM, 80% inhibition [18]) [18] desferrioxamine ( 0.010 mM or higher, irreversible loss of activity [10]) [10] dopamine ( noncompetitive vs. l-tryptophan and (6R)-l-erythro5,6,7,8-tetrahydrobiopterin [16]; 0.022 mM, 50% inhibition [20]) [16, 20] iron chelators [1] serotonin ( 1 mM, 7.5% inhibition, 10 mM, 70% inhibition [17]) [17] &

  2-mercaptoethanol ( essential for activation [4]) [4] CaCl2 ( 1 mM, 7% activation [18]) [18] l-cysteine [5] dithiothreitol ( or other sulfhydryl reagents like 2-mercaptoethanol and l-cysteine are essential for activation, 5fold increase in activity [5, 8]) [4, 5, 8] '(   Fe2+ ( not required for full activity [11]; enzyme from pineal gland, stimulation [1]; some brain enzymes are stimulated others not [1]; required for maximal activity [3]; 5fold increase in activity [5, 8]; 0.02 mM, 3.5fold increase in activity [9]; stimulates in the absence of catalase [12]; N- and C-terminal recombinant enzyme, iron is required for activity [19]; recombinant pineal enzyme contains 0.2-0.3 mol of iron/mol of enzyme [22]; Fe2+ concentration for half-maximal activation of recombinant N-terminal truncated enzyme: 0.0013 mM [23]) [1, 3, 5, 9, 12, 19, 22, 23] ) &  * +, 4.4 (6-methyltryptophan) [21] 12.4 (l-tryptophan) [21] 13 (2-isoazatryptophan) [21] 15.1 (7-azatryptophan) [21] 16.4 (4-methyltryptophan) [21] 18.9 (7-methyltryptophan) [21] 21.5 (4-azatryptophan) [21] 32 (6-azatryptophan) [21]

101

Tryptophan 5-monooxygenase

1.14.16.4

" & *-% , 0.000018 ( enzyme from carconoid tumor [11]) [11] 0.00018 ( partially purified enzyme from hindbrain [12]) [12] 0.0021 ( hindbrain enzyme [15]) [15] 0.015 [6] 0.0271 ( brain tryptophan hydroxylase [6]) [6] 0.045 ( brain tryptophan hydroxylase [14]) [14] 0.082 ( tryptophan hydroxylase form II [3,9]) [3, 9] 0.17 ( recombinant His-tagged enzyme [20]) [20] 0.235 ( tryptophan hydroxylase from pineal gland [7]) [7] 0.367 ( brain tryptophan hydroxylase [10]) [10, 17] 0.374 ( brain stem, tryptophan hydroxylase form I [3]) [3, 9, 17] 0.434 ( recombinant enzyme, activity in cell extracts [17]) [17] 0.6 ( recombinant pineal enzyme [22]) [22] 5.28 ( purified from mastocytoma cells [5,8]) [5, 8, 17] 13.3 ( liver enzyme [18]) [18] Additional information ( enzyme activity decreases in 4-, 8-, and 12week-old mice expressing exon 1 of human huntingtin gene by 62, 61 and 86% respectively compared to control animals [25]) [25] . / *', 0.002 (l-tryptophan, enzyme from crude mitochondrial fraction [13]) [13] 0.0021 (l-tryptophan) [21] 0.0058 (l-tryptophan, recombinant N- and C-terminal truncated enzyme, cofactor 6-methyltetrahydrobipterin [19]) [19] 0.006 (2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine, soluble enzyme from brain [13]) [13] 0.0067 (tetrahydrobiopterin, recombinant His-tagged enzyme [20]) [20] 0.00873 (l-tryptophan) [16] 0.0142 (tetrahydrobiopterin, recombinant N- and C-terminal truncated enzyme, substrate phenylalanine [19]) [19] 0.0146 (6-methyltetrahydrobiopterin, recombinant N- and C-terminal truncated enzyme, substrate phenylalanine [19]) [19] 0.0151 (6-methyltryptophan) [21] 0.016 (l-tryptophan, enzyme from pineal gland, cofactor tetrahydrobiopterin [7]) [7] 0.017 (l-tryptophan, F313W mutant, recombinant N-terminal truncated enzyme [23]) [23] 0.02 (l-tryptophan, cofactor (6R)-5,6,7,8-tetrahydrobipterin [22]) [22] 0.022 (l-phenylalanine, F313W mutant, recombinant N-terminal truncated enzyme [23]) [23] 0.022 (l-tryptophan, recombinant His-tagged enzyme [20]) [20] 0.023 (l-tryptophan) [18]

102

1.14.16.4

Tryptophan 5-monooxygenase

0.023 (l-tryptophan, cofactor 6-methyl-5,6,7,8-tetrahydrobipterin [22]) [22] 0.031 (tetrahydrobiopterine) [12] 0.031-0.033 (l-tryptophan, activated enzyme in desalted extracts [4]) [4] 0.032 (l-phenylalanine, enzyme from pineal gland, cofactor tetrahydrobiopterin [7]) [7] 0.032 (l-tryptophan, cofactor tetrahydrobiopterin [15]) [15] 0.033 (l-tryptophan, recombinant N-terminal truncated enzyme [23]) [23] 0.0344 ((6R)-l-erythro-5,6,7,8-tetrahydrobiopterin, biphasic kinetic, low Km -value [16]) [16] 0.0375 (7-methyltryptophan) [21] 0.045 ((6R)-5,6,7,8-tetrahydrobipterin, substrate tryptophan [22]) [22] 0.045 (l-tryptophan, cofactor 2-amino-4-hydroxy-6-methyltetrahydropteridine [5,8]) [5, 8] 0.0479 (l-tryptophan, recombinant N- and C-terminal truncated enzyme, cofactor tetrahydrobiopterin [19]) [19] 0.048 (l-phenylalanine, recombinant N-terminal truncated enzyme [23]) [23] 0.05 (2-amino-4-hydroxy-6-methyl-5,6,7,8-tetrahydropteridine) [11] 0.05 (tryptophan, cofactor tetrahydrobiopterine [12]) [12] 0.055 (2-amino-4-hydroxy-6-methyltetrahydropteridine, substrate l-tryptophan [5,8]) [5, 8] 0.0606 (l-phenylalanine, recombinant N- and C-terminal truncated enzyme, cofactor tetrahydrobiopterin [19]) [19] 0.0662 (7-azatryptophan) [21] 0.075 (6-methyl-5,6,7,8-tetrahydrobipterin, substrate tryptophan [22]) [22] 0.0752 (2-amino-4-hydroxy-6-methyltetrahydropteridine) [17] 0.078 (tryptophan, cofactor 2-amino-4-hydroxy-6-methyltetrahydropteridine [12]) [12] 0.0865 (2-isoazatryptophan) [21] 0.0865 (l-tryptophan) [10] 0.09 (phenylalanine, cofactor (6R)-5,6,7,8-tetrahydrobiopterin [22]) [22] 0.1 (l-phenylalanine, cofactor 2-amino-4-hydroxy-6-methyltetrahydropteridine [5,8]) [5, 8] 0.102 (l-phenylalanine, recombinant N- and C-terminal truncated enzyme, cofactor 6-methyltetrahydrobiopterin [19]) [19] 0.105 (2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine) [11] 0.119 (l-tryptophan, tryptophan hydroxylase form I [3]) [3, 9] 0.125 (2-amino-4-hydroxy-6-methyltetrahydropteridine, tryptophan hydroxylase form I [3]) [3, 9] 0.13 (2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine) [12]

103

Tryptophan 5-monooxygenase

1.14.16.4

0.135 (tetrahydrobiopterin, recombinant N- and C-terminal truncated enzyme, substrate tryptophan [19]) [19] 0.177 (6-methyltetrahydrobiopterin, recombinant N- and C-terminal truncated enzyme, substrate tryptophan [19]) [19] 0.262 (4-azatryptophan) [21] 0.286 (phenylalanine, cofactor tetrahydrobiopterin [15]) [15] 0.29 (tryptophan, cofactor 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8tetrahydropteridine [12]) [12] 0.294 (tetrahydrobiopterine, activated enzyme in desalted extracts [4]) [4] 0.295 (6-azatryptophan) [21] 0.3 (l-tryptophan, soluble enzyme from brain [13]) [13] 0.355 (l-tryptophan) [17] 0.796 ((6R)-l-erythro-5,6,7,8-tetrahydrobiopterin, biphasic kinetic, high Km value [16]) [16] 0.994 (4-methyltryptophan) [21] 2.1 ((7R)-5,6,7,8-tetrahydrobipterin, substrate tryptophan [22]) [22] Additional information ( 1.2% O2 with 0.2 mM 2-amino-4-hydroxy-6-methyl-5,6,7,8-tetrahydropteridine, 7.1% O2 with 0.4 mM 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine [11]; 20% O2, cofactor 2amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine, 2.5% O2, cofactor tetrahydrobiopterine [12]) [11, 12] . / *', 0.00082 (1,10-phenanthroline, vs. tryptophan [11]) [11] 0.0026 (1,10-phenanthroline, vs. 2-amino-4-hydroxy-6-methyl5,6,7,8-tetrahydropteridine [11]) [11] 0.007 (dopamine, vs. (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin, component with high affinity [16]) [16] 0.0162 (l-tyrosine, vs. (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin, component with high affinity [16]) [16] 0.017 (l-DOPA, vs. l-tryptophan [16]) [16] 0.0185 (l-tyrosine, vs. l-tryptophan [16]) [16] 0.021 (dl-4-chlorophenylalanine) [18] 0.026 (l-4-chlorophenylalanine) [8] 0.035 (l-5-hydroxytryptophan) [22] 0.0393 (dopamine, vs. (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin, component with low affinity [16]) [16] 0.0761 (d-DOPA, vs. l-tryptophan [16]) [16] 0.0803 (l-DOPA, vs. (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin, component with high affinity [16]) [16] 0.0887 (l-DOPA, vs. (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin, component with low affinity [16]) [16] 0.0943 (dopamine, vs. l-tryptophan [16]) [16] 0.15 (phenylalanine) [22] 0.161 (l-erythro-7,8-dihydrobiopterin, recombinant truncated enzyme [23]) [23]

104

1.14.16.4

Tryptophan 5-monooxygenase

0.197 (l-phenylalanine) [15] 0.202 (d-DOPA, vs. (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin, component with low affinity [16]) [16] 0.306 (l-tyrosine, vs. (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin, component with low affinity [16]) [16] 0.609 (d-DOPA, vs. (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin, component with high affinity [16]) [16] 0  6.4 ( in the presence of 0.2 mM Fe2+ and 2-mercaptoethanol [11]) [11] 6.8 ( in the presence of 0.2 mM Fe2+ , without 2-mercaptoethanol [11]) [11] 7.2 ( in the presence of 35.5 mM 2-mercaptoethanol [11]) [8, 11] 7.6 ( without 2-mercaptoethanol and Fe2+ [11]; tryptophan hydroxylase form I [3]) [3, 9, 11] 8 [18] 8-8.5 ( soluble enzyme from brain [13]) [13] 0 

 6-8.7 ( about 50% activity at pH 6.0 and 8.7 [8]) [8] 6-9.5 ( about 50% activity at pH 6 and 9.5 [9]) [9] )  *
, 30 ( assay at [3,9]) [3, 9] 40 [18]

  #   ' 1  30000 ( enzyme from pineal gland, gel filtration, sucrose density gradient ultracentrifugation [7]) [7] 200000 ( recombinant pineal enzyme, gel filtration [22]) [22] 220000-240000 ( enzyme from hindbrain [1]) [1] 230000 ( gel filtration, PAGE, sucrose density gradient centrifugation [15]) [15] 240000 ( recombinant His-tagged enzyme, gel filtration [20]) [20] 260000 ( enzyme from brain, gradient PAGE [10]) [10] 270000 ( enzyme from mastocytoma cells, gel filtration [5,8]) [5, 8] 280000 ( enzyme from mastocytoma cells, gradient PAGE [5,8]; liver enzyme, gel filtration [18]) [5, 8, 18] 288000 ( tryptophan hydroxylase form I, gradient PAGE [3]) [1, 3, 9] 300000 ( tryptophan hydroxylase form I, gel filtration [3]) [1, 3, 9]   ? ( x * 55000, brain enzyme, SDS-PAGE [10]) [10] tetramer ( 2 * 57700 + 2 * 60900, hindbrain enzyme, SDS-PAGE [15]; 2 * 57500 + 2 * 60000, midbrain enzyme, SDS-PAGE [1]; 105

Tryptophan 5-monooxygenase

1.14.16.4

4 * 59000, tryptophan hydroxylase form I from brain stem, SDS-PAGE [3,9]; 4 * 53000, enzyme from mastocytoma cells, SDS-PAGE [5,8]; 4 * 96000, SDS-PAGE [18]; 4 * 60000, His-tagged recombinant enzyme, immunoblot [20]; 4 * 50000, recombinant pineal enzyme, SDS-PAGE [22]) [1, 3, 5, 8, 9, 15, 18, 20, 22] $   "

Additional information ( less than 1% bound carbohydrate [15]) [15]

 2 %$ %' %

% brain ( activity in hypothalamus and midbrain-medulla regions, no activity in cerebellum or cortex [1]; stem [3,9]; hindbrain [12,15]; region of the raphe nucleus of rat midbrain [14]; serotonergic neurons [13]) [1, 3, 6, 9, 12-15] carcinoma cell [11] intestinal mucosa [1] liver [1, 18] mastocytoma cell [4, 5, 8] pineal gland [1, 7] 3#

Golgi apparatus ( associated with cell body [14]) [14] cytoplasm ( cell body [14]) [14] endoplasmic reticulum ( associated with cell body [14]) [14] microtubule ( dendrites and axons [14]) [14] particle-bound [13] soluble [1, 13] $"

(tryptophan hydroxylase form I and II, pH 4.8, ammonium sulfate, gel filtration, DEAE-Sepharose, 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine-agarose [1]; brain enzyme, calcium phosphate gel, dimethyltetrahydropteridine-agarose [6]) [1, 3, 6, 9, 10, 14] (partial [12]; brain enzyme, calcium phosphate gel, dimethyltetrahydropteridine-agarose [6]; hindbrain enzyme, calcium phosphate gel, polyethylene glycol 6000, Sepharose 6B, DEAE-cellulose [15]; recombinant Nand C-terminal truncated mutant enzyme [19,21]) [1, 6, 12, 15, 19, 21] (enzyme from pineal gland, ammonium sulfate, hydroxylapatite, P-cellulose, DEAE-cellulose, Sephadex G-200 [7]) [7, 13] (2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine-agarose [5]) [5, 8] (recombinant pineal enzyme, ammonium sulfate, pterin-agarose, Sepharose [22]; recombinant full-length and N-terminal truncated enzyme [23]) [11, 22, 23] [13]

106

1.14.16.4

Tryptophan 5-monooxygenase

(acid treatment, ammonium sulfate, Sepharose CL-6B, DEAE-sepharose, butyl-Sepharose, Toyopearl [18]) [18] (recombinant His-tagged enzyme, Nickel affinity chromatography [20]) [20]
 #

(modeled structure based on the known crystal structures of phenylalanine hydroxylase and tyrosine hydroxylase [24]) [24]
 

(expression of wild-type and N- and C-terminal truncated mutant enzyme in Escherichia coli [19]) [19] (expression in Escherichia coli [17]) [17] (expression in Escherichia coli [22]; expression of full-length and Nterminal truncated enzyme in Escherichia coli [23]) [22, 23] (His-tagged enzyme, expression in Escherichia coli [20]) [20] 

 

F313W ( N-terminal truncated enzyme [23]; F313W mutant shows no preference for tryptophan over phenylalanine as a substrate [26]) [23, 26]

4  0  5-10 ( enzyme retains more than 80% activity after 60 min at 4 C between pH 7.0 and pH 7.6, unstable above pH 10.0 and below pH 5.0 [18]) [18] )  4 ( complete loss of activity in absence of stabilizing agents after 24 h [6]; complete loss of activity in absence of stabilizing agents after 20 h [5,8]) [5, 6, 8] 35 ( stable below [18]) [18] 37 ( 80 min, no loss of activity, estimated half-life: 21 h [20]; half-life: 54 min [20]) [20] 5    "

, EDTA: 0.05 mM stabilizes [3] , l-tryptophan stabilizes [3] , Tween 20: 0.06% stabilizes [3] , catalase is necessary to protect the enzyme during purification [10] , glycerol, 10% stabilizes [3] , EDTA stabilizes [6] , Fe2+ protects against inactivation by H2 O2 in absence of catalase [12] , Tween 20 stabilizes [6] , glycerol stabilizes [6] , EDTA stabilizes [8]

107

Tryptophan 5-monooxygenase

1.14.16.4

, EDTA: 0.05 mM stabilizes [5] , NaCl: 1 M, in addition to other stabilizing agents brings a great improvement in stabilization [5, 8] , Tween 20 stabilizes [8] , Tween 20: 0.06% stabilizes [5] , catalase at a concentration of more than 1 mg/ml is required for stability during reaction at aerobic conditiones [4] , ethylene glycol, 50%, stabilizes [5, 8]    , -80 C, 1 month, 20% loss of activity [3] , 0 C, 20 h, 60% loss of activity [10] , -80 C, N2 atmosphere, dithiothreitol, 2 weeks, 20% loss of activity [12] , N- and C-terminal truncated recombinant enzyme, -70 C, 100 mM ammonium sulfate, ferrous iron, 10% glycerol, 2 mM dithiothreitol, pH 7.0, indefinitely, no loss of activity [19] , -20 C, 2 months, 40% loss of activity [13] , -20 C, 10% glycerol, 0.05 mM EDTA, 0.06% Tween 20, 28 d, 69% loss of activity [17] , -80 C, 1 month, 30% loss of activity [5] , 4 C, 0.06% Tween 20, 0.05 mM EDTA, 50% ethylene glycol, 1 M NaCl, 5 days, no loss of activity [5] , 4 C, 10% glycerol, 0.05 mM EDTA, 0.06% Tween 20, 28 d, 34% loss of activity [17] , 4 C, under N2 , 2 months, no loss of activity [11] , 4 C, under O2, 48 h, 90% loss of activity [11] , -20 C, 2 months, 40% loss of activity [13] , recombinant His-tagged enzyme, -80 C, 50 mM HEPES, pH 7.5, 200 mM NaCl, 10% glycerol, 0.05% Tween 20, 1 mM dithiothreitol, 4 days, no loss of activity [20] , recombinant His-tagged enzyme, 4 C, 50 mM HEPES, pH 7.5, 200 mM NaCl, 10% glycerol, 0.05% Tween 20, 1 mM dithiothreitol, 4 days, no loss of activity [20]

"  [1] Kaufman, S.: Aromatic amino acid hydroxylases. The Enzymes, 3rd Ed. (Boyer, P.D., Krebs, E.G., eds.), 18, 217-282 (1987) [2] Koizumi, S.; Matsushima, Y.; Nagatsu, T.; Linuma, H.; Takeuchi, T.; Umezawa, H.: 3,4-Dihydroxystyrene, a novel microbial inhibitor for phenylalanine hydroxylase and other pteridine-dependent monooxygenases. Biochim. Biophys. Acta, 789, 111-118 (1984) [3] Fujisawa, H.; Nakata, H.: Tryptophan 5-monooxygenase from rat brain stem. Methods Enzymol., 142, 83-87 (1987)

108

1.14.16.4

Tryptophan 5-monooxygenase

[4] Hasegawa, H.; Ichiyama, A.: Tryptophan 5-monooxygenase from mouse mastocytoma: high-performance liquid chromatography assay. Methods Enzymol., 142, 88-92 (1987) [5] Fujisawa, H.; Nakata, H.: Tryptophan 5-monooxygenase from mouse mastocytoma clone P815. Methods Enzymol., 142, 93-96 (1987) [6] Nakata, H.; Fujisawa, H.: Simple and rapid purification of tryptophan 5monooxygenase from rabbit brain by affinity chromatography. J. Biochem., 90, 567-569 (1981) [7] Nukiwa, T.; Tohyama, C.; Okita, C.; Kataoka, T.; Ichiyama, A.: Purification and some properties of bovine pineal tryptophan 5-monooxygenase. Biochem. Biophys. Res. Commun., 60, 1029-1035 (1974) [8] Nakata, H.; Fujisawa, H.: Tryptophan 5-monooxygenase from mouse mastocytoma P815. A simple purification and general properties. Eur. J. Biochem., 124, 595-601 (1982) [9] Nakata, H.; Fujisawa, H.: Purification and properties of tryptophan 5monooxygenase from rat brain-stem. Eur. J. Biochem., 122, 41-47 (1982) [10] Cash, C.D.; Vayer, P.; Mandel, P.; Maitre, M.: Tryptophan 5-hydroxylase. Rapid purification from whole rat brain and production of a specific antiserum. Eur. J. Biochem., 149, 239-245 (1985) [11] Hosoda, S.; Nakamura, W.; Takatsuki, K.: Properties of tryptophan hydroxylase from human carcinoid tumor. Biochim. Biophys. Acta, 482, 27-34 (1977) [12] Friedman, P.A.; Kappelman, A.H.; Kaufman, S.: Partial purification and characterization of tryptophan hydroxylase from rabbit hindbrain. J. Biol. Chem., 247, 4165-4173 (1972) [13] Ichiyama, A.; Nakamura, S.; Nishizuka, Y.; Hayaishi, O.: Enzymic studies on the biosynthesis of serotonin in mammalian brain. J. Biol. Chem., 245, 1699-1709 (1970) [14] Joh, T.H.; Shikimi, T.; Pickel, V.M.; Reis, D.J.: Brain tryptophan hydroxylase: purification of, production of antibodies to, and cellular and ultrastructural localization in serotonergic neurons of rat midbrain. Proc. Natl. Acad. Sci. USA, 72, 3575-3579 (1975) [15] Tong, J.H.; Kaufman, S.: Tryptophan hydroxylase. Purification and some properties of the enzyme from rabbit hindbrain. J. Biol. Chem., 250, 41524158 (1975) [16] Naoi, M.; Maruyama, W.; Takahashi, T.; Ota, M.; Parvez, H.: Inhibition of tryptophan hydroxylase by dopamine and the precursor amino acids. Biochem. Pharmacol., 48, 207-211 (1994) [17] Park, D.H.; Stone, D.M.; Kim, K.S.; Joh, T.H.: Characterization of recombinant mouse tryptophan hydroxylase expressed in Escherichia coli. Mol. Cell. Neurosci., 5, 87-93 (1994) [18] Nagai, T.; Hamada, M.; Kai, N.; Tanoue, Y.; Nagayama, F.: Characterization of yellowfin tuna (Thunnus albacares, Scombroidei) tryptophan hydroxylase. Comp. Biochem. Physiol. B, 116, 161-165 (1997) [19] Moran, G.R.; Daubner, S.C.; Fitzpatrick, P.F.: Expression and characterization of the catalytic core of tryptophan hydroxylase. J. Biol. Chem., 273, 12259-12266 (1998) 109

Tryptophan 5-monooxygenase

1.14.16.4

[20] Hamdan, F.F.; Ribeiro, P.: Characterization of a stable form of tryptophan hydroxylase from the human parasite Schistosoma mansoni. J. Biol. Chem., 274, 21746-21754 (1999) [21] Moran, G.R.; Phillips, R.S.; Fitzpatrick, P.F.: Influence of steric bulk and electrostatics on the hydroxylation regiospecificity of tryptophan hydroxylase: characterization of methyltryptophans and azatryptophans as substrates. Biochemistry, 38, 16283-16289 (1999) [22] Kowlessur, D.; Kaufman, S.: Cloning and expression of recombinant human pineal tryptophan hydroxylase in Escherichia coli: purification and characterization of the cloned enzyme. Biochim. Biophys. Acta, 1434, 317-330 (1999) [23] McKinney, J.; Teigen, K.; Froystein, N.A.; Salauen, C.; Knappskog, P.M.; Haavik, J.; Martinez, A.: Conformation of the substrate and pterin cofactor bound to human tryptophan hydroxylase. Important role of Phe313 in substrate specificity. Biochemistry, 40, 15591-15601 (2001) [24] Martinez, A.; Knappskog, P.M.; Haavik, J.: A structural approach into human tryptophan hydroxylase and its implications for the regulation of serotonin biosynthesis. Curr. Med. Chem., 8, 1077-1091 (2001) [25] Yohrling, I.G.; Jiang, G.C.; DeJohn, M.M.; Robertson, D.J.; Vrana, K.E.; Cha, J.H.: Inhibition of tryptophan hydroxylase activity and decreased 5-HT1A receptor binding in a mouse model of Huntington's disease. J. Neurochem., 82, 1416-1423 (2002) [26] Daubner, S.C.; Moran, G.R.; Fitzpatrick, P.F.: Role of tryptophan hydroxylase Phe313 in determining substrate specificity. Biochem. Biophys. Res. Commun., 292, 639-641 (2002)

110

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4

   
 1.14.16.5    1-alkyl-sn-glycerol,tetrahydrobiopterin:oxygen oxidoreductase    glyceryl-ether monooxygenase    EC 1.14.99.17 (formerly) O-alkylglycerol monooxygenase alkylglycerol monooxygenase glyceryl ether hydroxylase glyceryl ether monooxygenase glyceryl ether oxidase glyceryl ether oxygenase glyceryl ether-cleaving enzyme glyceryl etherase glyceryl-ether cleaving enzyme oxygenase, glyceryl ether mono
    37256-82-9

    





Rattus norvegicus [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15] Mus musculus [3, 10] Oryctolagus cuniculus [3, 10] Lima maximus (slug) [3] Canis familiaris [3, 10] Meriones sp. (gerbil [3, 10]) [3, 10] Cavia porcellus [3, 10] Mesocricetus auratus [3, 10]

111

Glyceryl-ether monooxygenase

1.14.16.5

!    "
 # 

1-alkyl-sn-glycerol + tetrahydrobiopterin + O2 = 1-hydroxyalkyl-sn-glycerol + dihydrobiopterin + H2 O (, mechanism [10])    oxidation redox reaction reduction       1-alkyl-sn-glycerol + tetrahydrobiopterin + O2 (, enzyme plays an essential role in conjugation with the cleavage enzyme in the regulation of cellular levels of -alkyl moieties in glycerolipids [7]) (Reversibility: ? [17]) [7] $ 1-hydroxyalkyl-sn-glycerol + dihydrobiopterin + H2 O      (2RS,1'R)-[1-3H1]-hexadecyloxypropane-1,2-diol + RS-6-methyl-5,6,7,8tetrahydropterin + O2 (, only 6.5% release of the tritium after 20 min, reaction is stereospecific for the pro-HS hydrogen [14]) (Reversibility: ? [14]) [14] $ ?  (2RS,1'S)-[1-3H1]-hexadecyloxypropane-1,2-diol + RS-6-methyl-5,6,7,8tetrahydropterin + O2 (, release of 37% of the tritium after 20 min, reaction is stereospecific for the pro-HS hydrogen [14]) (Reversibility: ? [14]) [14] $ ?  1-O-hexadecyl-sn-glycero-3-phosphocholine + 6-methyl-5,6,7,8-tetrahydropterin + O2 (Reversibility: ? [10]) [10] $ ?  2-(1-hexadecyloxy)propan-1,3-diol + RS-6-methyl-5,6,7,8-tetrahydropterin + O2 (Reversibility: ? [13]) [13] $ ?  2-(1-octadecyloxy)ethanol + RS-(cis)-6,7-dimethyl-5,6,7,8-tetrahydropterin + O2 (Reversibility: ? [13]) [13] $ ?  2-(1-octadecyloxy)ethanol + RS-6-methyl-5,6,7,8-tetrahydropterin + O2 (Reversibility: ? [13]) [13] $ ?  2-1'-n-hexadecyloxyethanol + 6-methyl-5,6,7,8-tetrahydropterin + O2 (Reversibility: ? [12]) [12] $ ?  2-1'-n-octadecyloxyethanol + 6-methyl-5,6,7,8-tetrahydropterin + O2 (Reversibility: ? [12]) [12] $ ?

112

1.14.16.5

Glyceryl-ether monooxygenase

 2-hexadecyloxy-ethan-1-ol + 5,6,7,8-tetrahydropterin + O2 (Reversibility: ? [10]) [10] $ ?  2-hexadecyloxy-ethan-1-ol + 6-methyl-5,6,7,8-tetrahydropterin + O2 (Reversibility: ? [10]) [10] $ ?  2-hexadecyloxyethan-1-ol + 5,6,7,8-tetrahydrofolic acid + O2 (Reversibility: ? [10]) [10] $ ?  2-hexadecyloxyethan-1-ol + 6-methyl-5,6,7,8-tetrahydropterin + O2 (Reversibility: ? [10]) [10] $ ?  2-hexadecyloxyethan-1-ol + 6R-tetrahydrobiopterin + O2 (Reversibility: ? [10]) [10] $ ?  2-hexadecyloxypropane-1,3-diol + 6-methyl-5,6,7,8-tetrahydropterin (Reversibility: ? [10]) [10] $ ?  2-hexadecylthioethan-ol + 6-methyl-5,6,7,8-tetrahydropterin + O2 (Reversibility: ? [10]) [10] $ ?  2-octadecyloxyethan-1-ol + 6-methyl-5,6,7,8-tetrahydropterin + O2 (Reversibility: ? [10]) [10] $ ?  2-octadecyloxyethan-1-ol + 6-methyl-5,6,7,8-tetrahydropterin + O2 (Reversibility: ? [10]) [10] $ ?  2-octadecylthioethan-ol + 6-methyl-5,6,7,8-tetrahydropterin + O2 (Reversibility: ? [10]) [10] $ ?  2R,3-1'-hexadecyloxypropan-1,2-diol + 2-amino-5,6,7,8-tetrahydropteridin-4-one + O2 (Reversibility: ? [7]) [7] $ ?  2R,3-1'-octadecyloxypropan-1,2-diol + 2-amino-5,6,7,8-tetrahydropteridin-4-one + O2 (Reversibility: ? [7]) [7] $ ?  2S,3-1'-hexadecyloxypropan-1,2-diol + 2-amino-5,6,7,8-tetrahydropteridin-4-one + O2 (, the oxygen transfer to 1'C of the aliphatic side-chain is highly stereospecific [7]) (Reversibility: ? [7]) [7] $ ?  2S,3-1'-octadecyloxypropan-1,2-diol + 2-amino-5,6,7,8-tetrahydropteridin-4-one + O2 (Reversibility: ? [7]) [7] $ ?  3-(1-octadecylthio)propan-1,2-diol +RS-6-methyl-5,6,7,8-tetrahydropterin + O2 (Reversibility: ? [13]) [13] $ ?

113

Glyceryl-ether monooxygenase

1.14.16.5

 3-1'-hexadecyloxy-2-methoxy-propan-1-ol + 6-methyl-5,6,7,8-tetrahydropterin + O2 (Reversibility: ? [12]) [12] $ ?  3-1'-hexadecyloxy-2-methoxy-propan-2-ol + 6-methyl-5,6,7,8-tetrahydropterin + O2 (Reversibility: ? [12]) [12] $ ?  3-1'-n-hexadecylthiopropane-1,2-diol + 6-methyl-5,6,7,8-tetrahydropterin + O2 (Reversibility: ? [12]) [12] $ ?  3-1'-n-octadecylthiopropane-1,2-diol + 6-methyl-5,6,7,8-tetrahydropterin + O2 (Reversibility: ? [12]) [12] $ ?  3-O-hexadecyl glycerol + 6-methyl-5,6,7,8-tetrahydropterin + O2 (Reversibility: ? [10]) [10] $ ?  3-hexadecyloxy-1-methoxypropan-1-ol + 6-methyl-5,6,7,8-tetrahydropterin (Reversibility: ? [10]) [10] $ ?  3-hexadecyloxy-2-methoxypropan-1-ol + 6-methyl-5,6,7,8-tetrahydropterin + O2 (Reversibility: ? [10]) [10] $ ?  R-3-(1-octadecyloxy)propan-1,2-diol + RS-6-methyl-5,6,7,8-tetrahydropterin + O2 (Reversibility: ? [13]) [13] $ ?  R-3-dodecyloxypropan-1,2-diol + 6-methyl-5,6,7,8-tetrahydropterin + O2 (Reversibility: ? [10]) [10] $ ?  R-3-eicosanyloxypropan-1,2-diol + 6-methyl-5,6,7,8-tetrahydropterin + O2 (Reversibility: ? [10]) [10] $ ?  R-3-heneicosanyloxypropan-1,2-diol + 6-methyl-5,6,7,8-tetrahydropterin + O2 (Reversibility: ? [10]) [10] $ ?  R-3-heptadecyloxypropan-1,2-diol + 6-methyl-5,6,7,8-tetrahydropterin + O2 (Reversibility: ? [10]) [10] $ ?  R-3-hexadecyloxypropan-1,2-diol + 6-methyl-5,6,7,8-tetrahydropterin + O2 (Reversibility: ? [10]) [10] $ ?  R-3-nonadecyloxypropan-1,2-diol + 6-methyl-5,6,7,8-tetrahydropterin + O2 (Reversibility: ? [10]) [10] $ ?  R-3-octadecyloxypropan-1,2-diol + 6-methyl-5,6,7,8-tetrahydropterin + O2 (Reversibility: ? [10]) [10] $ ?  R-3-pentadecyloxypropan-1,2-diol + 6-methyl-5,6,7,8-tetrahydropterin + O2 (Reversibility: ? [10]) [10] 114

1.14.16.5

Glyceryl-ether monooxygenase

$ ?  R-3-tridecyloxypropan-1,2-diol + 6-methyl-5,6,7,8-tetrahydropterin + O2 (Reversibility: ? [10]) [10] $ ?  R-3-undecyloxypropan-1,2-diol + 6-methyl-5,6,7,8-tetrahydropterin + O2 (Reversibility: ? [10]) [10] $ ?  RS-3-(1-heptyloxy)propan-1,2-diol + RS-6-methyl-5,6,7,8-tetrahydropterin (Reversibility: ? [13]) [13] $ ?  RS-3-(1-hexadecyloxy)-1-methoxypropan-2-ol + RS-6-methyl-5,6,7,8-tetrahydropterin + O2 (Reversibility: ? [13]) [13] $ ?  RS-3-(1-hexadecyloxy)-2-hydroxypropane-1-phosphocholine + RS-6methyl-5,6,7,8-tetrahydropterin + O2 (Reversibility: ? [13, 15]) [13, 15] $ ?  RS-3-(1-hexadecyloxy)propan-1,2-diol + RS-6-methyl-5,6,7,8-tetrahydropterin + O2 (Reversibility: ? [13]) [13] $ ?  RS-3-(1-hexadecyloxy)propan-1,2-diol + RS-6-methyl-5,6,7,8-tetrahydropterin + O2 (Reversibility: ? [13]) [13] $ ?  RS-3-(1-hexadecyloxy)propan-1-ol + RS-6-methyl-5,6,7,8-tetrahydropterin + O2 (Reversibility: ? [13]) [13] $ ?  RS-3-(1-oleyloxy)propan-1,2-diol + RS-6-methyl-5,6,7,8-tetrahydropterin + O2 (Reversibility: ? [13]) [13] $ ?  RS-3-docosanyloxypropan-1,2-diol + 6-methyl-5,6,7,8-tetrahydropterin + O2 (Reversibility: ? [10]) [10] $ ?  RS-3-dodecyloxypropan-1,2-diol + 6-methyl-5,6,7,8-tetrahydropterin + O2 (Reversibility: ? [10]) [10] $ ?  RS-3-heptyloxypropan-1,2-diol + 6-methyl-5,6,7,8-tetrahydropterin + O2 (Reversibility: ? [10]) [10] $ ?  RS-3-hexyloxypropan-1,2-diol + 6-methyl-5,6,7,8-tetrahydropterin + O2 (Reversibility: ? [10]) [10] $ ?  RS-3-nonyloxypropan-1,2-diol + 6-methyl-5,6,7,8-tetrahydropterin + O2 (Reversibility: ? [10]) [10] $ ?  RS-3-octyloxypropan-1,2-diol + 6-methyl-5,6,7,8-tetrahydropterin + O2 (Reversibility: ? [10]) [10] $ ? 115

Glyceryl-ether monooxygenase

1.14.16.5

 RS-3-pentyloxypropan-1,2-diol + 6-methyl-5,6,7,8-tetrahydropterin + O2 (Reversibility: ? [10]) [10] $ ?  RS-3-propyloxypropan-1,2-diol + 6-methyl-5,6,7,8-tetrahydropterin + O2 (Reversibility: ? [10]) [10] $ ?  RS-3-tetradecyloxypropan-1,2-diol + 6-methyl-5,6,7,8-tetrahydropterin + O2 (Reversibility: ? [10]) [10] $ ?  RS-batyl alcohol + 6-methyl-5,6,7,8-tetrahydropterin + O2 (, i.e. RS-3-octadecyloxypropane-1,2-diol, R(+)- and S(-)-6-methyl-5,6,7,8-tetrahydropterin are about equally effective [8]) (Reversibility: ? [8, 9, 10, 11]) [8, 9, 10, 11, 13] $ ?  RS-cyclohexylmethoxypropan-1,2-diol + 6-methyl-5,6,7,8-tetrahydropterin + O2 (Reversibility: ? [10]) [10] $ ?  RS-hexadecylthiopropane-1,2-diol + 6-methyl-5,6,7,8-tetrahydropterin + O2 (Reversibility: ? [10]) [10] $ ?  RS-octadecylthiopropane-1,2-diol + 6-methyl-5,6,7,8-tetrahydropterin + O2 (Reversibility: ? [10]) [10] $ ?  S-3-(1-hexadecyloxy)-2-hydroxypropane-1-phosphocholine + RS-6methyl-5,6,7,8-tetrahydropterin + O2 (Reversibility: ? [13]) [13] $ ?  S-3-(1-octadecyloxy)propan-1,2-diol + RS-6-methyl-5,6,7,8-tetrahydropterin + O2 (Reversibility: ? [13]) [13] $ ?  S-3-octadecyloxypropan-1,2-diol + 6-methyl-5,6,7,8-tetrahydropterin + O2 (Reversibility: ? [10]) [10] $ ?  batyl alcohol + 6R-5,6,7,8-tetrahydrobiopterin + O2 (, i.e. RS-3(1-octadecyloxy)propan-1,2-doiol [13]) (Reversibility: ? [13]) [13] $ ?  batyl alcohol + 6R-tetrahydrobiopterin + O2 (Reversibility: ? [10]) [10] $ ?  batyl alcohol + RS-(cis)-6,7-dimethyl-5,6,7,8-tetrahydropterin + O2 (, i.e. RS-3-(1-octadecyloxy)propan-1,2-diol [13]) (Reversibility: ? [13]) [13] $ ?  cis-RS-3-octadec-9'-enyloxypropane-1,2-diol + 6-methyl-5,6,7,8-tetrahydropterin + O2 (Reversibility: ? [10]) [10] $ ?  hexadecyl glycerol + 6-methyl-5,6,7,8-tetrahydropterin + O2 (Reversibility: ? [10]) [10] 116

1.14.16.5

Glyceryl-ether monooxygenase

$ ?  hexadecylglycerol + tetrahydrobiopterin + O2 (, 1-O-hexadecylglycerol [2]) (Reversibility: ? [1, 2, 3, 5, 10]) [1, 2, 3, 5, 10] $ hexadecanal + glycerol + dihydrobiopterin + H2 O [1, 2]  octadecylglycerol + tetrahydrobiopterin + O2 (Reversibility: ? [3]) [3] $ ? 2  (NH4 )2 Mo7 O24 [1, 10] 1,10-phenanthroline (, 1.0 mM, 68% inhibition, reaction with RS-batyl alcohol and 6-methyl-5,6,7,8-tetrahydropterin [13]) [10, 13] 2-(1-octadecylthio)ethanol (, reaction with RS-batyl alcohol and 6methyl-5,6,7,8-tetrahydropterin [13]) [13] 3-(1-octadecylthio)propan-1,2-diol (, 0.5 mM, 64% inhibition, reaction with RS-batyl alcohol and 6-methyl-5,6,7,8-tetrahydropterin [13]) [13] 8-hydroxyquinoline (, 1.0 mM, 15% inhibition, reaction with RS-batyl alcohol and 6-methyl-5,6,7,8-tetrahydropterin [13]) [10, 13] 8-hydroxyquinoline (, reaction with RS-batyl alcohol and 6-methyl5,6,7,8-tetrahydropterin [13]) [13] 8-hydroxyquinoline-5-sulfonic acid (, 1.0 mM, 13% inhibition, reaction with RS-batyl alcohol and 6-methyl-5,6,7,8-tetrahydropterin [13]) [13] CHAPS (, 0.2% or 0.5%, activity is reduced by ca. 15% after 30 min [13]) [13] CaCl2 [1] CsCl [1, 10] EDTA (, 5.0 mM, 16% inhibition [10]; , 5 mM, 16% inhibition, reaction with RS-batyl alcohol and 6-methyl-5,6,7,8-tetrahydropterin [13]) [10, 13] H2 O2 (, catalase protects from inactivation [6]) [6] KCN [2, 10] KCl [1, 10] Mega-10 (, noncompetitive [10,11]; , above 5.7 mM [10]) [10, 11, 15] MgSO4 [1] MnCl2 [1] NEM (, 8.0 mM; 91% inhibition, reaction with RS-batyl alcohol and 6-methyl-5,6,7,8-tetrahydropterin [13]) [2, 10, 13] Na2 SO4 [10] NaCl [1, 10] NaN3 [10] PCMB [2, 10] Triton X-100 (, 6% loss of activity at 0.1% detergent and 59% loss of activity at 0.3% detergent [13]) [13] ZnCl2 [1, 10] digitonin [10]

117

Glyceryl-ether monooxygenase

1.14.16.5

hexadecan-1,2-diol (, reaction with RS-batyl alcohol and 6-methyl5,6,7,8-tetrahydropterin [13]) [13] hexadecyl phosphocholine [9] hexadecyl-l-a-lyso-phosphatidylcholine (, competitive [10]) [10] hexadecylphosphocholine [10] myristoyl l-a-lyso-phosphatidylcholine [9] octadecan-1-ol (, competitive [10]; , 0.1 mM, 15% inhibition, reaction with RS-batyl alcohol and 6-methyl-5,6,7,8-tetrahydropterin [13]; , noncompetitive inhibition of the reaction with RS-3-(1-hexadecyloxy)2-hydroxypropane-1-phosphocholine and RS-6-methyl-5,6,7,8-tetrahydropterin [15]) [10, 13, 15] octadecanol (, competitive [11]) [11] palmitoyl l-a-lyso-phosphatidylcholine [9] tetradecanoyl-l-a-lyso-phosphatidylcholine (, competitive [10]) [10] tetradecylphosphocholine (, competitive [10]) [9, 10]
"%  (6R)-5,6,7,8-tetrahydrobiopterin [13] (6R)-tetrahydrobiopterin [10] (RS)-6,7-dimethyl-5,6,7,8-tetrahydropterin [10, 13] (RS)-6-methyl-5,6,7,8-tetrahydropterin (, R(+)- and S(-)-6-methyl5,6,7,8-tetrahydropterin are about equally effective [8]) [8, 10, 11, 13, 14, 15] 2-amino-5,6,7,8-tetrahydropteridin-4-one [7] 5,6,7,8-tetrahydrofolic acid [10] 5,6,7,8-tetrahydropterin [10] NAD+ (, stimulates [1]) [1] NADH (, stimulates [1]) [1] tetrahydrobiopterin (, optimal concentration: 1 mM [2]) [2, 10] &

  (NH4 )2 SO4 (, activates, optimal concentration is 1.25 mM [1]) [1] GSH (, 10 mM, increase reaction 5fold [1]; , required for expression of full activity, maximal stimulation at 1 mM [2]) [1, 2] NH4 Cl (, activates [1]) [1] Triton X-100 (, stimulates [2]) [2] phospholipids (, required for expression of full activity [2]) [2] " & *-% , 0.029 [3] . / *', 0.011 (RS-2-(1-hexadecyloxy)propan-1,2-diol, , with 6-methyl5,6,7,8-tetrahydropterin as cofactor [13]) [13] 0.012 (RS-3-hexadecyloxypropane-1,2-diol, , with 6-methyl-5,6,7,8tetrahydropterin as cofactor [10]) [10] 0.0183 (2-hexadecyloxypropane-1,3-diol, , with 6-methyl-5,6,7,8-tetrahydropterin as cofactor [10,13]) [10, 13]

118

1.14.16.5

Glyceryl-ether monooxygenase

0.0204 (S-3-octadecyloxypropane-1,2-diol, , with 6-methyl-5,6,7,8tetrahydropterin as cofactor [10,13]) [10, 13] 0.0246 (6R-5,6,7,8-tetrahydrobiopterin, , reaction with batyl alcohol [13]) [13] 0.0247 (RS-3-octadecyloxypropane-1,2-diol, , with 6-methyl-5,6,7,8tetrahydropterin as cofactor [10,13]) [10, 13] 0.025 (RS-batyl alcohol, , with 6-methyl-5,6,7,8-tetrahydropterin as cofactor [11]) [11] 0.0265 (RS-3-(1-hexadecyloxy)1-methoxypropan-2-ol, , with 6methyl-5,6,7,8-tetrahydropterin as cofactor [13]) [13] 0.0313 (2-octadecyloxyethan-1-ol, , with 6-methyl-5,6,7,8-tetrahydropterin as cofactor [10,13]) [10, 13] 0.0329 (R-3-hexadecyloxypropane-1,2-diol, , with 6-methyl-5,6,7,8tetrahydropterin as cofactor [10]) [10] 0.0375 (S-3-hexydecyloxypropane-1,2-diol, , with 6-methyl-5,6,7,8tetrahydropterin as cofactor [10]) [10] 0.0397 (R-3-pentadecyloxypropane-1,2-diol, , with 6-methyl-5,6,7,8tetrahydropterin as cofactor [10,13]) [10, 13] 0.0397 (cis-RS-3-octadec-9'-enyloxypropane-1,2-diol, , with 6methyl-5,6,7,8-tetrahydropterin as cofactor [10]) [10] 0.0415 (R-3-octadecyloxypropane-1,2-diol, , with 6-methyl-5,6,7,8tetrahydropterin as cofactor [10,13]) [10, 13] 0.0427 (R-3-tridecyloxypropane-1,2-diol, , with 6-methyl-5,6,7,8-tetrahydropterin as cofactor [10]) [10] 0.0474 (R-3-dodecyloxypropane-1,2-diol, , with 6-methyl-5,6,7,8-tetrahydropterin as cofactor [10]) [10] 0.0718 (R-3-heptadecyloxypropane-1,2-diol, , with 6-methyl-5,6,7,8tetrahydropterin as cofactor [10]) [10] 0.072 (RS-3-octadecyloxypropane-1,2-diol, , with 6,7-dimethyl5,6,7,8-tetrahydropterin as cofactor [10]) [10] 0.1 (RS-3-(1-hexadecyloxy)propan-1-ol, , with 6-methyl-5,6,7,8-tetrahydropterin as cofactor [13]) [13] 0.121 (RS-hexadecylthiopropane-1,2-diol, , with 6-methyl-5,6,7,8tetrahydropterin as cofactor [10]) [10] 0.137 (S-3-(1-hexadecyloxy)-2-hydroxypropane-1-phosphocholine, , with 6-methyl-5,6,7,8-tetrahydropterin as cofactor [13]) [13] 0.138 (6-methyl-5,6,7,8-tetrahydropterin, , reaction with RS-batyl alcohol [11]) [11] 0.173 (RS-3-(1-hexadecyloxy)-2-hydroxypropane-1-phosphocholine, , with 6-methyl-5,6,7,8-tetrahydropterin as cofactor [13]) [13] 0.228 (2-hexadecylthioethan-ol, , with 6-methyl-5,6,7,8-tetrahydropterin as cofactor [10]) [10] 0.265 (3-hexadecyloxy-1-methoxypropan-1-ol, , with 6-methyl5,6,7,8-tetrahydropterin as cofactor [10]) [10] 0.428 (RS-octadecylthiopropane-1,2-diol, , with 6-methyl-5,6,7,8-tetrahydropterin as cofactor [10]) [10]

119

Glyceryl-ether monooxygenase

1.14.16.5

0.521 (2-octadecylthioethan-1-ol, , with 6-methyl-5,6,7,8-tetrahydropterin as cofactor [10]) [10] 0.66 (1-O-hexadecylglycerol) [2] 0.66 (RS-3-hexadecyloxypropane-1,2-diol, , with 6,7-dimethyl5,6,7,8-tetrahydropterin as cofactor [10]) [10] 0.937 (R-3-nonadecyloxypropane-1,2-diol, , with 6-methyl-5,6,7,8tetrahydropterin as cofactor [10]) [10] 51.3 (RS-3-heptyloxypropane-1,2-diol, , with 6-methyl-5,6,7,8-tetrahydropterin as cofactor [10,13]) [10, 13] Additional information (, Km -values for batyl alcohol in presence of inhibitors [9]; , influence of detergents in Km -value for batyl alcohol [10]) [9, 10, 12, 13] . / *', 0.185 (Mega-10, , with respect to RS-6-methyl-5,6,7,8-tetrahydropterin [15]) [15] 0.316 (palmitoyl l-a-lyso-phosphatidylcholine) [9] 0.32 (hexadecyl-l-a-lyso-phosphatidylcholine) [1] 0.32 (tetradecyl phosphocholine) [9,10] 0.326 (myristoyl l-a-lyso-phosphatidylcholine) [9] 0.33 (tetradecanoyl-l-a-lyso-phosphatidylcholine) [10] 0.365 (hexadecylphosphocholine) [9] 0.37 (hexadecylphosphocholine) [10] 0.765 (octadecan-1-ol) [11,15] 0.77 (octadecan-1-ol) [10] 1.74 (Mega-10, , with respect to RS-3-(1-hexadecyloxy)-2-hydroxypropane-1-phosphocholine [15]) [10,11,15] 0  8.5 [2] 8.7 (, formation of glyceryl ether products [13]) [13] 9 [1] 0 

 7.5-9.5 (, pH 7.5: about 40% of maximal activity, pH 9.5: about 75% of maximal activity [2]) [2]

  #   ' 1  400000 (, gel filtration [2,5]) [2, 5]   ? (, x * 45000, SDS-PAGE [5]) [5]

120

1.14.16.5

Glyceryl-ether monooxygenase

 2 %$ %' %

% liver [1, 2, 3, 4, 5, 6, 7, 9, 10, 14, 15] 3#

membrane (, bound to [10]) [10] microsome [1, 2, 3, 4, 5, 6, 7, 9, 15] $"

(partial [2]) [2, 5]

4     , -20 C or -70 C, stable for many months [10]

"  [1] Soodsma, J.F.; Piantadosi, C.; Snyder, F.: Partial characterization of the alkylglycerol cleavage enzyme system of rat liver. J. Biol. Chem., 247, 39233929 (1972) [2] Ishibashi, T.; Imai, Y.: Solubilization and partial characterization of alkylglycerol monooxygenase from rat liver microsomes. Eur. J. Biochem., 132, 23-27 (1983) [3] Pfleger, R.C.; Piantadosi, C.; Snyder, F.: The biocleavage of isomeric glyceryl ethers by soluble liver enzymes in a variety of species. Biochim. Biophys. Acta, 144, 633-648 (1967) [4] Snyder, F.; Malone, B.; Piantadosi, C.: Tetrahydropteridine-dependent cleavage enzyme for O-alkyl lipids: substrate specificity. Biochim. Biophys. Acta, 316, 259-265 (1973) [5] Ishibashi, T.; Imai, Y.: Affinity purification of alkylglycerol monooxygenase from rat liver microsomes by chimyl alcohol-Sepharose 4B column chromatography. J. Lipid Res., 26, 393-395 (1985) [6] Rock, C.O.; Baker, R.C.; Fitzgerald, V.; Snyder, F.: Stimulation of the microsomal alkylglycerol monooxygenase by catalase. Biochim. Biophys. Acta, 450, 469-473 (1976) [7] Armarego, W.L.F.: Glyceryl-ether monooxygenase [EC 1.14.16.5] a mixed function oxidase requiring oxygen and a tetrahydropterin cofactor. Pteridines, 7, 90 (1996) [8] Kosar-Hashemi, B.; Taguchi, H.; Armarego, W.L.F.: Glyceryl-ether monooxygenase [EC 1.14.16.5]. Part V: Some aspects of the stoichiometry. Pteridines, 5, 1-7 (1994) [9] Kurisu, T.; Taguchi, H.; Paal, B.; Yang, N.; Armarego, W.L.F.: Glyceryl-ether monooxygenase [EC 1.14.16.5] Part VII. Effects of alkyl phosphocholines

121

Glyceryl-ether monooxygenase

[10] [11] [12] [13] [14] [15]

122

1.14.16.5

and acyl l-a-lyso-phosphatidylcholines detergents on enzyme activity. Pteridines, 5, 95-101 (1994) Taguchi, H.; Armarego, W.L.F.: Glyceryl-ether monooxygenase [EC 1.14.16.5]. A microsomal enzyme of ether lipid metabolism. Med. Res. Rev., 18, 43-89 (1998) Kosar-Hashemi, B.; Taguchi, H.; Armarego, W.L.F.: Glyceryl ether monooxygenase [EC 1.14.16.5]: stoichiometry and inhibition. Adv. Exp. Med. Biol., 338, 93-96 (1993) Taguchi, H.; Paal, B.; Armarego, W.L.F.: Glyceryl-ether monooxygenase [EC 1.14.16.5]. Part VIII. Probing the nature of the active site. Pteridines, 6, 4557 (1995) Kosar-Hashemi, B.; Armarego, W.L.F.: A convenient spectrophotometric method for measuring the kinetic parameters of glyceryl-ether monooxygenase (EC 1.14.16.5). Biol. Chem. Hoppe-Seyler, 374, 9-25 (1993) Taguchi, H.; Paal, B.; Armarego, W.L.F.: Glyceryl-ether monooxygenase [EC 1.14.16.5]. Part IX. Stereospecificity of the oxygenase reaction. J. Chem. Soc. Perkin Trans. I, 1997, 303-307 (1997) Taguchi, H.; Kosar-Hashemi, B.; Paal, B.; Yang, N.; Armarego, W.L.F.: Glyceryl-ether monooxygenase (EC 1.14.16.5), part VI.: nature of the glycerylether lipid substrates in aqueous buffer. Biol. Chem. Hoppe-Seyler, 375, 329-334 (1994)

'      

44

   
 1.14.16.6    (S)-2-hydroxy-2-phenylacetate,tetrahydrobiopterin:oxygen oxidoreductase (4hydroxylating)    mandelate 4-monooxygenase    l-mandelate-4-hydroxylase mandelic acid 4-hydroxylase oxygenase, mandelate 4-mono
    39459-82-0

     Pseudomonas convexa [1-3]

!    "
 # 

(S)-2-hydroxy-2-phenylacetate + tetrahydrobiopterin + O2 = (S)-4-hydroxymandelate + dihydrobiopterin + H2 O    redox reaction       (S)-2-hydroxy-2-phenylacetate + tetrahydropteridine + O2 (i.e. lmandelate, first step of oxidative degradation of l-mandelate by Pseudomonas convexa) [2]

123

Mandelate 4-monooxygenase

1.14.16.6

     (S)-2-hydroxy-2-phenylacetate + tetrahydropteridine (i.e. l-mandelate, highly substrate specific, d-mandelate is not hydroxylated, [2, 3]) [1-3] $ (S)-4-hydroxymandelate + dihydropteridine + H2 O [1-3] 2  1,10-phenanthroline [2] 2,2'-bipyridyl [2] 4-hydroxymercuribenzoate (partially reversible by thiol compounds) [2] 8-hydroxyquinoline [2] Ag2+ [2] Cd2+ [2] Cu2+ [2] EDTA (less effective) [2] Hg2+ [2] IAA (less effective) [2] NEM [2] SDS [2] amethopterine [2] aminopterine [2] guanidine hydrochloride [2] iodoacetate (less effective) [2] thiourea [2] urea [2]
"%  NADPH (requirement, cannot be replaced by NADH, [2]) [1-3] &

  2-amino-4-hydroxy-6,7-dimethyl-tetrahydropteridine (requirement, best pteridine compound tested, can be replaced by cell- and protein-free extract from Pseudomonas convexa, [2]) [1-3] THF (increase of activity) [2] '(   Fe2+ (requirement, cannot be replaced by Fe3+ , Mn2+ , Mg2+ , Cu2+ , Cu+ , Ni2+ , Cd2+ , [2]) [1-3] " & *-% , 0.021 [2] . / *', 0.1 ((S)-2-hydroxy-2-phenylacetate) [2] 0.19 (NADPH) [2] 0  5.4 [1, 3] 0 

 4.6-7.4 (half-maximal activity at pH 4.6 and pH 7.4) [2] 124

1.14.16.6

Mandelate 4-monooxygenase

)  *
, 38 (inactivation above) [2]

  #   ' 1  91000 ( gel filtration) [2]

 2 %$ %' %

3#

cytoplasm [1-3] $"

[2]

4  )  55 (5 min incubation, no loss of activity) [2] 5    "

, different buffers, pH-values, addition of substrate, Fe2+ , tetrahydropteridine or GSH, DTT and 2-mercaptoethanol do not enhance stability [2]    , -20 C, partially purified enzyme is stable for 36-40 h [2]

"  [1] Bhat, S.G.; Ramanarayanan, M.; Vaidyanathan, C.S.: Mandelic acid-4-hydroxylase, a new inducible enzyme from Pseudomonas convexa. Biochem. Biophys. Res. Commun., 52, 834-842 (1973) [2] Bhat, S.G.; Vaidyanathan, C.S.: Purifications and properties of l-mandelate4-hydroxylase from Pseudomonas convexa. Arch. Biochem. Biophys., 176, 314-323 (1976) [3] Bhat, S.G.; Vaidyanathan, C.S.: Involvement of 4-hydroxymandelic acid in the degradation of mandelic acid by Pseudomonas convexa. J. Bacteriol., 127, 1108-1118 (1976)

125

9  b    

8

   
 1.14.17.1    3,4-dihydroxyphenethylamine,ascorbate:oxygen oxidoreductase (b-hydroxylating)    dopamine b-monooxygenase    3,4-dihydroxyphenethylamine b-oxidase 3,4-dihydroxyphenylethylamine b-hydoxylase 4-(2-aminoethyl)pyrocatechol b-oxidase EC 1.14.2.1 (formerly) MDBH ( membrane-associated dopamine b-monooxygenase [1]) [1] SDBH ( soluble dopamine b-monooxygenase [1]) [1] dopa b-hydroxylase dopamine b-hydrolase dopamine b-hydroxylase dopamine b-oxidase dopamine hydroxylase dopamine(3,4-dihydroxyphenethylamine)b-mono-oxygenase dopamine-B-hydroxylase ( DBH [14]) [14] oxygenase, dopamine b-monophenylamine b-hydroxylase
    9013-38-1

    





126

Bos taurus [1, 3, 5, 8-28, 30-34, 38, 39] Rattus norvegicus (Sprague Dawley [40]) [2, 4, 37, 40, 41, 42] Homo sapiens [6, 35, 36] Gallus gallus [7] Acipenser baerii baerii [29] Felis catur [37]

1.14.17.1

Dopamine b-monooxygenase

!    "
 # 

3,4-dihydroxyphenethylamine + ascorbate + O2 = noradrenaline + dehydroascorbate + H2 O ( uni-uni bi-uni ping pong mechanism [7]; ping pong mechanism [8]; stoichiometry [11,24]; mechanism [12, 16, 17, 21, 24, 30]; new mechanism of enzyme [3])    oxidation redox reaction reduction       3,4-dihydroxyphenethylamine + ascorbate + O2 ( enzyme plays a key role in the biosynthetic interconversion of neurotransmitters [14, 17, 24, 25, 38]) (Reversibility: ? [1, 5, 14, 17, 18, 24, 25, 35, 36, 38]) [1, 5, 14, 17, 18, 24, 25, 35, 36, 38] $ noradrenaline + dehydroascorbate + H2 O ( norepinephrine [1, 5, 14, 17, 18, 24, 25]) [11, 5, 14, 17, 18, 24, 25]      1-(4-hydroxybenzyl)imidazole + ascorbate + O2 (Reversibility: ? [20]) [20] $ 4-hydroxybenzaldehyde + dehydroascorbate + H2 O + ? [20]  1-(4-hydroxybenzyl)imidazole + ascorbate + O2 (Reversibility: ? [20]) [20] $ ?  1-phenyl-1-aminomethylethene + ascorbate + O2 ( ascorbate and ferrocyanide can function as a electron donors [14]) (Reversibility: ? [14]) [14] $ 2,3-dihydroxy-2-phenylpropylamine + dehydroascorbate + H2 O + ? [14]  2-(4-hydroxyphenyl)prop-2-enylamine + ascorbate + O2 (Reversibility: ? [20]) [20] $ ?  2-bromo-3-(p-hydroxyphenyl)-1-propene + ascorbate + O2 (Reversibility: ? [23]) [23] $ 2-bromo-3-hydroxy-3-(p-hydroxyphenyl)-1-propene + H2 O [23]  2-chlorophenethylamine + ascorbate + O2 (Reversibility: ? [24]) [24] $ 2-amino-1-chloro-1-phenylethanol + dehydroascorbate + H2 O [24]  2-hydroxyphenethylamine + ascorbate + O2 (Reversibility: ? [24]) [24] $ 2-amino-1-phenylethane-1,1-diol + dehydroascorbate + H2 O [24]  2-phenylethylamine + ascorbate + O2 (Reversibility: ? [14]) [14] $ ?

127

Dopamine b-monooxygenase

1.14.17.1

 2-phenylprop-2-enylamine + ascorbate + O2 (Reversibility: ? [20]) [20] $ ?  3,4-dihydroxyphenethylamine + ascorbate + O2 ( 3,4-dihydroxyphenethylamine is identical with 4-(2-aminoethyl)-1,2-benzendiol i.e. dopamine, ferricyanide can replace ascorbate [7]; ferrocyanide can function as a electron donor [13]; N,N-dimethyl-1,4-phenylenediamine and ascorbic acid derivates are efficient reductants for the enzyme [30]; d-xyloascorbic acid, l-araboascorbic acid or d-araboascorbic acid can replace l-xyloascorbic acid [34]) (Reversibility: ? [1-24, 25, 28, 30, 31, 33-38, 40]) [1-24, 25, 28, 30, 31, 33-38, 40] $ noradrenaline + dehydroascorbate + H2 O ( norepinephrine [1-3, 5-7, 10-24, 25, 30, 31, 33, 37, 40]) [1-3, 5-7, 10-24, 25, 30-33, 35-38, 40]  3-phenylpropylamine + ascorbate + O2 (Reversibility: ? [20]) [20] $ ?  4-hydroxy-a-methylstyrene + ascorbate + O2 (Reversibility: ? [20]) [20] $ ?  4-hydroxybenzyl cyanide + ascorbate + O2 ( and benzyl cyanide analogs [22]) (Reversibility: ? [22]) [22] $ 4-hydroxymadelonitrile + dehydroascorbate + H2 O [22]  4-hydroxyphenyl-2-aminoethyl sulfide + ascorbate + O2 (Reversibility: ? [25]) [25] $ 4-hydroxyphenyl-2-aminoethyl sulfoxide + dehydroascorbate + H2 O [25]  4-hydroxyphenyl-2-aminopropyl selenide + ascorbate + O2 (Reversibility: ? [25]) [25] $ 4-hydroxyphenyl-2-aminopropyl selenoxide + dehydroascorbate + H2 O [25]  N-phenylethylenediamine + ascorbate + O2 (Reversibility: ? [14, 20]) [14, 20] $ ?  octopamine + ascorbate + O2 (Reversibility: ? [14, 24]) [14, 24] $ ?  phenyl 2-aminoethyl sulfide + ascorbate + O2 ( and derivates [25]) (Reversibility: ? [25]) [14, 20, 25] $ phenyl-2-aminoethyl sulfoxide + dehydroascorbate + H2 O [25]  phenylacetaldehyde + ascorbate + O2 (Reversibility: ? [21]) [21] $ ?  tyramine + ascorbate + O2 (Reversibility: ? [9, 12, 13, 15, 16, 20, 22, 25, 30, 32, 33, 35-38]) [9, 12, 13, 15, 16, 20, 22, 25, 30, 32, 33, 3538] $ octopamine + dehydroascorbate + H2 O [25, 33, 35-37]  Additional information ( enzyme with very broad substrate specifity [24]) [24] $ ? 128

1.14.17.1

Dopamine b-monooxygenase

2  (1H)-imidazole-4-acetic acid [16] 1(2H)-phthalazine hydrazone ( hydralazine [16]) [16] 1-(3,4-dihydroxybenzyl)imidazole [20] 1-(4-hydroxybenzyl)-2-methylimidazole [20] 1-(4-hydroxybenzyl)imidazole [20] 1-(4-hydroxybenzyl)imidazole-2-thiol [19] 1-(4-hydroxybenzyl)pyrazole [20] 1-(4-hydroxyphenyl)-1-(aminomethyl)-ethene [25] 1-benzimidazole [20] 1-benzylimidazole [20] 1-isoquinolinecarboxylic acid [16] 1-methylimidazole-2-thiol [19] 1-phenyl-1-aminomethylethene ( suicide inhibition [14]) [14] 1-phenylpropene [20] 2(1H)-pyridinone hydrazone ( 2-hydrazinopyridine [16]) [16] 2,2'-bi-(1H)-imidazole ( 2,2'-biimidazole [16]) [16] 2-(4-hydroxyphenyl)prop-2-enylamine [20] 2-bromo-3-(p-hydroxyphenyl)-1-propene ( mechanism-based inhibition [23]) [23] 2-chlorophenethylamine [24] 2-hydroxyphenylacetaldehyde [21] 2-phenylprop-2-enylamine [20] 3-phenylpropene [20] 4-hydroxy-a-methylstyrene [20] 4-hydroxybenzaldehyde [20] 4-hydroxybenzyl cyanide [22] CN- [15] CO [12] CaNa2 EDTA ( weak [36]) [36] KCN [11, 12] N-ethylaniline [20] N-phenylethylenediamine [20] N-3 [15] Na2 SO3 [9] ascorbate [5, 7] ascorbic acid oxidase [11] bathocuproine disulfonate [28] b-ethynyltyramine ( and enantiomers, mechanism of inhibition [17]) [17] catechol [13] diethyldicarbonate [8] diethyldithiocarbamate [8, 12] disulfiram [35, 36] ferrocyanide [13] histidine [26] hydroquinone [13] 129

Dopamine b-monooxygenase

1.14.17.1

malonate [5] norepinephrine [7] p-cresol ( mechanism of inhibition [18]) [18, 19] p-hydroxyphenylacetamide [21] phenylacetaldehyde [21] phenylacetamide [21] quinoline-2-carboxylic acid [16] sodium diethyldithiocarbamate [36] Additional information ( suicide inactivation by benzyl cyanides [22]; ascorbic acid derivates inhibit the enzyme at higher concentrations [30]; dithiocarbamate pesticides: increase of inhibitory potency from methyl-and dimethyldithiocarbamates to diethyldithiocarbamates up to the most potent ethylenbisdithiocarbamates [36]) [22, 30] &

  acetate [7] ascorbic acid ( complete dependence on added ascorbate, e.g. isoascorbate, glucoascorbate, d-ascorbate [11]) [11] dehydroascorbate [7] fumarate [7, 11, 30] '(   Cu2+ ( a copper protein [5, 8, 9, 12, 27, 32]; about 8 Cu2+ per tetramer [5]; 4 atoms of tightly bound copper per tetramer [6]; enzyme contains a constant amount of Cu2+ , 2 mol per mol of protein, and a variable amount of Cu2+ , copper content is a linear function of the purity of the enzyme [12]; 3 mol of copper per mol of tetramer, MW 290000 [27]; Km : 0.00003-0.0002 mM [28]; 1.1 Cu2+ /subunit, increasing stimulation of activity by addition of up to 1 Cu2+ /subunit, further additions up to at least 4 Cu/subunit gave neither stimulation nor inhibition [32]; enzyme not activated by exogenous copper but activity decreases at high concentrations [37]; 2,6-dimethylphenyl isocyanide as the isocyanide ligand demonstrated, first: the formation of a mono-DIMPI-four-coordinate complex at each copper, second: the formation of complexes containing more than one isocyanide per copper [39]) [5, 8, 12, 13, 27, 28, 32, 35-37, 39] Fe2+ ( 0.0004 mM per mol of enzyme [12]) [12] Mg2+ ( regulates the translation of enzyme and affects the ratio of the two glycosylated forms of the enzyme [41]) [41] NaCl [7] VO2+ ( 1 VO2+ /subunits during catalysis [32]) [32] Additional information ( Cu2+ , Mn2+ , Ni2+ , Co2+, Zn2+ , Pb2+ , Fe2+ , Fe3+ reduce translation of enzyme at concentrations above 1.5 mM, Ni2+ and Cu2+ inhibit the glycosylation [41]) [41] ) &  * +, 13.4 (2-bromo-3-(p-hydroxyphenyl)-1-propene) [23] 42 (4-hydroxy-a-methylstyrene) [20] 66 (2-chlorophenethylamine) [24]

130

1.14.17.1

Dopamine b-monooxygenase

102 (phenylacetaldehyde) [21] 252 (2-hydroxyphenethylamine) [24] 570 (1-(4-hydroxybenzyl)imidazole) [20] 600 (1-phenyl-1-aminomethylethene) [14] 720 (3-phenylpropylamine) [14] 780 (dopamine) [24] 840 (2-phenylprop-2-enylamine) [20] 1224 (3-phenylpropylamine) [20] 1260 (N-phenylethylenediamine) [20] 1980 (octopamine) [14] 2340 (phenylaminoethyl sulfide) [20] 3360 (2-(4-hydroxphenyl)prop-2-enylamine) [20] 3900 (1-phenylethylamine) [14] 4080 (phenylaminoethyl sulfide) [14] 6600 (dopamine) [20] 7260 (tyramine) [20] " & *-% , 1.5 [2] 3.25 [12] 3.9 [8] 69 [5] Additional information ( activity in the cat is twofold higher compared to rat [37]) [6, 14, 22, 27, 37, 38] . / *', 0.14 (O2, in presence of tyramine [22]) [22] 0.2 (dopamine) [7] 0.55 (tyramine) [8] 0.6 (ascorbate, in the absence of Cu2+ [37]) [37] 0.8 (ascorbate) [6] 1 (3,4-dihydroxyphenethylamine) [24] 1 (ascorbate, in the presence of Cu2+ [37]) [37] 1.1 (ascorbate, with or without Cu2+ [37]) [37] 1.25 (ascorbate) [8] 1.3 (2-(4-hydroxyphenyl)prop-2-enylamine) [20] 1.3 (tyramine) [22] 1.4 (d-araboascorbic acid) [34] 1.5 (l-xyloascorbic acid) [34] 1.6 (4-hydroxybenzyl cyanide) [22] 1.66 (tyramine, deglycosylated enzyme [10]) [10] 1.9 (1-(4-hydroxybenzyl)imidazole) [20] 2 (ascorbate) [7] 2 (tyramine) [37] 2 (tyramine) [6, 20] 2.17 (tyramine, native enzyme [10]) [10] 2.3 (d-xyloascorbic acid) [34] 2.3 (tyramine) [37] 131

Dopamine b-monooxygenase

1.14.17.1

2.7 (l-araboascorbic acid) [34] 2.8 (O2, in the presence of p-hydroxybenzyl cyanide [22]) [22] 3.7 (4-hydroxy-a-methylstyrene) [20] 5.1 (2-chlorophenethylamine) [24] 5.9 (2-bromo-3-(p-hydroxyphenyl)-1-propene) [23] 6 (ascorbate) [11] 6.7 (2-phenylprop-2-enylamine) [20] 7 (phenylethylamine) [14] 7.4 (phenylacetaldehyde) [21] 7.9 (phenylacetaldehyde) [21] 8.3 (1-phenyl-1-aminomethylethene) [14] 9.1 (N-phenylethylenediamine) [20] 12.2 (3-phenylpropylamine) [20] 14 (octopamine) [14] 17.2 (phenylaminoethyl sulfide) [20] 20.4 (3-phenylpropylamine) [14] 24 (2-hydroxyphenethylamine) [24] 26.5 (phenylaminoethyl sulfide) [14] Additional information ( Km of ascorbate, tyramine and O2 as a function of pH and fumarate activation [19]; Km of benzyl cyanide analogs with or without 6% dimethylformamide [22]; Km of N,N-dimethyl1,4-phenylenediamine and ascorbic acid derivates [30]; comparison of Km of native enzyme and dimeric and tetrameric species after treatment with dithiothreitol and iodoacetamide [33]) [19, 22, 30, 33] . / *', 0.0009 (1-isoquinolinecarboxylic acid) [16] 0.0019 (2(1H)-pyridinone hydrazone) [16] 0.0057 (1(2H)-phthalazine hydrazone) [16] 0.015 (b-ethynytyramine, R,S-1 [17]) [17] 0.021 (2,2'-bi-(1H)-imidazole) [16] 0.057 (b-ethynytyramine, S-1, in the presence of ascorbate [17]) [17] 0.14 (quinoline-2-carboxylic acid) [16] 0.16 (1-(4-hydroxybenzyl)pyrazole) [20] 0.208 (b-ethynytyramine, S-1 [17]) [17] 0.37 (hydroquinone, pH: 4.9 [13]) [13] 0.41 (histidine) [26] 0.52 (2-(4-hydroxyphenyl)prop-2-enylamine) [20] 0.8 (1-isoquinolinecarboxylic acid) [16] 1.05 (hydroquinone, pH: 5.9 [13]) [13] 1.1 (N-phenylethylenediamine) [20] 2.3 (4-hydroxybenzaldehyde) [20] 2.5 (1-phenylpropene) [20] 2.5 (4-hydroxy-a-methylstyrene) [20] 2.9 (1-(4-hydroxybenzyl)-2-methylimidazole) [20] 3.6 (3-phenylpropene) [20]

132

1.14.17.1

Dopamine b-monooxygenase

4.6 (1-(3,4-dihydroxybenzyl)imidazole) [20] 4.7 (2-phenylprop-2-enylamine) [20] 4.9 (2-bromo-3-(p-hydroxyphenyl)-1-propene) [23] 5 (norepinephrine) [7] 6.8 (N-ethylaniline) [20] 11.9 (1-benzylimidazole) [20] 13 (1-phenyl-1-aminomethylethene) [14] 20 (ascorbate) [7] Additional information ( Ki as function of pH and fumarate activation [19]) [15,19] 0  4.6 [37] 4.8-5 [37] 5 ( pI: 5.8 [6]) [6] 5-6 [7] 6 ( for the ferrocyanide as the sole electron donor [13]) [13] Additional information ( pI: 6.6 in the presence of 8 M urea [2]) [2] 0 

 4.5-6.5 ( sharp decrease in activity between pH 4.5 and 5.0 and between pH 6.0 and 6.5 [7]) [7] )  *
, 50 [8]

  #   ' 1  220000-250000 ( 220000 L208 homoenzyme, 230000 F208 homoenzyme, 250000 F208/L208 heteroenzyme, low-angle laser light scatteringphotometry [38]) [38] 260000 ( native enzyme, low-angle laser light scattering photometry coupled with high-performance gel filtration [33]) [33] 280000 ( SDS-PAGE with 0.01% SDS [8]) [8] 290000 ( sedimentation equilibrium analysis, 5 mM potassium phosphate buffer, pH 6.8, 0.1 M NaCl [3,12]) [3, 12, 27] 300000 ( dimeric form, SDS-PAGE [6]) [4, 6] 316000 ( gel filtration [7]) [7] 370000 ( in 5% ammonium sulfate buffer [2]) [2] 450000 ( in 0.2 M NaCl [2]) [2] 600000 ( in 1 M NaCl [2]) [2] 610000 ( tetrameric form, SDS-PAGE [6]) [6] 800000 ( in 5 mM sodium phosphate buffer, pH 7.0 [2]) [2] Additional information ( molecular forms change depending on concentration of salts and kind of salts [2]) [2]

133

Dopamine b-monooxygenase

1.14.17.1

  ? ( x * 75000, SDS-PAGE of reduced and carboxymethylated MDBH [1]; x * 70000-75000, Western blot analysis, recombinant enzyme [31]) [1, 31] tetramer ( 4 * 88000 SDS-PAGE [2]; 4 * 72000 SDS-PAGE [6]; 4 * 80000, SDS-PAGE after treatment with 2-mercaptoethanol, subunits joined in pairs by disulfide bonds [7]; 4 * 66000-74000, SDS-PAGE after cleavage of intersubunit disulfide bonds with dithiothreitol, tetramer consists of two disulfid-linked dimers [33]; 4 * 65000, deduced from cDNA [38]) [2, 6, 7, 33, 38] $   "

glycoprotein ( 5% carbohydrate by weight [10]; oligosaccharide moieties do not play a role in catalysis [10]; 3.5 residues of glucosamine per subunit, glucosamine is the only hexosamine detected [27]) [6, 7, 10, 27] phospholipoprotein ( hypothesis: completely processed enzyme may be anchored to cellular membranes by binding to phosphatidylserine [31]) [31]

 2 %$ %' %

% adrenal gland ( dietary copper deficiency is associated with increased formation of enzyme [40]) [2, 7, 9, 12, 14, 16, 26, 30, 37, 38, 40] adrenal medulla [1, 5, 8, 10, 11, 17-19, 21, 23, 25, 27, 28, 32-34, 39] blood serum [35, 36] brain ( chondrostean [29]) [29, 40] hypothalamus [40] pheochromocytoma cell [4] plasma [6] 3#

chromaffin granule [1, 5, 7, 8, 20, 21, 23, 25, 27, 30, 34] membrane ( recombinant enzyme [31]) [1, 30, 31] soluble ( recombinant enzyme [31]) [1, 16, 30, 31, 33] Additional information ( enzyme-immunoreactive cells in anterior tuberal nucleus, locus coeruleus and caudal rhombencephalon [29]) [29] $"

(partial [11]; large scale [27]) [1, 5, 8, 11, 12, 27, 38] (recombinant enzyme [31]) [31] [2] [6] [7]

134

1.14.17.1

Dopamine b-monooxygenase


 

(expression in Drosophila Schneider 2 cells [31]) [31] (expression in AtT-20 corticotrope tumor cells, PC12 pheochromocytoma cells and Chinese hamster ovary cells [42]) [42] 

 

L208F ( a natural variant - three phenotypes of enzyme isolated: 4 * L208-homoenzyme, 4 * F208-homoenzyme and 2 * F208, 2 * L208-heteroenzyme, no significant difference in kinetic properties [38]) [38] Additional information ( construction of chimera: dopamine-bmonooxygenase signal, residue 1-42 appended to peptidylglycine a-hydroxylating monoxygenase, result: the signal/anchor domain of enzyme is responsible for its membrane association and is likely to play a key role in the targeting of enzyme to secretory garnules in chromaffin cells and adrenergic neurons [42]) [42]

4  )  0-4 ( 24 h, 10-15% loss of activity [7]) [7] 50 ( 4 h, about 10% loss of activity [28]) [28] 60 ( 2 h, about 30% loss of activity [28]) [28] 5    "

, freezing and subsequent thawing results in rapid decrease of activity [6]    , -30 C, 10 mM potassium phosphate buffer, pH 6.5, stable for at least 1 month [8] , 0-4 C, 10-15% loss of activity in 24 h [8] , 4 C, stable for at least several days, dimeric species [33] , 4 C, 0.5 M NaCl in 0.02 M sodium phosphate, pH 7.0, several weeks [6] , -20 C, stable for at least 6 weeks [7]

"  [1] Slater, E.P.; Zaremba, S.; Hogue-Angeletti, R.A.: Purification of membranebound dopamine b-monooxygenase from chromaffin granules: relation to soluble dopamine b-monooxygenase. Arch. Biochem. Biophys., 211, 288296 (1981) [2] Okuno, S.; Fujisawa, H.: Purification and characterization of rat dopamine b-monooxygenase and monoclonal antibodies to the enzyme. Biochim. Biophys. Acta, 799, 260-269 (1984) [3] Tian, G.; Berry, J.A.; Klinman, J.P.: Oxygen-18 kinetic isotope effects in the dopamine b-monooxygenase reaction: Evidence for a new chemical me-

135

Dopamine b-monooxygenase

[4] [5] [6] [7] [8]

[9]

[10] [11] [12] [13] [14]

[15]

[16] [17]

136

1.14.17.1

chanism in non-heme, metallomonooxygenase. Biochemistry, 33, 226-234 (1994) Fong, J.C.; Shenkman, L.; Goldstein, M.: Purification and characterization of rat pheochromocytoma dopamine b-hydroxylase. J. Neurochem., 34, 346-350 (1980) Colombo, G.; Papadopoulos, N.J.; Ash, D.E.; Villafranca, J.J.: Characterization of highly purified dopamine b-hydroxylase. Arch. Biochem. Biophys., 252, 71-80 (1987) Frigon, R.P.; Stone, R.A.: Human plasma dopamine b-hydroxylase. Purification and properties. J. Biol. Chem., 253, 6780-6786 (1978) Long, R.A.; Weppelman, R.M.; Taylor, J.E.; Tolman, R. L.; Olson, G.: Purification and characterization of avian dopamine b-hydroxylase. Biochemistry, 20, 7423-7431 (1981) Aunis, D.; Miras-Portugal, M.T.; Mandel, P.: Bovine adrenal medullary dopamine b-hydroxylase: purification by affinity chromatography, kinetic studies and presence of essential histidyl residues. Biochim. Biophys. Acta, 327, 313-327 (1973) Merkler, D.J.; Kulathila, R.; Francisco, W.A.; Ash, D.E.; Bell, J.: The irreversible inactivation of two copper-dependent monooxygenases by sulfite: peptidylglycine a-amidating enzyme and dopamine b-monooxygenase. FEBS Lett., 366, 165-169 (1995) Hamos, J.; Desai, P.R.; Villafranca, J.J.: Characterization and kinetic studies of deglycosylated dopamine b-hydroxylase. FASEB J., 1, 143-148 (1987) Levin, E.Y.; Levenberg, B.; Kaufman, S.: The enzymatic conversion of 3,4dihydroxyphenylethylamine to norepinephrine. J. Biol. Chem., 235, 20802086 (1960) Friedman, S.; Kaufman, S.: 3,4-dihydroxyphenylethylamine b-hydroxylase. Physical properties, copper content, and role of copper in the catalytic acttivity. J. Biol. Chem., 240, 4763-4773 (1965) Rosenberg, R.C.; Gimble, J.M.; Lovenberg, W.: Inhibition of dopamine-bhydroxylase by alternative electron donors. Biochim. Biophys. Acta, 613, 62-72 (1980) May, S.W.; Mueller, P.W.; Padgette, S.R.; Herman, H. H.; Phillips, R.S.: Dopamine-B-hydroxylase: suicide inhibition by the novel olefinic substrate, 1phenyl-1-aminomethylethene. Biochem. Biophys. Res. Commun., 110, 161168 (1983) Blackburn, N.J.; Collison, D.; Sutton, J.; Mabbs, F. E.: Kinetic and E.P.R. studies of cyanide and azide binding to the copper sites of dopamine (3,4dihydroxyphenethylamine) b-mono-oxygenase. Biochem. J., 220, 447-454 (1984) Townes, S.; Titone, C.; Rosenberg, R.C.: Inhibition of dopamine b-hydroxylase by bidentate chelating agents. Biochim. Biophys. Acta, 1037, 240-247 (1990) DeWolf, W.E.; Chambers, P.A.; Southan, C.; Saunders, D.; Kruse, L.I.: Inactivation of dopamine b-hydroxylase by b-ethynyltyramine: kinetic characterization and covalent modification of an active site peptide. Biochemistry, 28, 3833-3842 (1989)

1.14.17.1

Dopamine b-monooxygenase

[18] DeWolf, W.E.; Carr, S.A.; Varrichio, A.; Goodhart, P. J.; Mentzer, M.A.; Roberts, G.D.; Southan, C.; Dolle, R.E.; Kruse, L.I.: Inactivation of dopamine b-hydroxylase by p-cresol: isolation and characterization of covalently modified active site peptides. Biochemistry, 27, 9093-9101 (1988) [19] Kruse, L.I.; DeWolf, W.E.; Chambers, P.A.; Goodhart, P.J.: Design and kinetic characterization of multisubstrate inhibitors of dopamine b-hydroxylase. Biochemistry, 25, 7271-7278 (1986) [20] Sirimanne, S.R.; Herman, H.H.; May, S.W.: Interaction of dopamine bmono-oxygenase with substituted imidazoles and pyrazoles. Catalysis and inhibition. Biochem. J., 242, 227-233 (1987) [21] Bossard, M.J.; Klinman, J.P.: Mechanism-based inhibition of dopamine bmonooxygenase by aldehydes and amides. J. Biol. Chem., 261, 1642116427 (1986) [22] Colombo, G.; Rajashekhar, B.; Giedroc, D.P.; Villafranca, J.J.: Alternate substrates of dopamine b-hydroxylase. I. Kinetic investigations of benzyl cyanides as substrates and inhibitors. J. Biol. Chem., 259, 1593-1600 (1984) [23] Colombo, G.; Rajashekhar, B.; Giedroc, D.P.; Villafranca, J.J.: Mechanismbased inhibitors of dopamine b-hydroxylase: inhibition by 2-bromo-3-(phydroxyphenyl)-1-propene. Biochemistry, 23, 3590-3598 (1984) [24] Klinman, J.P.; Krueger, M.: Dopamine b-hydroxylase: activity and inhibition in the presence of b-substituted phenethylamines. Biochemistry, 21, 67-75 (1982) [25] May, S.W.; Young, F.K.; Powers, J.L.; Gill-Woznichak, M.M.: Mechanismbased inactivation of dopamine b-monooxygenase in adrenal chromaffin cells. Biochem. Biophys. Res. Commun., 228, 278-284 (1996) [26] Izumi, H.; Hayakari, M.; Kondo, Y.; Takemoto, T.: Inhibition of dopamine bmonooxygenase by histidine. Hoppe-Seyler's Z. Physiol. Chem., 356, 18311833 (1975) [27] Ljones, T.; Skotland, T.; Flatmark, T.: Purification and characterization of dopamine b-hydroxylase from bovine adrenal medulla. Eur. J. Biochem., 61, 525-533 (1976) [28] Skotland, T.; Ljones, T.: The enzyme-bound copper of dopamine b-monooxygenase. Reaction with copper chelators, preparation of the apoprotein, and kinetics of the reconstitution by added copper. Eur. J. Biochem., 94, 145-151 (1979) [29] Adrio, F.; Anadon, R.; Rodriguez-Moldes, I.: Distribution of tyrosine hydroxylase (TH) and dopamine b-hydroxylase (DBH) immunoreactivity in the central nervous system of two chondrostean fishes (Acipenser baeri and Huso huso). J. Comp. Neurol., 448, 280-297 (2002) [30] Wimalasena, K.; Dharmasena, S.; Wimalasena, D.S.; Hughbanks-Wheaton, D.K.: Reduction of dopamine b-monooxygenase. A unified model for apparent negative cooperativity and fumarate activation. J. Biol. Chem., 271, 26032-26043 (1996) [31] Gibson, K.R.; Vanek, P.G.; Kaloss, W.D.; Collier, G.B.; Connaughton, J.F.; Angelichio, M.; Livi, G.P.; Fleming, P.J.: Expression of dopamine b-hydroxylase in Drosophila Schneider 2 cells. Evidence for a mechanism of mem-

137

Dopamine b-monooxygenase

[32] [33]

[34]

[35] [36] [37] [38]

[39]

[40] [41] [42]

138

1.14.17.1

brane binding other than uncleaved signal peptide. J. Biol. Chem., 268, 9490-9495 (1993) Abudu, N.; Banjaw, M.Y.; Ljones, T.: Kinetic studies on the activation of dopamine b-monooxygenase by copper and vanadium ions. Eur. J. Biochem., 257, 622-629 (1998) Ishida, T.; Narita, M.; Nozaki, M.; Horiike, K.: Selective cleavage and modification of the intersubunit disulfide bonds of bovine dopamine b-monooxygenase: conversion of tetramer to active dimer. J. Biochem., 120, 346352 (1996) Suzuki, E.; Kurata, T.; Shiabata, M.; Mori, M.; Arakawa, N.: Activities of dand l-xyloascorbic acid and d- and l-araboascorbic acid as a cofactor for dopamine b-hydroxylase reaction. J. Nutr. Sci. Vitaminol., 43, 491-496 (1997) Caroldi, S.; De Paris, P.; Zotti, S.; Zanella, I.; Brugnone, F.: Effects of disulfiram on serum dopamine-b-hydroxylase and blood carbon disulphide concentrations in alcoholics. J. Appl. Toxicol., 14, 77-80 (1994) De Paris, P.; Caroldi, S.: In vitro effect of dithiocarbamate pesticides and of CaNa2 EDTA on human serum dopamine-b-hydroxylase. Biomed. Environ. Sci., 8, 114-121 (1995) Fortin, D.; Coulon, J.F.; Roberge, A.G.: Comparative study of biochemical parameters and kinetic properties of dopamine-b-hydroxylase activity from cat and rat adrenals. Comp. Biochem. Physiol. B, 104, 567-575 (1993) Narita, M.; Ishida, T.; Tomoyoshi, T.; Nozaki, M.; Horiike, K.: A natural variant of bovine dopamine b-monooxygenase with phenylalanine as residue 208: purification and characterization of the variant homo- and heterotetramers of (F208)4 and (F208)2 (L208)2 . FEBS Lett., 396, 208-212 (1996) Reedy, B.J.; Murthy, N.N.; Karlin, K.D.; Blackburn, N.J.: Isocyanides as ligand-directed indicators of Cu(I) coordination in copper proteins. Probing the inequivalence of the Cu(I) centers in reduced dopamine-b-monooxygenase. J. Am. Chem. Soc., 117, 9826-9831 (1995) Prohaska, J.R.; Brokate, B.: Dietary copper deficiency alters protein levels of rat dopamine b-monooxygenase and tyrosine monooxygenase. Exp. Biol. Med., 226, 199-207 (2001) Feng, Z.; Sabban, E.L.: Regulation of the translation and processing of rat dopamine b-hydroxylase by metal ions in a cell free system. Biochem. Mol. Biol. Int., 36, 339-345 (1995) Oyarce, A.M.; Steveson, T.C.; Jin, L.; Eipper, B.A.: Dopamine b-monooxygenase signal/anchor sequence alters trafficking of peptidylglycine a-hydroxylating monooxygenase. J. Biol. Chem., 276, 33265-33272 (2001)


 !    

8

   
 1.14.17.2 (deleted, included in EC 1.14.18.1)    4-coumarate 3-monooxygenase

139

$        

8!

   
 1.14.17.3    peptidylglycine,ascorbate:oxygen oxidoreductase (2-hydroxylating)    peptidylglycine monooxygenase    PAM PAM-A PAM-B a-AE ( peptidylglycine a-amidating enzyme [30]) [28, 30] peptide a-amidating enzyme peptide a-amide synthase peptide-a-amide synthetase peptidyl a-amidating enzyme peptidylglycine 2-hydroxylase peptidylglycine a-amidating mono-oxygenase [4] peptidylglycine a-amidating monooxygenase peptidylglycine a-hydroxylase synthase, peptide a-amide Additional information ( EC 1.14.17.3 is often called peptidylglycine a-amidating monooxygenase (PAM) and the a-amidated product is mentioned as the product of the reaction, but the a-amidation of glycine-extended peptides is a two-step process catalyzed by 2 enzymes: 1. EC 1.14.17.3: production of peptidyl(2-hydroxyglycine) by a copper, molecular oxygen and ascorbate-dependent peptidyl-glycine a-hydroxylating monooxygenase (PHM), 2. conversion of the peptidyl-a-hydroxyglycine derivative into an a-amidated product at physiological pH by peptidyl-a-hydroxyglycine aamidating lyase (PHL) [6, 8, 19, 22-29, 31]; at alkaline pH spontaneous conversion [19]; the 2-step process is catalyzed by only 1 enzyme called type A peptidylglycine a-amidating enzyme [10, 30, 32, 33]) [6, 8, 10, 19, 2233]
    90597-47-0

140

1.14.17.3

Peptidylglycine monooxygenase

     Rattus norvegicus (bifunctional a-AE [30, 32-35]; PAM-1 and PAM-2 [26]; 2 forms: type A and B [10]) [1, 5, 10, 11, 13, 26-28, 30, 32-35] Sus scrofa [2, 3, 16, 18] Equus caballus [19] Ovis aries [4] Bos taurus (2 forms: A and B [6, 9]) [6, 7, 9, 14, 17, 21, 31] Xenopus laevis (peptidylglycine a-hydroxylating monooxygenase activity [29]; 3 genetic forms, 2 protein forms of amidating enzyme: protein form 1, gene AE-I, has only peptidylglycine a-hydroxylating activity, protein form 2, genes AE-III and AE-II, show peptidylglycine a-hydroxylating activity and peptidylhydroxylglycine N-C lyase activity [22-24]) [8, 12, 15, 22-24, 29] Homo sapiens [20] Aplysia californica (marine mollusc [25]; 2 forms: a soluble and a membrane-bound [25]) [25]

!    "
 # 

peptidylglycine + ascorbate + O2 = peptidyl(2-hydroxyglycine) + dehydroascorbate + H2 O (A copper protein. Peptidylglycines with a neutral amino acid residue in the penultimate position are the best substrates for the enzyme. The product is unstable and dismutates to glyoxylate and the corresponding desglycine peptide amide, a reaction catalysed by EC 4.3.2.5 peptidylamidoglycolate lyase. Involved in the biosynthesis of a-melanotropin and related biologically active peptides.; structural analysis of mutant H172A, binding of ligands CO, Cu2+ [34]; structural analysis [32]; kinetic analysis [33]; equilibrium ordered mechanism [33]; sequential activity of 2 enzymes contained within a bifunctional protein [26, 33]; ping-pongmechanism [24, 29]; kinetic model for slow-binding inhibition [28]; mechanism [2, 3, 8, 17, 29]; structure analysis [19])    oxidation redox reaction reduction       peptidylglycine + ascorbate + O2 ( amidation of neurohormonal peptides [20]; COOH-terminal glycine [6]) (Reversibility: ? [1-33]) [1-33] $ peptidyl(2-hydroxyglycine) + dehydroascorbate + H2 O [1-33]

141

Peptidylglycine monooxygenase

1.14.17.3

     4-dimethylaminoazobenzene-4'-sulfonyl-Gly-l-Phe-Gly(dabsyl-Gly-PheGly) + ascorbate + O2 (Reversibility: ? [21]) [21] $ 4-dimethylaminoazobenzene-4'-sulfonyl-Gly-l-Phe-NH2 (dabsyl-Gly-Phe2-hydroxyglycine) + dehydroascorbate + H2 O [21]  Ala-Ile-Gly-Val-Gly-Ala-Pro-Gly + ascorbate + O2 (Reversibility: ? [8, 23]) [8, 23] $ Ala-Ile-Gly-Val-Gly-Ala-Pro-2-hydroxyglycine + dehydroascorbate + H2 O [8, 23]  d-Tyr-Pro-Gly-Gly + ascorbate + O2 ( optimal activity in sulfonic acid buffers [4]; synthetic peptide [4]) (Reversibility: ? [4]) [4] $ d-Tyr-Pro-Gly-2-hydroxyglycine + dehydroascorbate + H2 O [4]  d-Tyr-Val-Gly + ascorbate + O2 ( synthetic peptide [11, 18, 25]) (Reversibility: ? [1-4, 9, 11, 18, 25, 28, 29]) [1-4, 9, 11, 18, 25, 28, 29] $ d-Tyr-Val-2-hydroxyglycine + dehydroascorbate + H2 O [1-4, 9, 11, 18, 25, 28, 29]  N-(4-nitrobenzyl)glycine + ascorbate + O2 (Reversibility: ? [17]) [17] $ nitrobenzylamine + glyoxylate + dehydroascorbate + H2 O [17]  Phe-Gly-Phe-Gly + ascorbate + O2 (Reversibility: ? [19]) [19] $ Phe-Gly-Phe-2-hydroxyglycine + dehydroascorbate + H2 O [19]  [(4-nitrobenzyl)oxy]acetic acid + ascorbate + O2 (Reversibility: ? [17]) [17] $ nitrobenzyl alcohol + glyoxylate + dehydroascorbate + H2 O [17]  adrenocorticotrophic hormone(9-14) + ascorbate + O2 (Reversibility: ? [1]) [1] $ adrenocorticotrophic hormone(9-13)-2-hydroxyglycine + dehydroascorbate + H2 O [1]  a-N-acetyl-Tyr-Val-Gly + ascorbate + O2 (Reversibility: ? [6, 20, 26, 27, 32]) [6, 20, 26, 27, 32] $ a-N-acetyl-Tyr-Val-2-hydroxyglycine + dehydroascorbate + H2 O [6, 26, 27, 32]  a-N-acetyl-adrenocorticotrophic hormone(1-14) + ascorbate + O2 ( inhibitory substrate [11]) (Reversibility: r [11]; ? [1]) [1, 11] $ a-N-acetyl-adrenocorticotrophic hormone(1-13)-2-hydroxyglycine + dehydroascorbate + H2 O [1, 11]  dansyl-d-Tyr-Val-Gly + ascorbate + O2 ( stereospecific [10]) (Reversibility: ? [5, 7, 8, 10, 13, 14, 16, 22-24, 28, 30, 32]) [5, 7, 8, 10, 13, 14, 16, 22-24, 28, 30, 32] $ dansyl-d-Tyr-Val-2-hydroxyglycine + dehydroascorbate + H2 O [22-24, 28, 32]  glyoxylic acid phenylhydrazone + ascorbate + O2 ( competitive substrate, inhibitory [2]) (Reversibility: ? [2]) [2] $ oxalate phenylhydrazide + dehydroascorbate + H2 O [2] 142

1.14.17.3

Peptidylglycine monooxygenase

 hippuric acid + ascorbate + O2 ( i.e. N-benzoylglycine [33, 35]) (Reversibility: ? [33, 35]) [33, 35] $ a-hydroxyhippuric acid + dehydroascorbate + H2 O [33, 35]  monoiodo-d-Tyr-Val-Gly + ascorbate + O2 ( synthetic peptide [11]) (Reversibility: ? [11]) [11] $ monoiodo-d-Tyr-Val-2-hydroxyglycine + dehydroascorbate + H2 O [11]  monoiodo-a-N-acetyl-Tyr-Val-Gly + ascorbate + O2 (Reversibility: ? [26, 27, 34]) [26, 27, 34] $ monoiodo-a-N-acetyl-Tyr-Val-2-hydroxyglycine + dehydroascorbate + H2 O [26, 27, 34]  peptidylglycine + ascorbate + O2 ( diverse synthetic substrates with C-terminal glycine [12, 18]; requirement for O2 [11, 19, 24, 25, 32-34]; COOH-terminal glycine [1-34]) (Reversibility: ? [1-35]) [1-35] $ peptidyl(2-hydroxyglycine) + dehydroascorbate + H2 O ( semidehydroascorbate [28]) [1-35]  trinitrophenyl-d-Tyr-Val-Gly + ascorbate + O2 ( COOH-terminal glycine [7]) (Reversibility: ? [7]) [7] $ trinitrophenyl-d-Tyr-Val-2-hydroxyglycine + dehydroascorbate + H2 O  Additional information ( cleavage of C-H bond in the second reaction step is irreversible [35]; tunneling of hydrogen ion, relatively flexible [35]; acetyl-l-Phe-Gly, acetyl-l-Phe-l-Phe-Gly, or (S)-O-acetyl-mandelyl-Gly, and stereoisomers are a-hydroxylated but not a-amidated [31]; endorphins are inhibitory substrates [11]; substrates are also physiologically relevant peptides related to a-melanotropine [1-3]; specificity [11]; PAM catalyzes: 1. sulfoxidation of e.g. (4-nitrobenzyl) thioacetic acid to the analogous sulfoxide, 2. amine Ndealkylation of e.g. N-(4-nitrobenzyl)glycine to 4-nitrobenzylamine and glyoxylate, 3. O-dealkylation of e.g. [(4-nitrobenzyl)oxy]acetic acid to 4nitrobenzyl alcohol and glyoxylate, 4. transformation of hippuric acid and several ring-substituted derivatives to the corresponding benzoamides and glyoxylic acid [17]; EC 1.14.17.3 is often called peptidylglycine a-amidating monooxygenase (PAM) and the a-amidated product is mentioned as the product of the reaction, but the a-amidation of glycine-extended peptides is a two-step process catalyzed by 2 enzymes: 1. EC 1.14.17.3: production of peptidyl(2-hydroxyglycine) by a copper, molecular oxygen and ascorbate-dependent peptidyl-glycine a-hydroxylating monooxygenase (PMH) and 2. conversion of the peptidyl-a-hydroxyglycine derivative into an a-amidated product at physiological pH by peptidyl-a-hydroxyglycine a-amidating lyase [6, 8, 19, 22-29, 31]; at alkaline pH spontaneous conversion [19]) [1-3, 6, 8, 11, 17, 19, 22-29, 31, 35] $ ? 2  4-phenyl-3-butenoic acid ( mechanism-based inhibition [17]) [17] Cu2+ ( at higher concentration [1]) [1]

143

Peptidylglycine monooxygenase

1.14.17.3

d-Tyr-Pro-Gly-Gly ( inhibition of activity with d-Tyr-Val-Gly [4]) [4] d-Tyr-Val-Gly ( inhibition above 20 mM of hydroxylation [23]; at 2 mM 82% inhibition, at 0.1 mM 45% inhibition [2]; inhibition of activity with d-Tyr-Pro-Gly [4]) [2, 4, 23] EDTA ( recombinant enzyme, a-hydroxylation activity can be restored by Mn2+ , Zn2+ , Cd2+ , and Co2+, but not by Ca2+ , Cu2+ , Mg2+ , and Fe3+ [30]) [4, 8, 12, 30] N-carboxymethyl N'-phenylhydrazone ( weak inhibition [2]) [2] NaN3 [4] Ni2+ [25] Zn2+ [25] [(4-methoxybenzoyl)oxy]acetic acid [17] acetyl-d-Leu-OCH2 COOH [31] acetyl-d-Phe-Gly [31] acetyl-d-Phe-OCH2 COOH [31] acetyl-dl-Phe-OCH2 COOH [31] acetyl-Gly-OCH2 COOH [31] acetyl-l-Leu-OCH2 COOH [31] acetyl-l-Phe-d-Phe-Gly [31] acetyl-l-Phe-Gly ( only substrate for a-hydroxylation [31]) [31] acetyl-l-Phe-OCH2 COOH [31] adrenocorticotrophic hormone(1-10) [11] a-N-acetyl-adrenocorticotrophic hormone(1-14) [11] ascorbate ( above 0.4 mM for the purified enzyme, above 1.5 mM for the enzyme in crude extract [1]) [1] diethyldithiocarbamate ( copper chelator [1, 21]) [1, 9, 21] dithiothreitol [12] endorphin(51-61)NH2 ( i.e. pro-adrenocorticotrophic hormone(110) [11]) [11] endorphins ( and proendorphins [11]) [11] glyoxylate 2-pyridylhydrazone [2] glyoxylate 4-carboxyphenylhydrazone [2] glyoxylate benzylhydrazone [2] glyoxylate phenylhydrazone ( strongly [2]) [2] glyoxylate phenylsemicarbazone [2] glyoxylate phenylthiosemicarbazone [2] glyoxylate semicarbazone [2] glyoxylate-N-hexyl-4-carbamoylphenylhydrazone ( strongly [2]) [2] imidazole [18] monoethyl fumarate ( mechanism-based inhibition [17]) [17] p-hydroxymercuribenzoate ( slightly [4]) [4] peptides ( especially those with COOH-terminal glycine residues [11]) [11] phosphate [4] sulfite ( irreversible inactivation is Cu2+ -dependent [28]) [28] trans-benzoylacrylic acid ( mechanism-based inhibition [17]) [17] 144

1.14.17.3

Peptidylglycine monooxygenase

Additional information ( form 1, AE-I, competitive inhibition by tripeptides with C-terminal glycine, most effective is methione within these peptides [24]; activity is not affected by pepstatin A, phenylmethylsulfonyl fluoride, and soybean trypsin inhibitor [21]) [21, 24]
"%  FAD ( slight activity with [11]) [11] ascorbate ( dependent on [1-26, 28-30, 33]) [1-26, 28-35] &

  acetate ( form 1, AE-I, activates below pH 6.0 [24]) [24] chloride ( form 1, AE-I, activates below pH 6.0 [24]) [24] dexamethasone ( increases gene expression 1.6fold, northern blot analysis [27]) [27] iodide ( form 1, AE-I, activates below pH 6.0 [24]) [24] '(   Cu2+ ( mutant H172A has reduced copper content: below 0.3 Cu2+ per protein molecule [34]; Cu2+ to protein ration is 0.97 [32]; required for a-hydroxylation step [30, 33]; 2fold increase in activity [25]; optimal activity at 0.016 mM [21]; Cu2+ is absolutely required for optimal activity [1, 8-10, 12, 19, 21, 24, 28]; Cu2+ strongly stimulates activity [4, 11]; a copper protein [1, 4, 8, 9, 12]) [1, 4, 812, 19-30, 32-35] Zn2+ ( required for a-amidation step [30]) [30] ) &  * +, 1.2 (acetyl-Tyr-Phe-Gly) [15] 8.6 (dansyl-Tyr-Val-Gly) [14] 59 (dansyl-Tyr-Val-Gly) [16] 180 (N-benzoylglycine, 15 C [35]) [35] 300 (N-dansyl-Tyr-Val-Gly) [13] 870 (N-benzoylglycine, 37 C [35]) [35] " & *-% , 0.0000000004 ( H9c2 cells [27]) [27] 0.0033 ( a-amidating lyase activity [7]) [7] 0.0124 ( skin, peptidylhydroxylglycine N-C lyase, highest activity of all tissues [22]) [22] 0.0206 ( skin, peptidylglycine a-hydroxylating activity, highest activity of all tissues [22]) [22] 0.038 ( purified PAM-A [9]) [9] 0.115 ( purified PAM-B [9]) [9] 1.15 ( recombinant purified enzyme [5]) [5, 9] 2.07 ( purified enzyme [13]) [13] 5.9 ( purified recombinant enzyme [32]) [32] 16 ( purified recombinant enzyme [8]) [8] Additional information ( very low activity in H9c2 cells, no ascorbate in assay mixture [27]; peptidylglycine a-amidating and a-hy-

145

Peptidylglycine monooxygenase

1.14.17.3

droxylating monooxygenase activities in several tumor tissue extracts, comparison with immunological analysis, overview, highest activity in medullary thyroid carcinoma [20]; very low activity in crude subcellular preparations [4, 11]) [1, 4, 11, 12, 18, 20, 25, 27, 32] . / *', 0.00035 (acetyl-Tyr-Phe-Gly, in presence of 0.25 mM ascorbate [12]) [12] 0.003 (dansyl-Tyr-Val-Gly, recombinant enzyme [5]) [5] 0.0032 (N-dansyl-Tyr-Phe-Gly, + 2 mM l-ascorbate [8]) [8] 0.007 (d-Tyr-Val-Gly, PAM-B, with 1.25 mM ascorbate [9]) [9] 0.022 (4-dimethylaminoazobenzene-4'-sulfonyl-Gly-l-Phe-Gly(dabsylGly-Phe-Gly), form 2 [21]) [21] 0.033 (d-Tyr-Pro-Gly-Gly, assayed without ascorbate and catalase, brain enzyme [4]) [4] 0.068 (d-Tyr-Pro-Gly-Gly, assayed without ascorbate and catalase, pituitary enzyme [4]) [4] 0.075 (d-Tyr-Pro-Gly-Gly, assayed with ascorbate and 0.1mg/ml catalase, pituitary enzyme [4]) [4] 0.143 (4-dimethylaminoazobenzene-4'-sulfonyl-Gly-l-Phe-Gly(dabsylGly-Phe-Gly), form 1 [21]) [21] 0.28 (l-ascorbate, + 0.02 mM N-dansyl-Tyr-Phe-Gly [8]) [8] 0.289 (d-Tyr-Pro-Gly-Gly, assayed with ascorbate and 0.1 mg/ml catalase, brain enzyme [4]) [4] 1.39 (acetyl-Tyr-Phe-Gly) [18] 3.5 (dansyl-Tyr-Phe-Gly(OH), recombinant mutant AE-II, form 2 [23]; reaction product of step 1, substrate of step 2 [23]) [23] 4.5 (dansyl-Tyr-Phe-Gly, recombinant mutant AE-II, form 2 [23]) [23] Additional information ( different assay methods [10]; Km for d-Tyr-Val-Gly depends on concentration of ascorbate, Km for ascorbate is constant over a wide range of d-Tyr-Val-Gly concentration [1]) [1, 10, 17, 31] . / *', 0.0006 (endorphin(51-61)NH2 ) [11] 0.001 (4-phenyl-3-butenoic acid, strictly ascorbate dependent [17]) [17] 0.001 (a-N-acetyl-adrenocorticotrophic hormone(1-14)) [11] 0.002 (acetyl-l-Phe-Gly) [31] 0.005 (adrenocorticotrophic hormone (1-10)) [11] 0.015 (glyoxylate phenylhydrazone) [2] 0.045 (acetyl-l-Phe-OCH2 COOH) [31] 0.0598 (acetyl-l-Leu-OCH2 COOH) [31] 0.11 (glyoxylate thiosemicarbazone) [2] 0.16 (trans-benzoylacrylic acid) [17] 0.48 ([(4-methoxybenzoyl)oxy]acetic acid) [17] 0.522 (acetyl-dl-Phe-OCH2 COOH) [31] 146

1.14.17.3

Peptidylglycine monooxygenase

0.58 (acetyl-l-Phe-d-Phe-Gly) [31] 1.25 (acetyl-Gly-OCH2 COOH) [31] 1.3 (acetyl-d-Phe-Gly) [31] 1.3 (monoethyl fumarate) [17] 2.11 (acetyl-d-Leu-OCH2 COOH) [31] 2.25 (acetyl-d-Phe-OCH2 COOH) [31] 0  4-4.5 ( membrane-bound peptidylglycine-a-hydroxylating activity [26]) [26] 4.5-5 ( soluble peptidylglycine-a-hydroxylating activity [26]; recombinant enzyme [5]) [5, 26] 5 ( assay at [26]) [26] 5-5.5 [13] 5.4 ( assay at, recombinant enzyme [8]) [8] 5.5-6 [8] 6 ( MES buffer, recombinant from 1, AE-I [24]; 2 optima: at pH 6.0 and at pH 7.5 [21]) [21, 24] 6-7 [12, 15] 6-7.5 [25] 6.5-7 [11] 6.5-7.5 [16] 7 ( phosphate buffer, recombinant form 1, AE-I [24]; assay at, recombinant enzyme [5]) [5, 11, 24] 7-8 ( dependent on Cu2+ -concentration [4]) [4] 7.5 ( 2 optima: at pH 6.0 and at pH 7.5 [21]) [18, 21] 8.5 ( PAM-A [9]) [9, 14] 9-9.5 ( PAM-B [9]) [9] 0 

 4-9 ( recombinant enzyme [5]) [5] 6-7.5 ( about 70% of activity maximum at pH 6.0 and 7.5 [11]) [11] 7.5-10 ( pH 7.5: about 50% of activity maximum, pH 10: about 60% of activity maximum, PAM-B [9]) [9] )  *
, 30 ( assay at [8, 22]) [8, 22] 37 ( assay at [1-3, 5, 9, 12, 13, 21, 25, 26]) [1-3, 5, 9, 12, 13, 21, 25, 26] Additional information ( above 40 C, temperature dependence [35]) [35]

  #   ' 1  38000 ( PAM-B, heterogeneous in size, gel filtration [9]) [9] 39000 ( native wild-type enzyme [8]; SDS-PAGE, gel filtration [8, 12]) [8, 12]

147

Peptidylglycine monooxygenase

1.14.17.3

40000 ( gel fltration, SDS-PAGE [2]) [2] 43000 ( recombinant form B, gel filtration and SDS-PAGE [10]; recombinant wild-type enzyme, SDS-PAGE, gel filtration [8]) [8, 10] 49000 ( form 1, gel filtration [21]) [21] 50000 ( gel filtration [1]) [1] 54000 ( PAM-A, heterogeneous in size, gel filtration [9]) [9] 60000 ( gel filtration [3, 25]; shows reversible aggregation with MW of 100000 [3]) [3, 25] 69000 ( form 2, gel filtration [21]) [21] 75000 ( gel filtration and native PAGE [13]; recombinant form A, gel filtration and SDS-PAGE [10, 28]; recombinant from mouse cells, gel filtration [5]) [5, 10, 13, 28] 78000 ( recombinant mutant AE-II, form 2, gel filtration [23]) [23] 92000 ( gel filtration [18]) [18] Additional information ( PAM in particulate fraction of H9c2 cells contains 86, 76, and 46 kDa proteins, soluble PAM fraction contains 110, 86, and 46 kDa proteins [27]) [6, 26, 27]   ? ( x * 75000, SDS-PAGE [13]) [13] monomer ( 1 * 38000, PAM-B, heterogeneous in size, SDSPAGE [9]; 1 * 39000, SDS-PAGE [12]; 1 * 40000, SDS-PAGE [2]; 1 * 43000, type B, SDS-PAGE [10]; 1 * 48000, PAM-A, heterogeneous in size, SDS-PAGE [9]; 1 * 50000, membrane-bound form, SDS-PAGE [25]; 1 * 74000, recombinant mutant AE-II, form 2, SDS-PAGE [23]; 1 * 75000, SDS-PAGE [13]; 1 * 75000, type A, SDS-PAGE [10]; 1 * 75000, recombinant enzyme, SDS-PAGE [5]; 1 * 92000, SDS-PAGE [18]) [2, 5, 9, 10, 12, 13, 18, 23, 25] $   "

glycoprotein ( partially glycosylated, at Asn 660, recombinant enzyme from mouse cells [5]) [5]

 2 %$ %' %

% atrial gland [25] brain ( predominantly membrane-associated, bifunctional enzyme [22]) [22] bronchial cancer cell [20] ganglion ( abdominal and pleuropedal [25]) [25] gastric cancer cell [20] gastrinoma cell [20] heart ( myoblast, H9c2 cells [27]; ventricle [22]; atrium [18, 22]; predominantly membrane-associated, bifunctional enzyme [22]) [18, 22, 25, 27] hippocampus ( PAM-1 and PAM-2 [26]) [26] 148

1.14.17.3

Peptidylglycine monooxygenase

hypothalamus ( PAM-1 and PAM-2 [26]; 2 forms, distinguishable by molecular weight [21]) [4, 21, 26] intestinal cancer cell [20] medullary thyroid carcinoma cell ( transplantable [13]) [13, 20, 28, 30, 32-35] neuroendocrine tumor cell [20] pancreatic cancer cell [20] pituitary gland ( anterior [11]; neurointermediate [6,7]; anterior, intermediate and posterior [1]) [1-7, 9, 11, 14, 16, 18, 29, 31] serum [19] skin ( soluble enzyme with peptidylglycine a-hydroxylating activity, AE-I [24]; soluble enzyme with peptidylglycine a-hydroxylating activity or peptidylhydroxylglycine N-C lyase activity [22, 23]) [8, 12, 15, 22-24] Additional information ( tissue distribution, overview [22, 25]) [22, 25] 3#

cytoplasm ( soluble form [25]; peptidylglycine a-hydroxylating activity and peptidylhydroxylglycine N-C lyase activity [22]) [22, 25, 26] extracellular ( recombinant enzyme secreted into medium [28, 30, 33]) [28, 30, 33] membrane ( from neurosecretory vesicles [26]; recombinant bifunctional enzyme form 2, uncleaved in insect cells [22]) [22, 26] membrane ( integral membrane [26]; transmembranal protein [23]) [18, 20, 23, 26] membrane ( particulate form, except for abdominal ganglia [25]) [25] secretory granule ( synaptosomal and neurosecretory granules [4]; chromaffin granules [7, 20]) [1, 3, 4, 7, 9, 11, 20] Additional information ( distribution of membrane-bound and soluble peptidylglycine a-hydroxylating activity [26]) [26] $"

(mutant H172A of type A a-AE, recombinant from chinese hamster ovary cells [34]; type A a-AE, recombinant from chinese hamster ovary cells [28, 30, 32, 33]; type A and B [10]; recombinant from mouse cells [5, 10]; partially [1]) [1, 5, 10, 13, 28, 30, 32-34] [2, 18] [19] (peptidylglycine a-hydroxylating monooxygenase and a peptidyl-a-hydroxyglycine a-amidating lyase [6]; forms A and B differing in MW and charge [9]) [6, 9, 31] (truncated mutant form 2 from gene AE-II, recombinant from Spodoptera frugiperda cell expression system [23]; native wild-type and recombinant from Spodoptera frugiperda cell expression system [8, 29]) [8, 12, 15, 23, 29] (membrane-bound form from atrial gland [25]) [25]

149

Peptidylglycine monooxygenase

1.14.17.3


 

(medullary thyroid a-AE, type A, expression in chinese hamster ovary cells [28, 30, 32-34]; myoblast enzyme, expression in H9c2 cells [27]; medullary thyroid carcinoma enzyme, expression in mouse C 127 cells via bovine papilloma virus vector, amino acid and DNA sequence analysis [5]; truncated type A enzyme, expression in mouse C127 cells [10]) [5, 10, 27, 28, 30, 32-34] (expression in Spodoptera fugiperda cells via baculovirus vector of membrane-associated, bifunctional enzyme form 2, AE-II, wild-type and truncated mutant, N-terminal amino acid sequence analysis [23]; expression in Spodoptera fugiperda cells via baculovirus vector of membrane-associated, bifunctional enzyme form 2, AE-III [22]; expression in Spodoptera fugiperda cells via baculovirus vector of soluble form 1, AE-I showing only peptidylglycine a-hydroxylating activity [24]; expression in Spodoptera fugiperda cells via baculovirus vector, amino acid sequence analysis [8, 29]) [8, 22-24, 29] 

 

H172A ( mutant of copper ligand of peptidylglycine a-hydroxylating enzyme, reduced copper content below 0.3 Cu2+ per protein molecule [34]) [34] Additional information ( construction of a truncated form of AE-II, lacking the transmembrane domain and leading to solubility of the fully active, truncated enzyme being secreted into the culture medium from Spodoptera frugiperda cells [23]) [23]

4  0  Additional information ( rapid inactivation of recombinant enzyme at acidic pH in vitro [5]) [5] )  4 ( incubation for 12-15 h, 47% loss of activity [1]) [1] 5    "

, Cu2+ can restore EDTA-treated only the a-hydroxylating activity [30] , repeated freeze-thaw cycles have no effect on enzyme activity [1] , substance-P, i.e. RPKPQQFFGLM-NH2 , protects against inactivation by sulfite [28] , stabilized by salts like KI and KCl, recombinant form 1, AE-I [24]

"  [1] Glembotski, C.C.: Further characterization of the peptidyl a-amidating enzyme in rat anterior pituitary secretory granules. Arch. Biochem. Biophys., 241, 673-683 (1985)

150

1.14.17.3

Peptidylglycine monooxygenase

[2] Bradbury, A.F.; Smyth, D.G.: Enzyme-catalysed peptide amidation. Isolation of a stable intermediate formed by reaction of the amidating enzyme with an imino acid. Eur. J. Biochem., 169, 579-584 (1987) [3] Bradbury, A.F.; Finnie, M.D.A.; Smyth, D.G.: Mechanism of C-terminal amide formation by pituitary enzymes. Nature, 298, 686-689 (1982) [4] Gale, J.S.; McIntosh, J.E.A.; McIntosh, R.P.: Peptidyl-glycine a-amidating mono-oxygenase activity towards a gonadotropin-releasing-hormone Cterminal peptide substrate, in subcellular fractions of sheep brain and pituitary. Biochem. J., 251, 251-259 (1988) [5] Beaudry, G.A.; Mehta, N.M.; Ray, M.I.; Bertelsen, A.H.: Purification and characterization of functional recombinant a-amidating enzyme secreted from mammalian cells. J. Biol. Chem., 265, 17694-17699 (1990) [6] Perkins, S.N.; Husten, E.J.; Eipper, B.A.: The 108-kDA peptidylglycine aamidating monooxygenase precursor contains two separable enzymatic activities involved in peptide amidation. Biochem. Biophys. Res. Commun., 171, 926-932 (1990) [7] Katapodis, A.G.; May, S.W.: A new facile trinitrophenylated substrate for peptide a-amidation and its use to characterize PAM activity in chromaffin granules. Biochem. Biophys. Res. Commun., 151, 499-505 (1988) [8] Suzuki, K.; Shimoi, H.; Iwasaki, Y.; Kawahara, T.; Matsuura, Y.; Nishikawa, Y.: Elucidation of amidating reaction mechanism by frog amidating enzyme, peptidylglycine a-hydroxylating monooxygenase, expressed in insect cell culture. EMBO J., 9, 4259-4265 (1990) [9] Murthy, A.S.N.; Mains, R.E.; Eipper, B.A.: Purification and characterization of peptidylglycine a-amidating monooxygenase from bovine neurointermediate pituitary. J. Biol. Chem., 261, 1815-1822 (1986) [10] Merkler, D.J.; Young, S.D.: Recombinant type A rat 75-kDa a-amidating enzyme catalyzes the conversion of glycine-extended peptides to peptide amides via an a-hydroxyglycine intermediate. Arch. Biochem. Biophys., 289, 192-196 (1991) [11] Glembotski, C.C.; Eipper, B.A.; Mains, R.E.: Characterization of a peptide aamidation activity from rat anterior pituitary. J. Biol. Chem., 259, 63856392 (1984) [12] Mizuno, K.; Sakata, J.; Kojima, M.; Kangawa, K.; Matsuo, H.: Peptide Cterminal a-amidating enzyme purified to homogeneity from Xenopus laevis skin. Biochem. Biophys. Res. Commun., 137, 984-991 (1986) [13] Mehta, N.M.; Gilligan, J.P.; Jones, B.N.; Bertelsen, A.H.; Roos, B.A.; Birnbaum, R.S.: Purification of a peptidylglycine a-amidating enzyme from transplantable rat medullary thyroid carcinomas. Arch. Biochem. Biophys., 261, 44-54 (1988) [14] Bradbury, A.F.; Smyth, D.G.: C-Terminal amide formation in peptide hormones. Biogenetics of Neurohormonal Peptides (Hakanson, R., Thorell, J., eds) Academic Press, London, 171-186 (1985) [15] Bendig, M.M.: Post-translational processing in Xenopus oocytes includes carboxyl-terminal amidation. J. Biol. Chem., 261, 11935-11937 (1986)

151

Peptidylglycine monooxygenase

1.14.17.3

[16] Sakata, J.; Mizuno, J.; Matsuo, H.: Tissue distribution and characterization of peptide C-terminal a-amidating activity in rat. Biochem. Biophys. Res. Commun., 140, 230-236 (1986) [17] Katapodis, A.G.; May, S.W.: Novel substrates and inhibitors of peptidylglycine a-amidating monooxygenase. Biochemistry, 29, 4541-4548 (1990) [18] Kojima, M.; Mizuno, K.; Kangawa, K.; Matsuo, H.: Purification and characterization of a peptide C-terminal a-amidating enzyme from porcine atrium. J. Biochem., 105, 440-443 (1989) [19] Tajima, M.; Iida, T.; Yoshida, S.; Komatsu, K.; Namba, R.; Yanagi, M.; Noguchi, M.; Okamoto, H.: The reaction product of peptidylglycine a-amidating enzyme is a hydroxyl derivative at a-carbon of the carboxyl-terminal glycine. J. Biol. Chem., 265, 9602-9605 (1990) [20] Scopsi, L.; Lee, R.; Gullo, M.; Collini, P.; Husten, E.J.; Eipper, B.A.: Peptidylglycine a-amidating monooxygenase in neuroendocrine tumors: its identification, characterization, quantification, and relation to the grade of morphologic differentiation, amidated peptide content, and granin immunocytochemistry. Appl. Immunohistochem., 6, 120-132 (1998) [21] Chikuma, T.; Kocha, T.; Hanaoka, K.; Kato, T.; Ishii, Y.; Tanaka, A.: Characterization of peptidylglycine a-amidating monooxygenase in bovine hypothalamus. Neurochem. Int., 25, 349-354 (1994) [22] Iwasaki, Y.; Shimoi, H.; Saiki, H.; Nishikawa, Y.: Tissue-specific molecular diversity of amidating enzymes (peptidylglycine a-hydroxylating monooxygenase and peptidylhydroxyglycine N-C lyase) in Xenopus laevis. Eur. J. Biochem., 214, 811-818 (1993) [23] Suzuki, K.; Ohta, M.; Okamoto, M.; Nishikawa, Y.: Functional expression and characterization of a Xenopus laevis peptidylglycine a-amidating monooxygenase, AE-II, in insect-cell culture. Eur. J. Biochem., 213, 93-98 (1993) [24] Shimoi, H.; Kawahara, T.; Suzuki, K.; Iwasaki, Y.; Jeng, A.Y.; Nishikawa, Y.: Characterization of a Xenopus laevis skin peptidylglycine a-hydroxylating monooxygenase expressed in insect-cell culture. Eur. J. Biochem., 209, 189194 (1992) [25] Boudreault, A.; Castellucci, V.F.; Chretien, M.; Lazure, C.: Identification, purification, and characterization of the molecular forms of Aplysia californica peptidylglycine a-amidating enzyme. J. Neurochem., 66, 2596-2605 (1996) [26] Oyarce, A.M.; Eipper, B.A.: Neurosecretory vesicles contain soluble and membrane-associated monofunctional and bifunctional peptidylglycine aamidating monoxygenase proteins. J. Neurochem., 60, 1105-1114 (1993) [27] Girard, B.; Ouafik, L.; Boudouresque, F.: Characterization and regulation of peptidylglycine a-amidating monooxygenase (PAM) expression in H9c2 cardiac myoblasts. Cell Tissue Res., 298, 489-497 (1999) [28] Merkler, D.J.; Kulathila, R.; Francisco, W.A.; Ash, D.E.; Bell, J.: The irreversible inactivation of two copper-dependent monooxygenases by sulfite: peptidylglycine a-amidating enzyme and dopamine b-monooxygenase. FEBS Lett., 366, 165-169 (1995)

152

1.14.17.3

Peptidylglycine monooxygenase

[29] Takahashi, K.; Onami, T.; Noguchi, M.: Kinetic isotope effects of peptidylglycine a-hydroxylating mono-oxygenase reaction. Biochem. J., 336, 131137 (1998) [30] Bell, J.; Ash, D.E.; Snyder, L.M.; Kulathila, R.; Blackburn, N.J.; Merkler, D.J.: Structural and functional investigations on the role of zinc in bifunctional rat peptidylglycine a-amidating enzyme. Biochemistry, 36, 16239-16246 (1997) [31] Ping, D.; Mounier, C.E.; May, S.W.: Reaction versus subsite stereospecificity of peptidylglycine a-monooxygenase and peptidylamidoglycolate lyase, the two enzymes involved in peptide amidation. J. Biol. Chem., 270, 2925029255 (1995) [32] Jaron, S.; Blackburn, N.J.: Characterization of a half-apo derivative of peptidylglycine monooxygenase. Insight into the reactivity of each active site copper. Biochemistry, 40, 6867-6875 (2001) [33] Francisco, W.A.; Merkler, D.J.; Blackburn, N.J.; Klinman, J.P.: Kinetic mechanism and intrinsic isotope effects for the peptidylglycine a-amidating enzyme reaction. Biochemistry, 37, 8244-8252 (1998) [34] Jaron, S.; Mains, R.E.; Eipper, B.A.; Blackburn, N.J.: The catalytic role of the copper ligand H172 of peptidylglycine a-hydroxylating monooxygenase (PHM): a spectroscopic study of the H172A mutant. Biochemistry, 41, 13274-13282 (2002) [35] Francisco, W.A.; Knapp, M.J.; Blackburn, N.J.; Klinman, J.P.: Hydrogen tunneling in peptidylglycine a-hydroxylating monooxygenase. J. Am. Chem. Soc., 124, 8194-8195 (2002)

153

     

8

   
 1.14.17.4    1-aminocyclopropane-1-carboxylate oxygenase (ethylene-forming)    aminocyclopropanecarboxylate oxidase    ACC oxidase ethylene-forming enzyme
    98668-53-2

     Lycopersicon esculentum (expressed in E. coli, purified and characterized [1]) [1, 2]

!    "
 # 

1-aminocyclopropane-1-carboxylate + ascorbate + O2 = ethylene + cyanide + dehydroascorbate + CO2 + 2 H2 O    oxidation redox reaction reduction       1-aminocyclopropane-1-carboxylate + ascorbate + O2 (Reversibility: ? [1, 2]) [1, 2] $ ethylene + cyanide + dehydroascorbate + CO2 + H2 O      1-aminocyclopropane-1-carboxylate + ascorbate + O2 (Reversibility: ? [1, 2]) [1, 2] $ ethylene + cyanide + dehydroascorbate + CO2 + H2 O 154

1.14.17.4

Aminocyclopropanecarboxylate oxidase

 2 %$ %' %

$"

(expressed in E. coli [1]) [1]
 

(expressed in E. coli [1]) [1]

"  [1] Zhang, Z.H.; Schofield, C.J.; Baldwin, J.E.; Thomas, P.; John, P.: Expression, purification and characterization of 1-aminocyclopropane-1-carboxylate oxidase from tomato in Escherichia coli. Biochem. J., 307, 77-85 (1995) [2] Zhang, Z.H.; Barlow, J.N.; Baldwin, J.E.; Schofield, C.J.: Metal-catalyzed oxidation and mutagenesis studies on the iron(II) binding site of 1-aminocyclopropane-1-carboxylate oxidase. Biochemistry, 36, 15999-16007 (1997) [3] Pirrung, M.C.: Ethylene biosynthesis from 1-aminocyclopropanecarboxylic acid. Acc. Chem. Res., 32, 711-718 (1999) [4] Charng, Y.; Chou, S.J.; Jiaang, W.T.; Chen, S.T.; Yang, S.F.: The catalytic mechanism of 1-aminocyclopropane-1-carboxylic acid oxidase. Arch. Biochem. Biophys., 385, 179-185 (2001) [5] Thrower, J.S.; Blalock, R.; Klinman, J.P.: Steady-state kinetics of substrate binding and iron release in tomato ACC oxidase. Biochemistry, 40, 97179724 (2001)

155

'      

7

   
 1.14.18.1    monophenol,l-dopa:oxygen oxidoreductase    monophenol monooxygenase    EC 1.10.3.1 (oxidation of o-diphenols to benzoquinones referred to as catecholase activity and hydroxylation of monophenols to o-diphenols referred to as cresolase activity are intrinsic properties of EC 1.14.18.1, accordingly a separation between both enzymes is imposssible) N-acetyl-6-hydroxytryptophan oxidase catechol oxidase catecholase chlorogenic acid oxidase chlorogenic oxidase cresolase diphenol oxidase dopa oxidase monophenol dihydroxyphenylalanine:oxygen oxidoreductase monophenol monooxidase monophenol oxidase monophenol, dihydroxy-l-phenylalanine oxygen oxidoreductase monophenolase o-diphenol oxidase o-diphenol oxidoreductase o-diphenol:O2 oxidoreductase o-diphenol:oxygen oxidoreductase o-diphenolase phenol oxidase phenolase polyaromatic oxidase polyphenol oxidase polyphenolase pyrocatechol oxidase tyrosinase tyrosine-dopa oxidase

156

1.14.18.1

Monophenol monooxygenase


    9002-10-2 (not distinguished from EC 1.10.3.1)

     Malus sp. (apple, var. red delicious [1, 76]) [1, 28, 47, 76] Aspergillus nidulans [3] marine bacterium (strain 2-40 isolated from a salt marsh grass, related to Alteromonas [2]) [2] Mus musculus (B16 melanoma cells [4,5,20,25,90]; isoenzymes T1, T2, T3 and T4 [25]; high-mobility- and low-mobility tyrosinase [90]) [4, 5, 20, 22, 25, 62, 63, 67, 81-83, 90, 93] Musca domestica (housefly [6]) [6, 18, 55, 60] mushroom (obtained from Sigma [7, 16]; 4 isoenzymes [80]) [7, 16, 34, 54, 61, 80, 97, 104, 106, 112] Mycobacterium avium-intracellulare (isolated from armadillo [8]) [8] Berberis stolonifera [9] Pleurotus ostreatus [10, 91] Tremella fuciformis [10] Lentinus edodes (6 isoenzymes [94]) [10, 94] Phanerochaete chrysosporium [10] Vanilla planifolia (probably 3 isoenzymes [115]) [115] Coriolus versicolor (white-rot basidomycete [11]) [10, 11] Spinacia oleracea [12, 17, 27] Neurospora crassa (enzyme is synthesized as a precursur [13]) [13, 26, 41, 51, 61, 52, 57, 78] Sarcophaga bullata [14, 105] Daucus carota (carrot [15]) [15, 33] Heterometrus bengalensis (scorpion [19]) [19] mammalia [25, 80, 113] Metroxylon sagus (sago palm, isoenzymes I and III [21]) [21] Vitis vinifera (monastrell grape [23]) [23, 24, 59] Vicia faba (c.v. long pod, broad bean [27]) [27, 49, 61, 84, 86] Phaseolus lunatus (bush bean [27]) [27] Lactuca sativa (iceberg lettuce [27]) [27, 110] Vigna radiata (mung bean [27]) [27, 85] Pisum sativum (pea [27]) [27] Glycine max (soybean [27]) [27] Nicotiana tabacum (tobacco) [27, 33] Lycopersicon esculentum (tomato [27]) [27, 65] Xenopus laevis [29, 32] Streptomyces antibioticus [30, 32, 113] Pyrus communis (d'anjou pear, 3 isoenzymes [31]) [31, 36] Streptomyces sp. (KY-453, isolated from soil [35]; REN-21, organic solvent resistant enzyme [106]) [35, 106] Dioscorea bulbifera (yam tubers [37]) [37, 38] 157

Monophenol monooxygenase























1.14.18.1

Mesocricetus auratus (tyrosinases A and B [39]) [39, 77] Gallus gallus (expressed in retinal pigment epithelium [95]) [42, 95] Mucuna pruriens [40] Populus nigra (black poplar [45]) [45] Papaver somniferum (opium, isoenzymes A and B [43]) [43] Rana esculenta ridibunda (frog [44]) [44] Triticum aestivum (wheat [46]) [46] Dioscorea rotundata (white yam from Nigeria [48]) [48] Homo sapiens [50, 66, 87, 103, 109, 111] Streptomyces glaucescens (intra- and extracellular enzymes are identical [53]) [53] Solanum melongena (eggplant [56]) [56, 107] Persea americana (avocado [58]) [58, 96] Theobroma cacao (cacao [64]) [64] Olea europaea (green olive [68]) [68] Polyporus dichrous [69] Musa cavendishii (banana [70]) [70] Calliphora erythrocephala (blowfly [71]) [71] Rana pipiens (frog, 2 probably identical enzymes [72]) [72] Vibrio tyrosinaticus (2 isoenzymes [73]) [73] Camellia sinensis (tea [75]) [75] Agaricus bisporus (white button mushroom, enzyme protects a human lymphoma cell line, Raji cells, against H2 O2 -induced oxidative damage to cellular DNA [114]; a-, b-, g-, and d-tyrosinase [79]) [79, 80, 88, 92, 100, 101, 114] Agaricus hortensis (mushroom [74]) [74] Haplopappus gracilis [33] Beta vulgaris (spinach-beet [89]) [89] Halocynthia roretzi [98] Pinus densiflora (3 isoenzymes [99]) [99] Prunus armeniaca (apricot, var Bergeron [102]) [102] Manduca sexta [105] Thermomicrobium roseum (ATCC 27502 [108]) [108] Solanum tuberosum (potato [112]) [112] insecta (enzyme is involved in sclerotisation, i.e., the hardening of the chitinous cuticle, and defense [113]) [113]

!    "
 # 

l-tyrosine + l-dopa + O2 = l-dopa + dopaquinone + H2 O ( monophenol + O2 = o-diphenol + H2 O, i.e. cresolase activity, proposed mechanism for sequence of hydroxylase and catecholase [89]; 2 o-diphenol + O2 = 2 oquinone + 2 H2 O i.e. catecholase activity, proposed mechanism for sequence of hydroxylase and catecholase [89]; proposed mechanism of hydroxylation and oxidation of phenols [41]; proposed interaction of the oxy 158

1.14.18.1

Monophenol monooxygenase

site effective structure with monophenolic and diphenolic substrates [57]; reaction mechanism of monophenolase and diphenolase activity [101]; proposed structural reaction mechanism for the monophenolase and diphenolase activity [104])    oxidation redox reaction reduction       l-3,4-dihydroxyphenylalanine + O2 (Reversibility: ? [5, 25]) [5, 25] $ l-dopaquinone + H2 O ( melanin precursur [5, 25]) [5, 25]  N-acetyl-6-hydroxytryptophan (Reversibility: ? [3]) [3] $ N-acetyltryptophan-6-one + H2 O [3]  tyrosine ( rate-limiting enzyme in melanin biosynthesis [5]; enzyme may be involved in reticuline synthesis [9]; enzyme initiates the formation of pigmentation, absence leads to forms of albinism [113]) (Reversibility: ? [5, 9, 25, 77]) [5, 9, 22, 25, 77, 113] $ l-3,4-dihydroxyphenylalanine + H2 O ( trivial name ldopa [5]) [5, 9, 22, 25, 77, 113]      (R)-coclaurine + O2 (Reversibility: ? [9]) [9] $ (R)-3-hydroxycoclaurine + H2 O [9]  (RS)-catechin + O2 ( isoenzymes 1-3, 96, 104, and 168% of activity with l-dopa respectively [99]; 83% of activity with l-dopa [108]) (Reversibility: ? [99, 108]) [99, 108] $ ?  (S)-coclaurine + O2 (Reversibility: ? [9]) [9] $ (S)-3-hydroxycoclaurine + H2 O [9]  2,4,5-trihydroxyphenethylamine + O2 (Reversibility: ? [67]) [67] $ ?  3,4,5-trihydroxybenzoic + O2 ( trivial name gallic acid [11,33]) (Reversibility: ? [11, 33]) [11, 33] $ ?  3,4,6-trihydroxyphenylalanine + O2 (Reversibility: ? [67]) [67] $ ?  3,4-dihydroxyanisol + O2 (Reversibility: ? [96]) [96] $ anisol o-quinone + H2 O [96]  3,4-dihydroxybenzoic acid + O2 ( isoenzymes 1-3, 22, 13, and 13% of l-dopa activity respectively [99]) (Reversibility: ? [99]) [99] $ benzoic acid o-quinone + H2 O [99]  3,4-dihydroxyphenethylamine + O2 (Reversibility: ? [67]) [67] $ ?

159

Monophenol monooxygenase

1.14.18.1

 3,4-dihydroxyphenylalanine methyl ester + O2 (Reversibility: ? [67]) [67] $ ?  3,4-dihydroxyphenylserine + O2 (Reversibility: ? [67]) [67] $ ?  3-(3,4-dihydroxyphenyl)-2-methylalanine + O2 (Reversibility: ? [67]) [67] $ ?  3-(4-hydroxyphenyl)propionic acid + O2 (Reversibility: ? [115]) [115] $ 3-(3,4-dihydroxyphenyl)propionic acid + H2 O [115]  3-hydroxyanthranilic acid + O2 (Reversibility: ? [100]) [100] $ cinnabarinic acid + H2 O [100]  3-hydroxyphloridzin + O2 (Reversibility: ? [47]) [47] $ ?  3-methylcatechol + O2 (Reversibility: ? [64]) [64] $ 3-methyl-o-benzoquinone + H2 O [64]  4-hydroxyanisol + O2 (Reversibility: ? [96]) [96] $ 3,4-dihydoxyanisol + H2 O [96]  4-hydroxybenzaldehyde + O2 (Reversibility: ? [115]) [115] $ 3,4-dihydroxybenzaldehyde + H2 O [115]  4-hydroxybenzoic acid + O2 ( 50% of activity with l-dopa [108]) (Reversibility: ? [108, 115]) [108, 115] $ 3,4-dihydroxybenzoic acid + H2 O [108, 115]  4-hydroxybenzyl alcohol + O2 (Reversibility: ? [115]) [115] $ 3,4-dihydroxybenzyl alcohol + H2 O [115]  4-methylcatechol + O2 ( no activity with tyrosine [21]; catecholase/cresolase activity ratio of 41 [107]) (Reversibility: ? [1-25, 33, 45, 46, 58, 68, 107, 115]) [1-25, 33, 45, 46, 58, 68, 107, 115] $ 4-methyl-o-benzoquinone + H2 O [125, 33, 45, 46, 58, 68, 107, 115]  4-nitrocatechol + O2 (Reversibility: ? [12]) [12] $ ?  4-tert-butylphenol + O2 (Reversibility: ? [100]) [100] $ 4-tert-butyl benzoquinone + H2 O [100]  d-tyrosine + O2 (Reversibility: ? [2]) [2] $ d-dopa + H2 O [2]  dl-epicatechin + O2 ( 91% of activity with l-dopa [108]) (Reversibility: ? [21, 108]) [21, 108] $ ?  l-adrenaline + O2 (Reversibility: ? [64]) [64] $ ?  l-epicatechin + O2 ( isoenzyme 1, 150% of ldopa activity, isoenzyme 2 and 3, 170 and 175% of activity with l-dopa respectively [99]; 155% of activity with l-dopa [106]) (Reversibility: ? [21, 47, 64, 99, 106]) [21, 47, 64, 99, 106] 160

1.14.18.1

Monophenol monooxygenase

$ ?  l-tyrosine + O2 ( 10% of activity with l-dopa [108]) (Reversibility: ? [9, 35, 40, 108]) [9, 35, 40, 108] $ l-dopa + H2 O [9, 35, 40, 108]  N-acetyl-3,4-dihydroxyphenethylamine + O2 (Reversibility: ? [67]) [67] $ ?  N-acetyl-6-hydroxytryptophan + O2 (Reversibility: ? [3]) [3] $ N-acetyltryptophan-6-one + H2 O [3]  N-acetyl-l-tyrosine + O2 (Reversibility: ? [100]) [100] $ N-acetyl-dopaquinone + H2 O [100]  N-acetyldopamine + O2 ( enzyme has both o-diphenoloxidase and N-acetyldopamine quinone:N-acetyldopamine quinone methide isomerase activity [14]) (Reversibility: ? [14, 18]) [14, 18] $ N-acetyldopamine quinone + H2 O [14, 18]  N-acetyldopamine quinone + O2 ( enzyme has both o-diphenoloxidase and N-acetyldopamine quinone:N-acetyldopamine quinone methide isomerase activity [14]) (Reversibility: ? [14]) [14] $ 1,2-dehydro-N-acetyldopamine + H2 O [14]  N-b-alanyldopamine + O2 (Reversibility: ? [18]) [18] $ N-b-alanyldopamine quinone + H2 O [18]  N-formyl-l-tyrosine + O2 (Reversibility: ? [40]) [40] $ ?  N-methyl-3,4-dihydroxyphenethylamine + O2 (Reversibility: ? [67]) [67] $ ?  N-methylcoclaurine + O2 (Reversibility: ? [9]) [9] $ 3-hydroxy-N-methylcoclaurine + H2 O [9]  adrenaline bitartrate + O2 (Reversibility: ? [40]) [40] $ ?  a-methyl-dopa + O2 (Reversibility: ? [40]) [40] $ a-methyldopaquinone + H2 O [40]  caffeic acid + O2 ( 17% of activity with ldopa [108]) (Reversibility: ? [12, 45, 47, 89, 99, 108, 115]) [12, 45, 47, 89, 99, 108, 115] $ caffeoyl o-quinone [45, 47, 89, 99, 108, 115]  catechol + O2 ( no activity with p-cresol and tyrosine [43]; no activity with tyrosine [45]; 125% of activity with l-dopa [108]) (Reversibility: ? [3, 11, 12, 21, 28, 33, 37, 38, 43, 45, 48, 56, 64, 68, 99, 108]) [3, 11, 12, 21, 28, 33, 37, 38, 43, 45, 48, 56, 64, 68, 99, 108] $ o-benzoquinone + H2 O [3, 11, 12, 21, 28, 33, 37, 38, 43, 45, 48, 56, 64, 68, 99, 108]  chlorogenic acid + O2 ( 108% of activity with l-dopa [108]) (Reversibility: ? [1, 3, 12, 21, 33, 48, 68, 99, 108, 115]) [1, 3, 12, 21, 33, 48, 68, 99, 108, 115] 3-[3-(3,4-benzoquinone)1-oxo-2-propenyl]-1,4,5-trihydroxycyclohexanecarboxylic acid + H2 O [1, 3, 12, 21, 33, 48, 68, 99, 108, 115] d-catechin + O2 ( isoenzymes 1, 2 and 3, 90, 86 and 188% of l-dopa activity respectively [99]; 67% of activity with l-dopa [108]) (Reversibility: ? [3, 21, 47, 64, 68, 99, 108]) [3, 21, 47, 64, 68, 99, 108] ? dopa + O2 ( ddopa [40]; l-dopa [2, 5, 40]; no activity with p-cresol and l-tyrosine [38]) (Reversibility: ? [2, 5, 6, 8, 12, 13, 15, 18, 33, 35, 38, 40, 48, 53, 56, 58, 99, 108, 115]) [2, 5, 6, 8, 12, 15, 18, 33, 35, 38, 40, 48, 53, 56, 58, 99, 108, 115] dopaquinone + H2 O [2, 5, 6, 8, 12, 15, 18, 33, 35, 38, 40, 48, 53, 56, 58, 99, 108, 115] dopamine + O2 ( preferred substrates in terms of affinity in descending order: N-b-alanyldopamine, dopamine, N-acetyldopamine, norepnephrine, epinephrine, dopa [18]) (Reversibility: ? [18, 40, 58, 68]) [18, 40, 58, 68] dopamine quinone + H2 O [18, 40, 58, 68] epicatechin gallate + O2 (Reversibility: ? [75]) [75] ? epigallocatechin gallate + O2 (Reversibility: ? [75]) [75] ? epinephrine + O2 (Reversibility: ? [18]) [18] ? esculetin + O2 (Reversibility: ? [12]) [12] ? g-l-glutaminyl-3,4-dihydroxybenzene + O2 (Reversibility: ? [101]) [101] g-l-glutaminyl-3,4-benzoquinone + H2 O [101] g-l-glutaminyl-4-hydroxybenzene + O2 (Reversibility: ? [101]) [101] g-l-glutaminyl-3,4-dihydroxybenzene + H2 O [101] hydrocaffeic acid + O2 (Reversibility: ? [12]) [12, 115] ? hydroquinone monomethylether + O2 (Reversibility: ? [3]) [3] quinone monomethylether [3] noradrenaline + O2 (Reversibility: ? [40]) [40] ? norepinephrine + O2 (Reversibility: ? [18]) [18] ? o-coumaric acid + O2 ( 58% of activity with l-dopa [108]) (Reversibility: ? [108]) [108] ?

1.14.18.1

Monophenol monooxygenase

 o-methoxyphenol + O2 ( trivial name guaiacol [3,10]) (Reversibility: ? [3, 10]) [3, 10] $ 1,2-dihydroxy-3-methoxybenzene [3, 10]  orcin + O2 ( isoenzymes 1-3, 41, 33, and 25% of activity with l-dopa respectively [99]; 42% of activity with l-dopa [108]) (Reversibility: ? [99, 108]) [99, 108] $ ?  p-coumaric acid + O2 ( artificial electron donors: NADH, dimethyltetrahydropteridine and ascorbic acid [89]) (Reversibility: ? [45, 47, 89, 99, 115]) [45, 47, 89, 99, 115] $ caffeic acid + H2 O [45, 47, 89, 99, 115]  p-cresol + O2 (Reversibility: ? [17, 24, 58]) [17, 24, 58] $ 4-methylpyrocatechol + H2 O [17, 24, 58]  phenol + O2 (Reversibility: ? [35]) [35] $ catechol + H2 O [35]  phenol + O2 (Reversibility: ? [35]) [35] $ o-dihydroxybenzene [35]  phloridzin + O2 (Reversibility: ? [47]) [47] $ ?  phloroglucin + O2 ( 87% of activity with l-dopa [108]) (Reversibility: ? [108]) [108] $ ?  protocatechuic acid + O2 (Reversibility: ? [12, 68, 115]) [12, 68, 115] $ ?  protocatechuic aldehyde + O2 (Reversibility: ? [12, 115]) [12, 115] $ ?  pyrogallol + O2 (Reversibility: ? [35, 37, 38, 48, 64]) [35, 37, 38, 48, 64] $ ?  pyrogallol + O2 ( isoenzymes 1-3, 210, 263, and 225% of activity with l-dopa respectively [99]; 417% of activity with l-dopa [108]) (Reversibility: ? [99, 108]) [99, 108] $ ?  quinol + O2 (Reversibility: ? [64]) [64] $ quinone + H2 O [64]  resorcinol + O2 ( isoenzymes 1-3, 96, 129, and 100% of activity with l-dopa respectively [99]; 67% of activity with l-dopa [108]) (Reversibility: ? [99, 108]) [99, 108] $ ?  syringic acid + O2 (Reversibility: ? [11]) [11] $ ?  tyramine + O2 ( isoenzymes 1, 20% of l-dopa activity, isoenzyme 2, 13% of activity with l-dopa, isoenzyme 3, 25% of activ-

163

Monophenol monooxygenase

1.14.18.1

ity with l-dopa [99]; 67% of activity with l-dopa [108]) (Reversibility: ? [9, 40, 99, 108]) [9, 40, 99, 108] $ 3,4-dihydroxyphenylethylamine [9, 40, 99, 108] 2  1,10-phenanthroline ( 1 mM, inactivation, half-life: 30 min [45]) [45] 1-phenyl-2-thiourea ( 1 mM, 94% inhibition [3]) [3, 26, 78, 90, 98] 2,3-dimercapto-1-propanol ( 2 mM, 93% inhibition [56]) [56] 2,4,6-cycloheptatriene-1-one ( copper chelator, trivial name tropolone, mixed inhibition, 90% reversible by dialysis, approx. 70% recovery by addition of CuSO4 [34]; 1 mM, 91% inhibition of catecholase activity [107]) [34, 107] 2-mercaptoethanol ( 1 mM, complete inhibition [48]; 2 mM, 93% inhibition [56]; 0.029 mM, 50% inhibition [68]; 0.1 mM, complete inhibition [108]) [2, 26, 38, 48, 56, 68, 108] 3,4-dihydroxybenzoic acid [89] 4-hexylresorcinol ( 1 mM, 65% inhibition of catecholase activity [107]) [107] 4-methylcatechol ( substrate inhibition [7]) [7] 4-nitrophenol ( competitive to catechol [37]) [37] 5-hydroxy-2-(hydroxymethyl)-2H-pyran-4-one ( trivial name kojic acid, 1 mM, complete inhibition of l-dopa oxidation, 94% inhibition of tyrosinase activity [35]; 1.12 mM, 50% inhibition of recombinant enzyme [103]; 1 mM, 50% inhibition of l-dopa oxidation [108]) [35, 103, 106, 108] 5-hydroxyindole [29] 5-hydroxytryptophan [29] 5-methyl-1,3-benzenediol ( competitive to catechol [37]) [37] 8-hydroxyquinoline ( 3 mM, 44% inhibition [3]; 1.3 mM, 50% inhibition [68]) [3, 68] CN- ( 1 mM, complete inhibition [9]; 0.2 mM, 50% inhibition of isoenzyme I, 0.13 mM, 50% inhibition of isoenzyme III [21]; noncompetitive vs. dopa and tyrosine [29]; 0.2 mM, 70% inhibition of catechol oxidation, 63% inhibition of pyrogallol oxidation, 50% inhibition of dopa oxidation [38]; 1 mM, 59% inhibition of isoenzyme a, 83% inhibition of isoenzyme B [43]; 0.6 mM, 50% inhibition [45]; 1 mM, 96% inhibition [48]) [2, 9, 21, 29, 37, 38, 43, 45, 48, 61, 73, 78] CO [26, 89] Cl- ( 200 mM, 13% inhibition at pH 6.0, 85% inhibition at pH 5.0, 96% inhibition at pH 4.0 [28]; 6 mM, 50% inhibition at pH 4.5, 34 mM, 50% inhibition at pH 5 [68]; 1 mM, 35% inhibition of l-dopa oxidation [108]) [28, 68, 108] Cu2+ ( inhibition at concentrations higher than 5 mM [45]) [45]

164

1.14.18.1

Monophenol monooxygenase

dl-dithiothreitol ( 1 mM, complete inhibition [48]; very slight inhibition at 300 pmol/unit of enzyme [92]; 0.1 mM, complete inhibition [98]) [12, 48, 92, 98, 106, 115] EDTA ( 1 mM, 71% inhibition of l-dopa oxidation [108]) [108] Hg2+ ( inhibition of tyrosine hydroxylation [35]) [35] KCl [112] l-ascorbic acid ( 1 mM, complete inhibition of purified enzyme, 20% inhibition of crude enzyme [48]; 2 mM, 78% inhibition [56]; 0.04 mM, complete inhibition [99]) [40, 43, 48, 56, 99] l-cysteine ( 0.5 mM, 61% inhibition of catechol oxidation [38]; 1 mM, complete inhibition [48]; 2 mM, 95% inhibition [56]; 0.1 mM, 86% inhibition [98]) [26, 38, 48, 56, 98, 106] l-phenylalanine ( 8.34 mM, 50% inhibition of recombinant enzyme [103]) [38, 103] Na2 S2 O4 ( 0.1 mM, 30% inhibition [98]) [98] NaCl [112] O2 ( at concentrations above 30% [47]) [47] acetylacetone ( 0.1 mM, 75% inhibition [12]) [12] arbutin ( 3.7 mM, 50% inhibition of recombinant enzyme [103]) [103] azide ( 12 mM, 50% inhibition [9]; 1 mM, 60% inhibition of catecholase activity [40]) [9, 26, 37, 38, 40] bathocuproine [9, 12] bathocuproine sulfonate [89] benzhydroxamic acid [26] benzoic acid ( 0.2 mM, 50% inhibition [68]) [26, 68, 89] b-(N-3-hydroxypyridone-4)-a-aminopropionic acid ( copper chelator, trivial name l-mimosine [26, 34]; 0.1 mM, 50% inhibition [34]) [26, 34] catechol ( irreversible inactivation [26]) [26] citrate [68] cuprizone [12] cuproine [12] diethyldithiocarbamate ( competitive vs. tyrosine, addition of Cu2+ restores activity [2]; 0.1 mM, complete inhibition [3]; 0.3 mM, complete inhibition [9]; 0.1 mM, 80% inhibition [12]; 0.5 mM, 50% inhibition of isoenzyme I, 0.19 mM, 50% inhibition of isoenzyme III [21]; noncompetitive vs. dopa and tyrosine [29]; 1 mM, complete inhibition of l-dopa oxidation, 21% inhibition of tyrosinase activity [35]; 0.05 mM, 97% inhibition of tyrosinase activity, 83% inhibition of catecholase activity [40]; 1 mM, 34% inhibition of isoenzyme A, 95% inhibition of isoenzyme B [43]; 0.072 mM, 50% inhibition [46]; 1 mM, complete inhibition [48]; 0.166 mM, 90% inhibition [58]; 0.0051 mM, 50% inhibition [68]; 10 mM, 80% inactivation, activity is restored by incubation with 0.01 mM Cu2+ , Mn2+ , Cd2+ or Fe2+ [73]; 2 mM, complete inhibition, 1 mM Cu2+ restores activity to original level [98]; 1 mM, 165

Monophenol monooxygenase

1.14.18.1

complete inhibition of l-dopa oxidation [108]) [2, 3, 8, 9, 12, 21, 26, 29, 35, 37, 40, 43, 46, 48, 58, 68, 73, 78, 89, 98, 106, 108, 115] dimethylsulfoxide [31] dipicolinic acid [9] dithioerythritol ( 0.05 mM, 82% inhibition of catechol oxidation, 95% inhibition of pyrogallol oxidation and 62% inhibition of dopa oxidation [38]) [38] ferulic acid [45] gallic acid [45] glutathione ( 0.01 mM, 50% inhibition of recombinant enzyme [103]; 1 mM, 86% inhibition of l-dopa oxidation [108]) [103, 108, 115] inhibitor peptide ( 2 natural occuring inhibitors: a 1200 Da peptide that inhibits tyrosinase competitively and second uncharacterized peptide [74]) [74] inhibitor protein from human skin [50] iodobenzoic acid ( 78% inhibition [48]) [48] lactic acid ( 3.73 mM, 50% inhibition of recombinant enzyme [103]) [103, 115] metabisulfite ( 0.1 mM, 99% inhibition [3]; 1 mM, complete inhibition [48]; 0.1 mM, 99% inhibition, 1 mM Cu2+ restores activity to original level [98]) [3, 38, 46, 48, 98] o-nitrophenol ( competitive to catechol [37]) [37] o-phenanthroline hydrate ( 3 mM, 30% inhibition [3]) [3] p-cresol ( competitive to catechol [37]) [37, 38] p-hydroxybenzoic acid [89] phenylhydrazine ( 1 mM, 86% inhibition, noncompetitive inhibition [3]) [3] poly(9)-oxyethylenelauryl ether [17] polyvinylpyrrolidone ( 1.1%, 50% inhibition [46]) [46] potassium metabisulfite ( 0.092 mM, 50% inhibition [46]) [46] quinone isomerase ( part of a complex consisting of phenoloxidase, quinone isomerase and quinone methide isomerase [105]) [105] resorcine ( competitive to catechol [37]) [37] salicylaldoxime ( 0.43 mM, 50% inhibition [68]) [68] sodium bisulfite ( 0.062 mM, 50% inhibition [68]) [68] sodium dodecylsulfate ( above 10 mM inhibition of catecholase activity [17]) [17] sodium sulfate [21] thiourea ( 13 mM, 50% inhibition [9]; 0.5 mM, 85% inhibition of catechol oxidation [38]; 0.8 mM, 50% inhibition [68]) [9, 37, 38, 68, 78] Additional information ( not inhibited by a,a'-dipyridyl [12, 29]; not inhibited by EDTA [9, 12, 21]; product inhibition of o-quinones [64]; not inhibited by sodium azide [3]; tyrosinase activity is inhibited by an unknown epidermal protein

166

1.14.18.1

Monophenol monooxygenase

[50]; not inhibited by 1 mM arbutin, sodium azide, EDTA, or 0.2 mM Cu2+ , Fe3+ , Zn2+ , Mg2+ and Ca2+ [106]) [3, 12, 21, 29, 50, 61, 64, 106]
"%  ascorbate ( activation of tyrosine hydroxylase activity [40]) [40] &

  1-decanesulfonic acid ( slight activation of latent enzyme [17]) [17] 3-hydroxyanthranilic acid ( higher than 0.005 mM, 600% activation of N-acetyl-tyrosine hydroxylation, activation of 4-tert-butylphenol oxidation, shortens lag time of the enzyme [100]) [100] l-dopa ( and analogs, activation [47, 67]; low-mobility enzyme form shows an absolute requirement of l-dopa for tyrosine hydroxylation in contrast to high-mobility enzyme form [90]) [47, 67, 90] acid ( activation by short exposure to pH 3.0-3.5 [61]) [61] alkali ( activation by short exposure to pH 11.5 [61]) [61] anionic detergents ( activation [34, 62]) [29, 34, 62] dimethylsulfoxide ( 5% DMSO, 180% activation [31]) [31] dithiothreitol ( small amounts abolish the characteristic lag phase of monohydric phenol oxidation without effect on the maximum rate of reaction or on the total O2 consumption [92]) [92] ferulic acid ( activation [45]) [45] gallic acid ( activation [45]) [45] monomeric glycoprotein ( approx. 3fold activation [19]) [19] organic solvents ( activation [62]) [62] phosphate ( increase of activity [45]) [45] photoactivation [72] sodium deoxycholate ( approx. 60% activation [29]) [29] sodium dodecylsarcosinate ( approx. 70% activation [29]) [29] sodium dodecylsulfate ( approx. 1.5fold activation of latent enzyme [17]; 1 mM, 90% activation [29]; 0.6 mM, maximal activation [32]) [17, 29, 31, 32] trypsin ( activation of proenzyme [15]; latent enzyme form, 4.5fold activation by trypsin [17]) [15, 17] Additional information ( it is suggested that the activation of the proenzyme is accompanied by a conformational change in which the tryptophyl residues move to the active site [44]) [44] Additional information ( activity depends on buffer system: highest activity in citrate-phosphate buffer [112]; activation of latent enzyme under frosty conditions [17]) [17, 112] '(   Ca2+ ( inactive proenzyme is maximally activated by 1 mM Ca2+ [15]; inactive enzyme is maximally activated by 10 mM Ca2+ [33]) [15, 33] Co2+ ( 0.9 mM, slight activation of isoenzymes I and III [21]; 50 mM, 360% activation [45]) [21, 45]

167

Monophenol monooxygenase

1.14.18.1

Fe3+ ( 1 mM, increase in activity [99]) [99] K+ ( 50% activation of l-dopa oxidation [108]) [108] Mg2+ ( 1 mM, increase in activity [99]; 33% activation of l-dopa oxidation [108]) [99, 108] Mn2+ ( activation of proenzyme [15]; activation [45]; slight activation of isoenzymes I and III [21]; activation above 1 mM Mn2+ [33]) [15, 21, 33, 45] Ni2+ ( 0.9 mM, slight activation of isoenzymes I and III [21]; 50 mM, 20% activation [45]) [21, 45] Zn2+ ( 0.9 mol per polypeptide [3]; 0.9 mM, approx. 2fold activation of isoenzymes I and III [21]) [3, 21] copper ( 2 gatom per mol [26, 52, 66]; 2.1 copper atoms per polypeptide, enzyme form B [3]; 1 gatom per mol [35]; 4 gatom per mol [48]; 0.2% total copper content, probably 1 copper atom per subunit [46]; 2 atoms of copper per 150000 Da [12]; extended X-ray absorption fine structure, EXAFS, studies [41]; chemical and spectroscopic studies of binuclear copper site [57]; 0.1 mM, approx. 3fold activation of isoenzymes I and III [21]; enzyme contains a binuclear copper complex at the active site which is responsible for the interaction with phenolic substrates and the binding and activation of molecular O2, depending on the oxidation state of the copper, 3 different forms are known: met-, oxy-, and deoxytyrosinase [26]; activation at low concentrations, inhibition above 5 mM [45]; 0.22% copper, probably 4 copper atoms per enzyme molecule [48]; 0.21% copper, intra- and extracellular enzyme [53]; 2 copper atoms per functional unit of 42000 Da [61]; 1 mM, increase in activity [99]; enzyme contains 2 copper-binding domains [102]; 50% activation of l-dopa oxidation [108]; 1 H-NMR spectra, each copper atom is coordinated by the Ne atoms of 3 histidine residues [113]) [2, 3, 12, 21, 26, 33, 35, 41, 45, 46, 48, 52, 53, 57, 61, 66, 80, 98, 99, 102, 108, 113] ) &  * +, 0.7 (tyrosine, enzyme from malignant melanocytes [67]) [67] 1.3 (tyrosine, enzyme from normal melanocytes [67]) [67] 36 (l-a-methyl tyrosine) [104] 136.2 (d-tyrosine) [106] 150 (l-tyrosinase, under 100 kPa O2 [97]) [97] 192 (phenol) [35] 200 (tyrosine, tyrosine hydroxylase activity [29]) [29] 432 (l-tyrosine) [35] 474 (l-tyrosine) [104] 582 (pyrogallol) [35] 762 (phenol) [104] 1170 (l-tyrosinase, under 21 kPa O2 [97]) [97] 1554 (tyramine) [104] 1600 (dopa, dopa oxidase activity [29]) [29] 1764 (l-isoproterenol) [104]

168

1.14.18.1

Monophenol monooxygenase

2130 (dopa methyl ester) [104] 2658 (4-hydroxyphenyl acetic acid) [104] 2658 (l-a-methyldopa) [104] 4002 (4-hydroxyphenyl propionic acid) [104] 4452 (dl-tyrosine) [106] 4500 (l-dopa, isoenzyme A [73]) [73] 4884 (l-tyrosine) [106] 5400 (l-dopa, isoenzyme B [73]) [73] 6000 (l-tyrosine, isoenzyme A and B [73]) [73] 6444 (l-dopa) [104] 7020 (d-dopa) [106] 7050 (l-dopa) [35] 7380 (esculetin) [12] 7920 (4-ethoxyphenol) [104] 11050 (4-hydroxyanisole) [104] 19000 (l-dopa) [78] 20160 (protocatechuic aldehyde) [12] 26340 (dopamine) [104] 33210 (3,4-dihydroxyphenyl propionic acid) [104] 37900 (3,4-dihydroxyphenyl acetic acid) [104] 38510 (4-tert-butylcatechol) [104] 50500 (4-methylcatechol) [104] 51600 (protocatechuic acid) [12] 52660 (catechol) [104] 67200 (l-dopa) [106] 70200 (dl-dopa) [106] 87000 (caffeic acid) [12] 99000 (chlorogenic acid) [12] 177600 (catechol) [12] 277800 (hydrocaffeic acid) [12] 296400 (dopa) [12] 433200 (4-methylcatechol) [12] " & *-% , 0.019 ( partially purified enzyme, tyrosine hydroxylation [58]) [58] 0.032 [67] 0.065 ( with tyrosine [9]) [9] 0.228 ( tyrosine hydroxylation [50]) [50] 0.277 ( with catechol [12]) [12] 1.43 ( tyrosine hydroxylase activity [29]) [29] 2.34 ( tyrosine hydroxylation [42]) [42] 2.49 [108] 2.5 ( dopa oxidation [93]) [93] 7.2 ( with 4-methylcatechol [17]) [17] 10.4 ( dopa oxidation [29]) [29] 17.3 ( isoenzyme 2, tyrosine hydroxylation [72]) [72]

169

Monophenol monooxygenase

1.14.18.1

17.5 ( isoenzyme 1, tyrosine hydroxylation [72]) [72] 26 ( recombinant enzyme [103]) [103] 32 ( with 4-methylcatechol, trypsin activation [17]) [17] 45 [25] 45 ( enzyme fraction II [77]) [77] 46 ( isoenzyme A [73]) [73] 64.2 ( tyrosine hydroxylation [114]) [114] 68 ( partially purified isoenzyme 3 [31]) [31] 69.5 ( isoenzyme 2, dopa oxidation [72]) [72] 72 ( isoenzyme 1, dopa oxidation [72]) [72] 105 [75] 106 ( l-dopa oxidation [42]) [42] 130 [106] 137 ( isoenzyme B [73]) [73] 143 [71] 215 [35] 336 [56] 442 ( 4-hydroxyanisol hydroxylation [96]) [96] 540 [78] 658 ( l-dopa oxidation [40]) [40] 1030 ( isoenzyme 2 [31]) [31] 1205 [26] 1301 ( catecholase activity [110]) [110] 1480 ( isoenzyme 1 [31]) [31] 3432 [102] 6889 [5] 9854 [24] 11280 [1] 45000 [25] Additional information ( assay methods [25]; enzyme form A, 480.0 D absorbance 470 nm/min/mg, enzyme form B, 258.6 D absorbance 470 nm/min/mg [3]; 4359.0 D absorbance/min x 1000/mg [11]; 1064.0 D absorbance 490 nm/min/mg [15]; 3314 units, 1 unit is the amount of enzyme causing an increase in absorbance at 490 nm of 0.001/min/ml [33]; 0.0088 units, 1 unit is defined as the amount of enzyme that causes a change in absorbance of 0.001/min [38]; isoenzyme A: 92.0 units, isoenzyme B: 97 units, 1 unit is defined as absorbance change of 0.001 at 430 nm per min [43]; 9900 units, 1 unit is defined as the amount of enzyme that causes a change in absorbance of 0.001/min [48]; 35263 units, 1 unit is defined as absorbance change of 0.001 at 430 nm per min [59]; 0.281 units, 1 unit is defined as 0.0024 ml O2 /min [68]; 8.4 ml O2 /min/mg [76]) [3, 11, 15, 25, 33, 38, 43, 48, 59, 68, 76] . / *', 0.001 (p-cresol, isoenzyme A3 [94]) [94] 0.001 (p-cresol, isoenzyme B1 [94]) [94]

170

1.14.18.1

Monophenol monooxygenase

0.001 (p-cresol, isoenzyme B2 [94]) [94] 0.001 (p-cresol, isoenzyme B3 [94]) [94] 0.002 (p-cresol, isoenzyme A1 [94]) [94] 0.003 (p-cresol, isoenzyme A2 [94]) [94] 0.014 (l-tyrosine, isoenzyme A2 [94]) [94] 0.014 (l-tyrosine, isoenzyme A3 [94]) [94] 0.014 (l-tyrosine, isoenzyme B1 [94]) [94] 0.014 (l-tyrosine, isoenzyme B2 [94]) [94] 0.014 (l-tyrosine, isoenzyme B3 [94]) [94] 0.014 (N-acetyl-3,4-dihydroxyphenethylamine) [67] 0.014 (catechol, isoenzyme A3 [94]) [94] 0.015 (l-tyrosine, isoenzyme A1 [94]) [94] 0.016 (4-tert-butylphenol) [100] 0.016 (l-dopa) [67] 0.018 (catechol, isoenzyme B3 [94]) [94] 0.02 (3,4-dihydroxyphenylethanol) [67] 0.022 (dl-dopa) [67] 0.024 (N-methyl-3,4-dihydroxyphenethylamine) [67] 0.026 (d-dopa) [67] 0.046 (3,4-dihydroxyphenylalanine methyl ester) [67] 0.047 (3,4-dihydroxyphenethylamine) [67] 0.067 (O2, electron donor ascorbic acid [89]) [89] 0.074 (l-dopa, isoenzyme B3 [94]) [94] 0.08 (4-hydroxyanisole) [104] 0.085 (l-dopa, isoenzyme A3 [94]) [94] 0.087 (catechol, isoenzyme B1 [94]) [94] 0.089 (l-dopa, isoenzyme A2 [94]) [94] 0.09 (catechol, isoenzyme A1 [94]) [94] 0.091 (catechol, isoenzyme A2 [94]) [94] 0.094 (3,4,5-trihydroxyphenethylamine) [67] 0.1 (N-b-alanyldopamine) [18] 0.102 (2,4,5-trihydroxyphenethylamine) [67] 0.104 (catechol, isoenzyme B2 [94]) [94] 0.11 (dopamine) [18] 0.12 (l-tyrosine, high-mobility enzyme form [90]) [90] 0.133 (ascorbic acid, electron donor ascorbic acid [89]) [89] 0.145 (3,4-dihydroxyphenylserine) [67] 0.17 (4-ethoxyphenol) [104] 0.17 (4-methylcatechol) [16] 0.17 (4-methylcatechol, in H2 O [16]) [16] 0.17 (l-tyrosine, recombinant enzyme [103]) [103] 0.18 (l-dopa) [108] 0.18 (tyramine) [40] 0.185 (l-dopa, isoenzyme B2 [94]) [94] 0.194 (3-(3,4-dihydroxyphenyl)-2-methylalanine) [67] 0.2 (3-hydroxyphloridzin) [47] 0.2 (4-methylcatechol, in MOPS buffer, pH 7.0 [7]) [7] 171

Monophenol monooxygenase

1.14.18.1

0.2 (4-methylcatechol, measured in bis(2-ethylhexyl)sodium sulfosuccinate-isooctane reverse micelles [16]) [16] 0.2 (caffeic acid) [47] 0.2 (catechol) [75] 0.21 (l-tyrosine) [104] 0.23 (3,4-dihydroxyphenylglycol) [67] 0.23 (l-tyrosine, low-mobility enzyme form [90]) [90] 0.25 (dopamine) [58] 0.268 (p-coumaric acid, electron donor ascorbic acid [89]) [89] 0.3 (catechol) [104] 0.3 (g-l-glutaminyl-4-hydroxybenzene) [101] 0.305 (3,4,6-trihydroxyphenylalanine) [67] 0.36 (l-dopa, recombinant enzyme [103]) [103] 0.37 (protocatechuic acid) [115] 0.4 (tyrosine) [29] 0.42 (caffeic acid) [115] 0.42 (p-coumaric acid) [115] 0.44 (4-hydroxyphenyl propionic acid) [104] 0.48 (dl-dopa) [34] 0.48 (N-acetyldopamine) [18] 0.49 (chlorogenic acid) [115] 0.5 (l-dopa) [5] 0.5 (l-dopa, in B16/C3 tumor cell homogenates [5]) [5] 0.5 (l-tyrosine) [101] 0.5 (p-cresol) [24] 0.51 (l-dopa, high-mobility enzyme form [90]) [90] 0.51 (tyramine) [104] 0.55 (tyramine) [58] 0.59 (protocatechuic aldehyde) [115] 0.6 (dopa) [29] 0.61 (norepinephrine) [18] 0.65 (p-cresol) [58] 0.67 (hydrocaffeic acid) [115] 0.69 (phenol) [35] 0.7 (catechol) [56] 0.7 (phenol) [104] 0.7 (pyrogallol) [75] 0.77 (dopa, enzyme fraction II [77]) [77] 0.78 (3-hydroxyanthranilic acid) [100] 0.8 (l-dopa) [104] 0.9 (4-methylcatechol) [58] 1 (dopa methyl ester) [104] 1 (tert-butylcatechol) [107] 1.09 (dopamine) [40] 1.11 (pyrogallol) [35] 1.13 (epinephrine) [18] 1.15 (4-methylcatechol) [115] 172

1.14.18.1

Monophenol monooxygenase

1.2 (l-a-methyl tyrosine) [104] 1.2 (l-dopa, isoenzyme B1 [94]) [94] 1.25 (l-tyrosine) [106] 1.4 (epicatechin) [75] 1.45 (adrenaline bitartrate) [40] 1.5 (l-dopa) [101] 1.5 (O2, substrate g-l-glutaminyl-4-hydroxybenzene [101]) [101] 1.6 (catechin) [75] 1.64 (dl-tyrosine) [106] 1.7 (epicatechin, pyrogallol [64]) [64] 1.8 (l-tyrosine) [40] 1.89 (3,4-dihydroxyphenyl propionic acid) [104] 1.9 (l-dopa, low-mobility enzyme form [90]) [90] 1.91 (4-hydroxyphenyl acetic acid) [104] 1.96 (3,4-dihydroxyphenylalanine) [115] 1.98 (d-tyrosine) [106] 2 (d- and l-tyrosine) [58] 2 (epicatechin gallate) [75] 2 (epigallocatechin gallate) [75] 2 (noradrenalin) [40] 2 (p-cresol) [17] 2-14 (hydroquinone monomethylether, range of estimated values for crude or purified extracts [3]) [3] 2.1 (O2, substrate l-tyrosine [101]) [101] 2.1 (catechin) [64] 2.2 (d-tyrosine, isoenzyme A [73]) [73] 2.2 (l-dopa, isoenzyme A1 [94]) [94] 2.2 (dopamine) [104] 2.2 (epigallocatechin) [75] 2.36 (4-methylcatechol) [104] 2.5 (4-methylcatechol) [64] 2.5 (a-methyl-dopa) [40, 64] 2.7 (4-methylcatechol) [107] 2.7 (l-dopa) [47] 2.8 (4-tert-butylcatechol) [104] 2.87 (protocatechuic aldehyde) [12] 2.89 (3-(p-hydroxyphenyl)propionic acid) [115] 3 (d-catechin, isoenzyme III [21]) [21] 3 (tyrosine) [35] 3.03 (dl-epicatechin, isoenzyme I [21]) [21] 3.1 (l-tyrosine, isoenzyme A and B [73]) [73] 3.2 (4-methylcatechol) [47] 3.22 (d-epicatechin, isoenzyme III [21]) [21] 3.24 (esculetin) [12] 3.3 (4-methylcatechol) [68] 3.3 (l-dopa) [17] 3.85 (l-epicatechin, isoenzyme I [21]) [21] 173

Monophenol monooxygenase

1.14.18.1

3.93 (dopa) [18] 4 (4-methylcatechol, in ternary solution composed of hexane-isopropanol-water [7]) [7] 4 (dopa) [35] 4 (p-coumaric acid) [47] 4-5 (4-methyl catechol, in poly-(10)-oxyethylene oleyl ether-cyclohexane reverse micelles [23]) [23] 4.1 (chlorogenic acid, at pH 4.0, slight increase in Km above pH 5.0 [1]) [1] 4.14 (l-dopa) [106] 4.2 (4-methylcatechol) [17] 4.7 (catechin) [47] 4.7 (dopa, in cuticles [6]) [6] 4.75 (4-methylcatechol) [12] 4.8 (dl-dopa) [38] 4.9 (chlorogenic acid, at pH 5.5, slight increase in Km above pH 5.0 [1]) [1] 5 (4-methylcatechol) [45] 5 (d-catechin, isoenzyme I [21]) [21] 5 (catechol) [43, 45] 5 (catechol, isoenzymes A and B [43]) [43] 5 (chlorogenic acid) [68] 5.1 (3,4-dihydroxyphenyl propionic acid) [104] 5.13 (4-methylcatechol) [46] 5.15 (chlorogenic acid) [12] 5.2 (4-methylcatechol, at pH 3.5-5.0, strong increase in Km above pH 5.0 [1]) [1] 5.26 (dl-epicatechin, isoenzyme III [21]) [21] 5.39 (l-dopa) [112] 5.55 (d-dopa) [40] 5.7 (epicatechin) [47] 5.9 (chlorogenic acid) [47] 6 (l-dopa) [58] 6.1 ((+)-catechin, at pH 4.0, slight increase in Km above pH 4.0 [1]) [1] 6.25 (dopa) [56] 6.3 (pyrogallol) [38] 6.33 (4-hydroxybenzoic acid) [115] 6.37 (4-hydroxybenzaldehyde) [115] 6.43 (caffeic acid) [12] 6.6 ((+)-catechin, at pH 5.5, slight increase in Km above pH 4.0 [1]) [1] 6.66 (l-dopa) [40] 6.67 (d-epicatechin, isoenzyme I [21]) [21] 6.8 (l-a-methyldopa) [104] 7.1 (l-isoproterenol) [104] 7.8 (g-l-glutaminyl-3,4-dihydroxybenzene) [101] 7.95 (catechol) [12] 174

1.14.18.1

Monophenol monooxygenase

8 (N-formyl-l-tyrosine) [40] 8.3 (catechol) [68] 8.63 (dopa) [12] 8.7 (l-dopa, isoenzyme 1 [99]) [99] 9 (4-methyl catechol, in buffer [23]) [23] 9.1 (catechol) [38] 9.3 (l-dopa, isoenzyme 2 [99]) [99] 9.56 (dl-dopa) [106] 10 (l-dopa, isoenzyme 3 [99]) [99] 11.1 (l-epicatechin, isoenzyme III [21]) [21] 13 (catechol) [48] 13.7 (l-adrenaline) [64] 14.77 (4-hydroxybenzyl alcohol) [115] 15 (4-methylcatechol, in ternary solution composed of toluene-isopropanol-water [7]) [7] 20 (3-methylcatechol) [64] 20.5 (4-methylcatechol, at pH 6.3, strong increase in Km above pH 5.0 [1]) [1] 27.4 (hydrocaffeic acid) [12] 28.7 (d-dopa) [106] 36 (l-dopa, isoenzyme A [73]) [73] 38.2 (O2, substrate l-dopa [101]) [101] 67 (l-dopa, isoenzyme B [73]) [73] 79 (quinol) [64] 100.2 (O2, substrate g-l-glutaminyl-3,4-dihydroxybenzene [101]) [101] 189 (protocatechuic acid) [12] Additional information ( 4.3% O2 [47]) [47] . / *', 0.00034 (diethyldithiocarbamate, non-competitive vs. l-dopa [35]) [35] 0.001 (kojic acid, isoenzyme A1 [94]) [94] 0.001 (kojic acid, isoenzyme B3 [94]) [94] 0.0013 (diethyldithiocarbamate, vs. O2 [89]) [89] 0.003 (kojic acid, isoenzyme A3 [94]) [94] 0.003 (kojic acid, isoenzyme B1 [94]) [94] 0.005 (5-hydroxyindole, dopa oxidase inhibition [29]) [29] 0.006 (kojic acid, isoenzyme A2 [94]) [94] 0.0062 (diethyldithiocarbamate, non-competitive vs. l-tyrosine [35]) [35] 0.007 (EDTA, isoenzyme A3 [94]) [94] 0.008 (kojic acid, isoenzyme B2 [94]) [94] 0.0119 (diethyldithiocarbamate, vs. p-coumaric acid [89]) [89] 0.013 (5-hydroxy-2-(hydroxymethyl)-2H-pyran-4-one, non-competitive vs. l-dopa [35]) [35] 0.015 (2,4,6-cycloheptatriene-1-one) [34]

175

Monophenol monooxygenase

1.14.18.1

0.016 (quinol) [64] 0.0162 (CO, vs. O2 [89]) [89] 0.025 (l-cysteine) [64] 0.03 (diethyldithiocarbamate, dopa oxidase inhibition [29]) [29] 0.03 (diethyldithiocarbamate, tyrosine hydroxylase [29]) [29] 0.04 (diethyldithiocarbamate, noncompetitive vs. catechol [37]) [37] 0.057 (5-hydroxy-2-(hydroxymethyl)-2H-pyran-4-one, non-competitive vs. l-tyrosine [35]) [35] 0.062 (CO, vs. p-coumaric acid [89]) [89] 0.07 (thiourea, mixed inhibition vs. catechol [37]) [37] 0.075 (EDTA, isoenzyme B3 [94]) [94] 0.08 (CN- , tyrosine hydroxylase inhibition [29]) [29] 0.08 (diethyldithiocarbamate, noncompetitive vs. pyrogallol [37]) [37] 0.09 (CN- , dopa oxidase inhibition [29]) [29] 0.09 (hydroquinone) [45] 0.095 (CN- , competitive vs. tyrosine [73]) [73] 0.1 (l-cysteine) [48] 0.15 (CO, vs. ascorbic acid [89]) [89] 0.16 (KCN, noncompetitive vs. catechol [37]) [37] 0.17 (p-nitrophenol, competitive vs. catechol [37]) [37] 0.2 (CN- ) [48] 0.2 (KCN, noncompetitive vs. pyrogallol [37]) [37] 0.2 (benzoic acid) [68] 0.23 (4-methylcatechol, in bis(2-ethylhexyl)sodium sulfosuccinateisooctane reverse micelles [16]) [16] 0.24 (4-hydroxycinnamic acid) [45] 0.25 (p-nitrophenol, competitive vs. pyrogallol [37]) [37] 0.267 (diethyldithiocarbamate, noncompetitive vs. tyrosine [73]) [73] 0.277 (bathocuproinesulfonate, vs. caffeic acid [89]) [89] 0.284 (bathocuproinesulfonate, vs. O2 [89]) [89] 0.298 (EDTA, isoenzyme A1 [94]) [94] 0.338 (CO, vs. caffeic acid [89]) [89] 0.35 (2,5-dihydroxybenzoic acid) [45] 0.35 (4-hydroxybenzoic acid, vs. O2 [89]) [89] 0.375 (4-hydroxybenzoic acid, vs. ascorbic acid [89]) [89] 0.45 (benzoic acid, vs. O2 [89]) [89] 0.5 (2-mercaptoethanol) [48] 0.5 (4-hydroxybenzoic acid, vs. p-coumaric acid [89]) [89] 0.5 (thiourea, noncompetitive vs. pyrogallol [37]) [37] 0.52 (3,4-dihydroxybenzoic acid, vs. O2 [89]) [89] 0.561 (bathocuproinesulfonate, vs. p-coumaric acid [89]) [89] 0.58 (EDTA, isoenzyme B1 [94]) [94] 0.668 (EDTA, isoenzyme A2 [94]) [94] 0.7 (benzoic acid, vs. p-coumaric acid [89]) [89] 176

1.14.18.1

Monophenol monooxygenase

0.75 (4-hydroxybenzoic acid, vs. caffeic acid [89]) [89] 0.75 (benzoic acid, vs. caffeic acid [89]) [89] 0.825 (bathocuproinesulfonate, vs. ascorbic acid [89]) [89] 0.88 (p-cresol, competitive vs. catechol [37]) [37] 0.9 (p-cresol, uncompetitive vs. pyrogallol [37]) [37] 0.97 (orcinol, competitive vs. pyrogallol [37]) [37] 1.25 (NaN3 , noncompetitive vs. catechol [37]) [37] 1.39 (EDTA, isoenzyme B2 [94]) [94] 1.4 (cinnamic acid, noncompetitive vs. pyrogallol [37]) [37] 1.43 (orcinol, mixed inhibition vs. catechol [37]) [37] 1.62 (KCl, competitive vs. l-DOPA [112]) [112] 1.75 (2-hydroxybenzoic acid) [45] 1.8 (N-3 ) [48] 1.8 (NaN3 , noncompetitive vs. pyrogallol [37]) [37] 1.82 (NaCl, competitive vs. l-DOPA [112]) [112] 2 (4-hydroxybenzoic acid) [45] 2 (cinnamic acid, noncompetitive vs. catechol [37]) [37] 2.5 (3-hydroxycinnamic acid) [45] 3.6 (4-methylcatechol, in H2 O, substrate inhibition [16]) [16] 4.25 (quinoline, competitive vs. catechol [37]) [37] 4.3 (3,4-dihydroxybenzoic acid) [45] 4.33 (l-phenylalanine, noncompetitive vs. catechol [37]) [37] 4.7 (quinoline, noncompetitive vs. pyrogallol [37]) [37] 6.4 (resorcinol, noncompetitive vs. pyrogallol [37]) [37] 7.9 (resorcinol, competitive vs. catechol [37]) [37] 28.6 (l-phenylalanine, uncompetitive vs. pyrogallol [37]) [37] 104 (4-methylcatechol, weak substrate inhibition [17]) [17] 0  3 ( oxidation of 4-methylcatechol [115]) [115] 3.5-5 ( catecholase activity [24]) [24] 4 ( oxidation of protocatechuic acid [115]) [115] 4.2 ( optima at pH 4.2 and pH 7.0 [76]) [76] 4.5 ( minimal activity at pH 5.5 [47]) [47, 68] 4.5-5 ( with 4-methylcatechol, chlorogenic acid and (+)-catechin [1]) [1] 5 ( significant decrease above and below pH 5, 79% activity at pH 5.3, 62% activity at pH 4.3 [28]; 4-hydroxyanisol hydroxylation [96]) [28, 96] 5-6.5 ( catecholase activity entrapped in reverse micelles [23]) [23] 5-8 ( dopa oxidation [78]) [78] 5.1 ( 50% activity at pH 3.7 and pH 7.7 respectively [36]) [36] 5.5 ( phenol oxidase activity [45]; catecholase activity [107]) [45, 69, 107] 5.5-6.5 ( catecholase activity [40]) [40] 5.5-7.3 ( dopa oxidase activity [29]) [29] 5.7 ( substrate catechol [75]) [75]

177

Monophenol monooxygenase

1.14.18.1

6 ( 50% activity at pH 4.5 and pH 7.5 respectively [9]; isoenzymes A3 and B3 [94]) [9, 94] 6.2 ( free enzyme [16]) [16] 6.4 [2] 6.5 ( isoenzymes I and III [21]; isoenzymes A1, A2, B1 and B2 [94]) [12, 21, 94] 6.5-7.5 ( substrate catechol, 2 pH maxima at 6.5 and 7.5 [56]) [56] 6.5-7.9 ( substrate dopa, 2 pH maxima at pH 6.5 and pH 7.9 [56]) [56] 6.6 [65] 6.6-7.8 [73] 6.6-7.8 ( 50% activity at pH 5.7 [73]) [73] 6.7-7.2 [15] 6.8 ( with l-dopa [35]) [35, 48] 6.8-7.8 ( tyrosine hydroxylase activity [29]) [29] 7 ( with hydroquinone monomethylether [3]; optima at pH 4.2 and pH 7.0 [76]; with l-dopa [106]) [3, 38, 76, 106] 7.5 ( tyrosine hydroxylase [40]; o-diphenolase [45]; l-dopa oxidation, recombinant enzyme, less than 50% activity below pH 6.5 and above pH 8.5 [103]; cresolase activity [107]) [40, 45, 103, 107] 9 ( cuticle-bound enzyme [6]) [6] 9-9.6 ( isoenzymes 1-3 [99]) [99] 9.5 ( l-dopa oxidation at 50 C, less than 50% activity below pH 7.5 and above pH 10.5 [108]) [108] 0 

 2.5-5.5 ( oxidation of protocatechuic acid [115]) [115] 3-5.5 ( oxidation of 4-methylcatechol [115]) [115] 3.5-6 ( marked decrease above pH 6.0 [24]) [24] 3.5-7.5 ( cresolase activity rises in this range without reaching a defined maximum [24]) [24, 68] 4-7.5 ( substrate catechol, sharp decrease in activity above [56]) [56] 4-8 ( substrate dopa, sharp decrease in activity above [56]) [56] 4-9 ( isoenzymes I and III, no activity above or below [21]) [21, 107] 4.5-7.2 ( free enzyme [16]) [16] 5-7 ( low activity above and below [23]) [23] 5-8 [12] 5-9 ( in reversed micelles [16]) [16] 5.5 ( no activity below [47]) [47] 6-7.8 [2] 6-8 [65] )  *
, 20-30 [9] 25 [48] 178

1.14.18.1

Monophenol monooxygenase

25-40 ( catecholase activity [23, 24]) [23, 24] 30 ( in bis(2-ethylhexyl)sodium sulfosuccinate-isooctane reverse micelles [16]) [16] 35 ( isoenzyme I [21]; with l-dopa [35, 106]) [21, 35, 106] 40 [38, 106] 40-60 ( cresolase activity [24]) [24] 42 [2] 45 ( in H2 O [16]; isoenzyme III [21]; cuticle-bound enzyme [6]) [6, 16, 21] 50 ( l-dopa oxidation, recombinant enzyme [103]) [103] 55-60 ( isoenzymes 1-3 [99]) [99] ) 

 *
, 20-50 ( polyphenol oxidase I [21]) [21] 25-70 [2] 30-60 ( polyphenol oxidase II [21]) [21] 45 ( no activity above [23]) [23]

  #   ' 1  12000-60000 ( gel filtration, multiple forms [70]) [70] 29000-36000 (Streptomyces sp. KY-453, gel filtration, SDS-PAGE [35]) [35] 29500 ( gel filtration [30]) [30] 30610 ( deduced from nucleotide sequence [30]) [30] 31000 ( disc PAGE [60]) [60] 34000 ( sedimentation analysis [60]) [60] 36000 ( gel filtration [35]) [35] 38500 ( isoenzyme B, gel filtration [73]) [73] 40000 ( isoenzyme A, gel filtration [73]; isoenzymes I and III, gel filtration [21]) [21, 73] 42000 ( gel filtration [68]) [68] 45000 ( gel filtration [65]) [65] 46000 ( gel filtration [1]; deduced from amino acid sequence [52]) [1, 51, 52] 60000 ( PAGE [45]; gel filtration [9]; gel filtration [98]) [9, 45, 98] 66000 ( multiple isoforms, PAGE [42]) [42] 67000 ( immunoblot, anti-mushroom tyrosinase antibodies [2]) [2] 69000 ( tyrosinase A and B, PAGE [39]) [39] 90000 ( gel filtration [40]; gel filtration [108]) [40, 108] 107000 ( gel filtration [48]) [48] 115000 ( gel filtration [38, 37]; gel filtration [46]) [37, 38, 46]

179

Monophenol monooxygenase

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116000-128000 [61] 118600-119500 ( b-tyrosinase, sedimentation equilibrium [79]) [79] 118800 ( g-tyrosinase, sedimentation equilibrium [79]) [79] 122500 ( gel filtration [54]) [54] 123800 ( a, b and g isoenzymes, sedimetation equilibrium [80]) [80] 150000 ( gel filtration [12]) [12] 175000-230000 ( SDS-PAGE in the presence of urea [29]) [29] 188000 ( major peak, minor peak at 49000 Da, gel filtration [110]) [110] 220000-229000 ( gel filtration in the absence and presence of SDS [29]) [29] 330000 ( gel filtration [18]) [18]   ? ( x * 48000, enzyme form B, SDS-PAGE [3]; x * 50000, enzyme form A, SDS-PAGE [3]; x * 66000, isoenzyme 1, immunoblot [5]; x * 68000, isoenzyme 2, immunoblot [5]; x * 70000, isoenzyme 4, immunoblot [5]; x * 80000, isoenzyme 4, immunoblot [5]; x * 69000, SDS-PAGE [39]; x * 26000, SDS-PAGE [47]; x * 66700, trypsin cleaved enzyme, SDSPAGE [66]; x * 30000, isoenzyme 1 and 2, high speed sedimentation equilibrium in presence of SDS [72]; x * 33000, isoenzyme 1 and 2, SDS-PAGE [72]; x * 32000, calculated from amino acid analysis [72]; x * 45000, immunoprecipitation with anti-broad bean polyphenoloxidase antibodies [27]; x * 29000, intra- and extracellular enzyme, SDS-PAGE [53]; x * 75000, heating to 100 C, SDS-PAGE [71]; x * 67000, phenol oxidases A and B, SDS-PAGE [91]; x * 54000 + x * 49000, isoenzymes A1 and A2, SDS-PAGE [94]; x * 54000 + x * 15000, isoenzyme A3, SDS-PAGE [94]; x * 55000 + x * 50000, isoenzymes B1 and B2, SDS-PAGE [94]; x * 55000 + x * 15000, isoenzyme B3, SDS-PAGE [94]; x * 58000, mature enzyme, deduced from amino acid sequence [95]; x * 65000, isoenzyme 1, SDS-PAGE [99]; x * 55000, isoenzyme 2, SDS-PAGE [99]; x * 45000, isoenzyme 3, SDS-PAGE [99]; x * 56200, mature enzyme, deduced from amino acid sequence [102]; x * 66000, recombinant enzyme, SDS-PAGE [103]; x * 32000, SDS-PAGE [106]; x * 60000, SDS-PAGE [110]) [3, 5, 27, 39, 47, 53, 66, 71, 72, 91, 94, 95, 99, 102, 103, 106, 110] dimer ( 2 * 42000, SDS-PAGE [40]; 2 * 36000, SDSPAGE [9]; 2 * 43000, SDS-PAGE [108]) [9, 40, 108] monomer ( 1 * 29000-36000, SDS-PAGE [35]; 1 * 36000, SDS-PAGE [30]; 1 * 60000, SDS-PAGE [45]; 1 * 36000, proenzyme, SDS-PAGE [15]; 1 * 46000, deduced from amino acid sequence [25, 52]; 1 * 175000, SDS-PAGE [29]; 1 * 29500, SDS-PAGE [30]; 1 * 66000, multiple isoforms, SDS-PAGE [42]; 1 * 62000, SDS-PAGE [98]) [15, 25, 29, 30, 35, 42, 45, 51, 52, 98]

180

1.14.18.1

Monophenol monooxygenase

tetramer ( 4 * 40000, SDS-PAGE [12]; 4 * 31000, SDS-PAGE [37]; 3 * 30000 + 1 * 23500, SDS-PAGE [46]; 4 * 30000 [61]; 4 * 30000, SDS-PAGE [80]) [12, 37, 46, 61, 80] $   "

glycoprotein ( isoenzymes are most probably due to post-translational glycosylation, isoenzymes are immunological similar and possess the same amino acid content, 6 potential glycosylation sites in cDNA [22]; tyrosinases A and B, approx. 4 asparagine-linked sugar chains, 25-34% of those are high-mannose types [39]; carbohydrate content: 3-4% [49]; trypsin cleaved enzyme, carbohydrate content approx. 13% [66]; enzyme contains 9 potential N-glycosylation sites, Asn-X-Thr/Ser [95]; enzyme contains 1 putative glycosylation site [102]; glycosylation sites N86, N111, N337 and N371 are required for recognition by individual T cell clones [111]) [22, 25, 29, 39, 42, 49, 66, 95, 102, 111] no glycoprotein [53] ribonucleoprotein [76] Additional information ( isoenzymes with molecular weights of 58000 and 68000 Da are highly resistant to proteinase K digestion [5]) [5]

 2 %$ %' %

% cell culture [15, 33, 40] commercial preparation [16, 34, 54] conidiophore [3] culture filtrate [3] epidermis [44] feather [42] fruit ( peel and cortex [1]; main activity in pulp and peel [70]; expressed at the immature-green stage of fruit development [102]) [1, 28, 47, 56, 58, 59, 65, 68, 70, 84-86, 88, 102, 114] hemocyte ( enzyme is released in response to foreign materials such as sheep red blood cells and yeast cells as well as to allogenic hemocytes [98]) [98] hemolymph [19] husk [64] larva [14, 55, 60, 71] leaf [12, 17, 27, 45] melanoma cell [4, 5, 20, 25, 39, 63, 66, 67, 77, 81-83, 87, 90, 95] mycelium [26, 30] peel [1, 76] pith [21]

181

Monophenol monooxygenase

1.14.18.1

pupa [6, 18] shoot (culture) [115] skin [29, 50, 72] tuber [37, 38, 48] 3#

chloroplast [110] extracellular ( less than 3% intracellular activity [35]) [3, 10, 11, 35, 53, 61] membrane [66] particle-bound [56, 76] plastid [49] soluble [33, 56] thylakoid [17] $"

(ammonium sulfate, Phenyl Sepharose [1]; Triton X-100, butanol extraction, calcium phosphate gel [76]) [1, 47, 76] (DEAE-Sepharose, Phenyl Sepharose, enzyme form A and B [3]) [3] (proteinase K digest, DEAE-52 cellulose, 4 isozymes [5]; ammonium sulfate, S-300 chromatography, preparative PAGE [25]; preparative PAGE [67]; high-mobility tyrosinase form, ammonium sulfate, hydroxyapatite, gel filtration, low-mobility tyrosinase form, DEAE-Sephadex, partial purification [90]) [5, 25, 67, 90, 93] (affinity chromatography, Sephadex G-200 [18]; hydroxylapatite, Sephadex G-200 [60]) [18, 55, 60] (Sephadex G-25, hydoroxylapatite-DEAE-Sephacel, Q Sepharose, Mono Q, isoelectric focusing [9]) [9] (protamine sulfate, ammonium sulfate, Superdex 75, DEAE-Sephacel, superdex 75, Q-Sepharose, isoenzymes A1-A3 and B1-B3 [94]) [94] (extracellur enzyme, partial [11]) [11] (salting out and Sephadex G-75, DEAE-cellulose, DEAE-Sephadex A-25, hydroxylapatite [12]) [12, 17] (ammonium sulfatem CM-cellulose, DEAE-Sephadex, hydroxylapatite [26]; ammonium sulfate, Sephadex G-100, celite [78]) [26, 78] (proenzyme, ammonim sulfate, DEAE-cellulose, Cu2+ -Sepharose, Sephacryl S-200 [15]; ammonium sulfate, DEAE-cellulose, partially purified [33]) [15, 33] [19] [25] (isoenzyme I, hydroxylapatite, DEAE-Cellulose, Sephadex G-100, isoenzyme III, hydroxylapatite, Sephadex G-100 [21]) [21] (Triton X-100, ammonium sulfate, partial purification [24]; Phenyl Sepharose [59]) [24, 59] (ammonium sulfate, Sephacryl HR S200, DEAE-mensep 1000 [110]) [110] (ammonium sulfate, DEAE-cellulose, Sephadex G 100 [65]) [65] [29] (recombinant enzyme [30]) [30] 182

1.14.18.1

Monophenol monooxygenase

(isoenzymes 1 and 2, ammonium sulfate, phenyl-Sepharose, DEAE-cellulose, hydroxylapatite, isoenzyme 3, partially purified [31]) [31, 36] (ammonium sulfate, CM-cellulose, Sephadex G-100 [35]; ammonium sulfate, DEAE-cellulose, QAE-Sephadex A-50, Toyopearl HW-55 [106]) [35, 106] [38] (tyrosinase A and B [39]) [39, 77] [42] (ammonium sulfate, Celite chromatography, DEAE-Sepharose [40]) [40] (ammonium sulfate, Sephadex G-75, DEAE-cellulose [45]) [45] (isoenzymes A and B, ammonium sulfate, CM-cellulose, Sephacryl S200 [43]) [43] [44] (ammonium sulfate, DEAE-Sephadex, Sephadex G-20, DEAE-Sephadex [48]) [48] (ammonium sulfate, DEAE-cellulose, Sephadex G150, trypsin digestion, Sephadex G-150, DEAE-cellulose, partial purification [66]; recombinant Histagged enzyme, DEAE-Sephacel, Ni2+ -affinity column [103]) [50, 66, 103] [53] (ammonium sulfate, Sephadex G-200 [56]) [56] (TX-114 extraction, partial purification [96]) [96] [64] (ammonium sulfate, Sephadex G-100, DEAE cellulose, partially purified [68]) [68] [70] (zonal centrifugation on a sucruse gradient in the presence of SDS [71]) [71] (2 probably identical isozymes, ammonium sulfate, DEAE-cellulose, CM-sephadex, Bio-gel P300 [72]) [72] [73] [75] (a-, b-, g- and d-tyrosinase, ammonium sulfate, hydroxylapatite [79]) [79] (ammonium sulfate, DEAE-Sepharose, Phenyl-Sepharose, hydroxyapatite [114]) [114] [74] (Sp-Sephadex, Sephadex G-100 [98]) [98] (Sephacryl S-200, Sephadex G-75, DEAE-Sephacel, isoenzymes 1-3 [99]) [99] (ammonium sulfat, Phenyl Sepharose, DEAE-Sepharose [102]) [102] (DEAE-Sephacel, Mono Q [108]) [108] (ammonium sulfate, DEAE-cellulose, partially purified [33]) [33]
 

(in vitro transcription translation of 2 different cDNA clones encoding tyrosinase 1 and 2 [4]; high molecular weight isoenzyme [20]) [4, 20]

183

Monophenol monooxygenase

1.14.18.1

(cloning of cDNA [13]) [13] (overexpression in Streptomyces antibioticus [30]) [30] (cloning of cDNA [95]) [95] (expression in Escherichia coli [103]; expression of wild-type, N86Q, N111Q, N337Q, N371Q and several double and triple mutant enzymes derived from this single mutants in CHO cells [109]) [103, 109] (cloning of cDNA [102]) [102] 

 

N111Q ( mutation in potential N-glycosylation site, 95% of wildtype l-dopa oxidase activity [109]; glycosylation site is required for recognition by individual T cell clones [111]) [109, 111] N111Q/N337Q ( mutations in potential N-glycosylation site, 95% of wild-type l-dopa oxidase activity [109]) [109] N111Q/N337Q/N371Q ( mutations in potential N-glycosylation site, no l-dopa oxidase activity [109]) [109] N111Q/N371Q ( mutations in potential N-glycosylation site, 59% of wild-type l-dopa oxidase activity [109]) [109] N161Q ( similar properties as wild-type [111]) [111] N230Q ( similar properties as wild-type [111]) [111] N290Q ( similar properties as wild-type [111]) [111] N337Q ( mutation in potential N-glycosylation site, 93% of wildtype l-dopa oxidase activity [109]; glycosylation site is required for recognition by individual T cell clones [111]) [109, 111] N337Q/N3711Q ( mutations in potential N-glycosylation site, 37% of wild-type l-dopa oxidase activity [109]) [109] N371Q ( mutation in potential N-glycosylation site, 64% of wildtype l-dopa oxidase activity [109]; glycosylation site is required for recognition by individual T cell clones [111]) [109, 111] N86Q ( mutation in potential N-glycosylation site, 70% of wildtype l-dopa oxidase activity [109]; glycosylation site is required for recognition by individual T cell clones [111]) [109, 111] N86Q/N111Q ( mutations in potential N-glycosylation site, 68% of wild-type l-dopa oxidase activity [109]) [109] N86Q/N111Q/N337Q ( mutations in potential N-glycosylation site, no l-dopa oxidase activity [109]) [109] N86Q/N111Q/N337Q/N371Q ( mutations in potential N-glycosylation site, no l-dopa oxidase activity [109]) [109] N86Q/N111Q/N371Q ( mutations in potential N-glycosylation site, no l-dopa oxidase activity [109]) [109] N86Q/N337Q ( mutations in potential N-glycosylation site, 35% of wild-type l-dopa oxidase activity [109]) [109] N86Q/N337Q/N371Q ( mutations in potential N-glycosylation site, no l-dopa oxidase activity [109]) [109] N86Q/N371Q ( mutations in potential N-glycosylation site, 30% of wild-type l-dopa oxidase activity, contains at least 3 times less copper than wild-type [109]) [109]

184

1.14.18.1

Monophenol monooxygenase

4  0  3-8 ( isozyme PPO III, 90% activity after 18 h at pH 3.0 [21]) [21] 3.5-4.5 ( optimum at pH 4.5, less stable above pH 6.0 [47]) [47] 4-6 ( unstable at pH 6.5, loss of 80% activity at pH 3.0 [36]) [36] 5-10 ( isozyme PPO I, 90% activity after 18 h at pH 10.0 [21]) [21] 6 ( 4 days at 2 C, 70% inactivation [47]) [47] 6-9.5 ( 80% activity after 1 h incubation [35]) [35] 7-9.5 ( cuticle-bound enzyme [6]) [6] 8.5-10 ( more than 70% activity, loss of approx. 75% activity below pH 6.0 and above pH 11.0 [108]) [108]    &   acetone ( no loss of activity after 20 h in 20% acetone [106]) [106] dimethyl sulfoxide ( no loss of activity after 20 h in 20% dimethylsulfoxide [106]) [106] ethanol ( 44% activity in 50% ethanol, no lss in activity after 20 h in 30% ethanol [106]) [106] methanol ( 50% activity in 50% methanol [106]) [106] 5    "

, highly resistant to SDS and proteinase K [5] , maximal stability in Tween 20, 0.25 mg/ml melanosomal lipids stabilize [93] , half-life depends on O2 pressure, 46 h under 21 kPa O2, 7.7 h under 100 kPa O2 [97] , inactivation by freezing/thawing [17] , maximal stabilization in the presence of 0.6 mM sodium dodecylsulfate [32] , tyrosine hydroxylation activity is inactivated faster than catecholase activity [40] , trypsin causes no inactivation [72]    , 4 C, 15 days, 10% loss of activity [1] , 4 C, 3 weeks, no loss of activity [3] , 4 C, saturated ammonium sulfate solution [55, 60] , 4 C, achieves full activity in two days without addition of trypsin, 2.5fold activation at -20 C in 2 months [17] , -20 C, 10 mM phosphate buffer, pH 7.2, 500 mM NaCl, 30% glycerol, more than 6 months, no loss of activity [26] , 4 C, 60% ammonium sulfate, several months, no loss of activity [78] , as microcrystals or as ammonium sulfate precipitate [57] , in polypropylene vials [25] , -20 C, 0.02% bovine serum albumin [49] , liquid N2 or -70 C, phosphate buffer pH 7.2, 150 mM NaCl, several months, no loss of activity [29]

185

Monophenol monooxygenase

1.14.18.1

, 4 C, 1 mM phosphate buffer, pH 7.0 [30] , -40 C, enzyme extract, pH 5.0, over 3 months, no loss of activity [36] , 4 C, 36 mM phosphate, 720 mM KCl, pH 7.0, 15 days, 25% loss of activity, remaining activity stable for 120 days, 50% inactivation by thawing after freezing at -40 C [31] , -20 C, acetone powder, long term storage [38] , 4 C, 2 weeks, 10% loss of activity [48] , -20 C, 0.02% bovine serum albumine, no loss of activity [50] , -20 C, 50 mM phosphate buffer, pH 7.0, more than 3 months, no loss of activity [107] , 0 C, presence of SDS protects against decomposition [56] , -40 C, 2 months, 60% loss of activity [75] , -20 C, several weeks, no loss of activity [113] , -20 C, crude enzyme, several days [27] , liquid N2 [73, 77]

"  [1] Janovitz-Klapp, A.; Richard, F.; Nicholas, J.: Polyphenoloxidase from apple, partial purification and some properties. Phytochemistry, 28, 29032907 (1989) [2] Kelley, S.K.; Coyne, V.E.; Sledjeski, D.D.; Fuqua, W. C.; Weiner, R.M.: Identification of a tyrosinase from periphytic marine bacterium. FEMS Microbiol. Lett., 67, 275-280 (1990) [3] Birse, C.E.; Clutterbuck, A.J.: N-acetyl-6-hydroxytryptophan oxidase, a developmentally controlled phenol oxidase from Aspergillus nidulans. J. Gen. Microbiol., 136, 1725-1730 (1990) [4] Terao, M.; Tabe, L.; Garattini, E.; Sartori, D.; Studer, M.; Mintz, B.: Isolation and characterization of variant cDNAs encoding mouse tyrosinase. Biochem. Biophys. Res. Commun., 159, 848-853 (1989) [5] Yurkow, E.J.; Laskin, J.D.: Purification of tyrosinase to homogeneity based on its resistance to sodium dodecyl sulfate-proteinase K digestion. Arch. Biochem. Biophys., 275, 122-129 (1989) [6] Hara, T.; Tsukamoto, T.; Watanabe, K.; Yamasaki, N.; Funatsu, M.: Properties of cuticular phenoloxidase from pupae of the housefly, Musca domestica L.. Agric. Biol. Chem., 55, 13-17 (1991) [7] Vulfson, E.N.; Ahmed, G.; Gill, I.; Kozlov, I.A.; Goodenough, P.W.; Law, B.A.: Alterations to the catalytic properties of polyphenoloxidase in detergentless microemulsions and ternary water-organic solvent mixtures. Biotechnol. Lett., 13, 91-96 (1991) [8] Harris, E.B.; Sanchez, R.M.; Job, C.K.; Prabhakaran, K.; Hastings, R.C.: Isolation and characterization of an environmental acid-fast organism producing diphenoloxidase activity in vivo. FEMS Microbiol. Lett., 70, 95-100 (1990)

186

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Monophenol monooxygenase

[9] Loeffler, S.; Zenk, M.H.: The hydroxylation step in the biosynthetic pathway leading from norcolaurine to reticuline. Phytochemistry, 29, 34993503 (1990) [10] Tanabe, N.; Sagawa, I.; Ohtsubo, K.i.; Iijima, Y.; Yanagi, S.O.: Comparison of phenoloxidase activities during the cultivation of several basidomycetes. Agric. Biol. Chem., 53, 3061-3063 (1989) [11] Moore, N.L.; Mariam, D.H.; Williams, A.L.; Dashek, W. V.: Substrate specificity, de novo synthesis and partial purification of polyphenol oxidase derived from the wood-decay fungus, Coriolus versicolor. J. Ind. Microbiol., 4, 349-364 (1989) [12] Oda, Y.; Kato, H.; Isoda, Y.; Takahashi, N.; Yamamoto, T.; Takada, Y.; Kudo, S.: Purification and properties of phenoloxidase from spinach leaves. Agric. Biol. Chem., 53, 2053-2061 (1989) [13] Kupper, U.; Niedermann, D.M.; Travaglini, G.; Lerch, K.: Isolation and characterization of the tyrosinase gene from Neurospora crassa. J. Biol. Chem., 264, 17250-17258 (1989) [14] Saul, S.J.; Sugumaran, M.: Characterization of a new enzyme system that desaturates the side chain of N-acetyldopamine. FEBS Lett., 251, 69-73 (1989) [15] Söderhäll, I.; Söderhäll, K.: Purification of prophenol oxidase from Daucus carota cell cultures. Phytochemistry, 28, 1805-1808 (1989) [16] Bru, R.; Sanchez-Ferrer, A.; Garcia-Carmona, F.: Characteristics of tyrosinase in AOT-isooctane reverse micelles. Biotechnol. Bioeng., 34, 304-308 (1989) [17] Sanchez-Ferrer, A.; Villalba, J.; Garcia-Carmona, F.: Triton X-114 as a tool for purifying spinach polyphenol oxidase. Phytochemistry, 28, 1321-1325 (1989) [18] Hara, T.; Tsukamoto, T.; Maruta, K.; Funatsu, M.: Purification and kinetic properties of phenoloxidase from pupae of the housefly. Agric. Biol. Chem., 53, 1387-1393 (1989) [19] Datta, T.K.; Basu, P.S.; Datta, P.K.; Banerjee, A.: Purification of a unique glycoprotein that enhances phenol oxidase activity in scorpion (Heterometrus bengalensis) haemolymph. Biochem. J., 260, 525-529 (1989) [20] Jimenez, M.; Maloy, W.L.; Hearing, V.J.: Specific identification of an authentic clone for mammalian tyrosinase. J. Biol. Chem., 264, 3397-3403 (1989) [21] Okamoto, A.; Imagawa, H.; Arai, Y.; Ozawa, T.: Partial purification and some properties of polyphenoloxidases from sago palm. Agric. Biol. Chem., 52, 2215-2222 (1988) [22] Takeuchi, S.; Yamamoto, H.; Takeuchi, T.: Expression of tyrosinase gene in amelanotic mutant mice. Biochem. Biophys. Res. Commun., 155, 470-475 (1988) [23] Sanchez-Ferrer, A.; Bru, R.; Garcia-Carmona, F.: Kinetic properties of polyphenoloxidase in organic solvents. A study in Brij 96-cyclohexane reverse micelles. FEBS Lett., 233, 363-366 (1988)

187

Monophenol monooxygenase

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[24] Sanchez-Ferrer, A.; Bru, R.; Cabanes, J.; Garcia-Carmona, F.: Characterization of catecholase and cresolase activities of monastrell grape polyphenol oxidase. Phytochemistry, 27, 319-321 (1988) [25] Hearing, V.J.: Mammalian monophenol monooxygenase (tyrosinase): purification, properties, and reactions catalyzed. Methods Enzymol., 142, 154-165 (1987) [26] Lerch, K.: Monophenol monooxygenase from Neurospora crassa. Methods Enzymol., 142, 165-169 (1987) [27] Flurkey, W. H.: Polyphenoloxidase in higher plants. Immunological detection and analysis of in vitro translation products. Plant Physiol., 81, 614618 (1986) [28] Rouet-Mayer, M.A.; Philippon, J.: Inhibition of catechol oxidases from apples by sodium chloride. Phytochemistry, 25, 2717-2719 (1986) [29] Wittenberg, C.; Triplett, E.L.: A detergent-activated tyrosinase from Xenopus laevis. I. Purification and partial characterization. J. Biol. Chem., 260, 12535-12541 (1985) [30] Bernan, V.; Filpula, D.; Herber, W.; Bibb, M.; Katz, E.: The nucleotide sequence of the tyrosinase gene from Streptomyces antibioticus and characterization of the gene product. Gene, 37, 101-110 (1985) [31] Wissemann, K.W.; Montgomery, M.W.: Purification of d'anjou pear (Pyrus communis L.) polyphenol oxidase. Plant Physiol., 78, 256-262 (1985) [32] Wittenberg, C.; Triplett, E.L.: A detergent-activated tyrosinase from Xenopus laevis. II. Detergent activation and binding. J. Biol. Chem., 260, 1254212546 (1985) [33] Söderhäll, K.; Carlberg, I.; Eriksson, T.: Isolation and partial purification of prophenoloxidase from Daucus carota L. cell cultures. Plant Physiol., 78, 730-733 (1985) [34] Kahn, V.; Andrawis, A.: Inhibition of mushroom tyrosinase by tropolone. Phytochemistry, 24, 905-908 (1985) [35] Yoshimoto, T.; Yamamoto, K.; Tsuru, D.: Extracellular tyrosinase from Streptomyces sp. KY-453: purification and some enzymatic properties. J. Biochem., 97, 1747-1754 (1985) [36] Smith, D.M.; Montgomery, M.W.: Improved methods for the extraction of polyphenol oxidase from d'anjou pears. Phytochemistry, 24, 901-904 (1985) [37] Anosike, E.O.; Ayaebene, A.O.: Properties of polyphenol oxidase from tubers of the yam Dioscora bulbifera. Phytochemistry, 21, 1889-1893 (1982) [38] Anosike, E.O.; Ayaebene, A.O.: Purification and some properties of polyphenol oxidase from the yam tubers, Dioscorea bulbifera. Phytochemistry, 20, 2625-2628 (1981) [39] Ohkura, T.; Yamashita, K.; Mishima, Y.; Kobata, A.: Purification of hamster melanoma tyrosinases and structural studies of their asparagine-linked sugar chains. Arch. Biochem. Biophys., 235, 63-77 (1984) [40] Wichers, H.J.; Peetsma, G.J.; Malingre, T.M.; Huizing, H.J.: Purification and properties of a phenol oxidase derived from suspension cultures of Mucuna pruriens. Planta, 162, 334-341 (1984)

188

1.14.18.1

Monophenol monooxygenase

[41] Woolery, G.L.; Powers, L.; Winkler, M.; Solomon, E.I.; Lerch, K.; Spiro, T.G.: Extended X-ray absorption fine structure study of the coupled binuclear copper active site of tyrosinase from Neurospora crassa. Biochim. Biophys. Acta, 788, 155-161 (1984) [42] Yamamoto, H.; Brumbaugh, J.A.: Purification and isoelectric heterogeneity of chicken tyrosinase. Biochim. Biophys. Acta, 800, 282-290 (1984) [43] Hsu, A.F.; Kalan, E.B.; Bills, D.D.: Partial purification and characterization of the soluble polyphenol oxidases from suspension cultures of Papaver somniferum. Plant Sci. Lett., 34, 315-322 (1984) [44] Manjon, A.; Ferragut, J.A.; Garcia-Borron, J.C.; Iborra, J.L.: Conformational studies of soluble and immobilized frog epidermis tyrosinase by fluorescence. Appl. Biochem. Biotechnol., 9, 173-185 (1984) [45] Tremolieres, M.; Bieth, J.G.: Isolation and characterization of the polyphenoloxidase from senescent leaves of black poplar. Phytochemistry, 23, 501-505 (1984) [46] Interesse, F.S.; Ruggiero, P.; Dvella, G.; Lamparelli, F.: Characterization of wheat o-diphenolase isoenzyme. Phytochemistry, 22, 1885-1889 (1983) [47] Goodenough, P.W.; Kessell, S.; Lea, A.G.H.; Loeffler, T.: Mono- and diphenolase activity from fruit of Malus pumila. Phytochemistry, 22, 359-363 (1983) [48] Ikediobi, C.O.; Obasuyi, H.N.: Purification and some properties of o-diphenolase from white yam tubers. Phytochemistry, 21, 2815-2820 (1982) [49] Mayer, A.M.: Polyphenol oxidases in plants - recent progress. Phytochemistry, 26, 11-20 (1987) [50] Vijayan, E.; Husain, I.; Ramaiah, A.; Madan, N.C.: Purification of human skin tyrosinase and its protein inhibitor: properties of the enzyme and the mechanism of inhibition by protein. Arch. Biochem. Biophys., 217, 738747 (1982) [51] Lerch, K.; Longoni, C.; Jordi, E.: Primary structure of tyrosinase from Neurospora crassa. I. Purification and amino acid sequence of the cyanogen bromide fragments. J. Biol. Chem., 257, 6408-6413 (1982) [52] Lerch, K.: Primary structure of tyrosinase from Neurospora crassa. II. Complete amino acid sequence and chemical structure of a tripeptide containing an unusual thioether. J. Biol. Chem., 257, 6414-6419 (1982) [53] Crameri, R.; Ettlinger, L.; Hutter, R.; Lerch, K.; Suter, M.A.; Vetterli, J.A.: Secretion of tyrosinase in Streptomyces glaucescens. J. Gen. Microbiol., 128, 371-379 (1982) [54] Sharma, R.C.; Ali, R.: Hydrodynamic properties of mushroom tyrosinase. Phytochemistry, 20, 399-401 (1981) [55] Yonekura, M.; Shimoda, T.; Funatsu, M.: Reinvestigation of purification of phenoloxidase from larvae of housefly. Agric. Biol. Chem., 45, 101-104 (1981) [56] Sharma, R.C.; Ali, R.: Isolation and characterization of catechol oxidase from Solanum melongena. Phytochemistry, 19, 1597-1600 (1980) [57] Himmelwright, R.S.; Eickman, N.C.; LuBien, C.D.; Lerch, K.; Solomon, E.I.: Chemical and spectroscopic studies of the binuclear copper active

189

Monophenol monooxygenase

[58] [59] [60] [61] [62] [63] [64] [65] [66] [67]

[68] [69] [70] [71] [72] [73] [74]

190

1.14.18.1

site of Neurospora tyrosinase: comparison to hemocyanins. J. Am. Chem. Soc., 102, 7339-7344 (1980) Kahn, V.; Pomerantz, S.H.: Monophenolase activity of avocado polyphenol oxidase. Phytochemistry, 19, 379-385 (1980) Wissemann, K.W.; Lee, C.Y.: Purification of grape polyphenoloxidase with hydrophobic chromatography. J. Chromatogr., 192, 232-235 (1980) Yamaura, I.; Yonekura, M.; Katsura, Y.; Ishiguro, M.; Funatsu, M.: Purification and some physico-chemical properties of phenoloxidase from the larvae of housefly. Agric. Biol. Chem., 44, 55-59 (1980) Mayer, A.M.; Harel, E.: Polyphenol oxidases in plants. Phytochemistry, 18, 193-215 (1979) Menon, I.A.; Haberman, H.F.: Activation of tyrosinase in microsomes and melanosomes from B16 and Harding-Passey melanomas. Arch. Biochem. Biophys., 137, 231-242 (1970) Yamamoto, H.; Takeuchi, S.; Kudo, T.; Makino, K.; Nakata, A.; Shinoda, T.; Takeuchi, T.: Cloning and sequencing of mouse tyrosinase cDNA. Jpn. J. Genet., 62, 271-274 (1987) Chaplin, M.F.: Preparation and properties of an o-diphenol:O2 oxidoreductase from cocoa husk. Phytochemistry, 17, 1897-1899 (1978) Signoret, A.; Crouzet, J.: Activites polyphenoloxydasique et peroxydasique du fruit de la tomate (Lycopersicum esculentum) purification et quelques proprietes. Agric. Biol. Chem., 42, 1871-1877 (1978) Nishioka, K.: Particulate tyrosinase of human malignant melanoma. Solubilization, purification following trypsin treatment, and characterization. Eur. J. Biochem., 85, 137-146 (1978) Hearing, V.J.; Ekel, T.M.; Montague, P.M.; Hearing, E.D.; Nicholson, J.M.: Mammalian tyrosinase: isolation by a simple new procedure and characterization of its steric requirements for cofactor activity. Arch. Biochem. Biophys., 185, 407-418 (1978) Ben-Shalom, N.; Kahn, V.; Harel, E.; Mayer, A.M.: Catechol oxidase from green olives: properties and partial purification. Phytochemistry, 16, 1153-1158 (1977) Räihä, M.; Sundman, V.: Characterization of lignosulfonate-induced phenol oxidase a in the atypical white-rot fungus Polyporus dichrous. Arch. Microbiol., 105, 73-76 (1975) Padron, M.P.; Lozano, J.A.; Gonzales, A.G.: Properties of o-diphenol:O2 oxidoreductase from Musa cavendishii. Phytochemistry, 14, 1959-1963 (1975) Pau, R.N.; Eagles, P.A.M.: The isolation of an o-diphenal oxidase from third-instar larvae of the blowfly Calliphora erythrocephala. Biochem. J., 149, 707-712 (1975) Mikkelsen, R.B.; Triplett, E.L.: Tyrosinases in Rana pipiens. Purification and physical properties. J. Biol. Chem., 250, 638-643 (1975) Pomerantz, S.H.; Murthy, V.V.: Purification and properties of tyrosinases from Vibrio tyrosinaticus. Arch. Biochem. Biophys., 160, 73-82 (1974) Madhosingh, C.; Sundberg, L.: Purification and properties of tyrosinase inhibitor from mushroom. FEBS Lett., 49, 156-158 (1974)

1.14.18.1

Monophenol monooxygenase

[75] Coggon, P.; Moss, G.A.; Sanderson, G.W.: Tea catechol oxidase: isolation, purification and kinetic characterization. Phytochemistry, 12, 1947-1955 (1973) [76] Stelzig, D.A.; Akhtar, S.; Ribeiro, S.: Catechol oxidase of red delicious apple peel. Phytochemistry, 11, 535-539 (1972) [77] Pomerantz, S.H.; Li, J.P.C.: Tyrosinases (hamster melanoma). Methods Enzymol., 17A, 620-626 (1970) [78] Horowitz, N.H.; Fling, M.; Horn, G.: Tyrosinase (Neurospora crassa). Methods Enzymol., 17A, 615-620 (1970) [79] Nelson, R.M.; Mason, H.S.: Tyrosinase (mushroom). Methods Enzymol., 17A, 626-632 (1970) [80] Vanneste, W.H.; Zuberbuhler, A.: Copper-containing oxygenases. Mol. Mech. Oxygen Activ. (Hayaishi, O., ed.) Academic Press, New York, 371404 (1974) [81] Kwon, B.S.; Walkulchik, M.; Haq, A.K.; Halaban, R.; Kestler, D.: Sequence analysis of mouse tyrosinase cDNA and the effect of melanotropin on its gene expression. Biochem. Biophys. Res. Commun., 153, 1301-1309 (1988) [82] Muller, G.; Ruppert, S.; Schmid, E.; Schutz, G.: Functional analysis of alternatively spliced tyrosinase gene transcripts. EMBO J., 7, 2723-2730 (1988) [83] Fuller, B.B.; Lundsford, J.B.; Iman, D.S.: a-Melanocyte-stimulating hormone regulation of tyrosinase in Cloudman S-91 mouse melanoma cell cultures. J. Biol. Chem., 262, 4024-4033 (1987) [84] Flurkey, W.H.: In vitro biosynthesis of Vicia faba polyphenoloxidase. Plant Physiol., 79, 564-567 (1985) [85] Vaughn, K.C.; Duke, S.O.: Tentoxin-induced loss of plastidic polyphenol oxidase. Physiol. Plant., 53, 421-428 (1981) [86] King, R.S.; Flurkey, W.H.: Effects of limited proteolysis on broad bean polyphenoloxidase. J. Sci. Food Agric., 41, 231-240 (1987) [87] Kwon, B.S.; Haq, A.K.; Pomerantz, S.H.; Halaban, R.: Isolation and sequence of a cDNA clone for human tyrosinase that maps at the mouse calbino locus [published erratum appears in Proc Natl Acad Sci U S A 1988 Sep;85(17):6352]. Proc. Natl. Acad. Sci. USA, 84, 7473-7477 (1987) [88] Strothkamp, K.Jolly, R.; Mason, H.S.: Quaternary structure of mushroom tyrosinase. Biochem. Biophys. Res. Commun., 70, 519-524 (1976) [89] McIntyre, R.J.; Vaughan, P.F.T.: Kinetic studies on the hydroxylation of pcoumaric acid to caffeic acid by spinach-beet phenolase. Biochem. J., 149, 447-461 (1975) [90] Jimenez-Cervantes, C.; Garcia-Borron, J.C.; Valverde, P.; Solano, F.; Lozano, J.A.: Tyrosinase isoenzymes in mammalian melanocytes. 1. Biochemical characterization of two melanosomal tyrosinases from B16 mouse melanoma. Eur. J. Biochem., 217, 549-556 (1993) [91] Palmieri, G.; Giardina, P.; Marzullo, L.; Desiderio, B.; Nitti, G.; Cannio, R.; Sannia, G.: Stability and activity of a phenol oxidase from the ligninolytic fungus Pleurotus ostreatus. Appl. Microbiol. Biotechnol., 39, 632-636 (1993)

191

Monophenol monooxygenase

1.14.18.1

[92] Naish-Byfield, S.; Cooksey, C.J.; Riley, P.A.: Oxidation of monohydric phenol substrates by tyrosinase: effect of dithiothreitol on kinetics. Biochem. J., 304, 155-162 (1994) [93] Jimenez-Cervantes, C.; Garcia-Borron, J.C.; Lozano, J.A.; Solano, F.: Effect of detergents and endogenous lipids on the activity and properties of tyrosinase and its related proteins. Biochim. Biophys. Acta, 1243, 421-430 (1995) [94] Kanda, K.; Sato, T.; Ishii, S.; Enei, H.; Ejiri, S.: Purification and properties of tyrosinase isozymes from the gill of Lentinus edodes fruiting body. Biosci. Biotechnol. Biochem., 60, 1273-1278 (1996) [95] April, C.S.; Franz, T.; Kidson, S.H.: The cloning and characterization of chick tyrosinase from a novel embryonic cDNA library. Exp. Cell Res., 224, 372-378 (1996) [96] Espin, J.C.; Trujano, M.F.; Tudela, J.; Garcia-Canovas, F.: Monophenolase activity of polyphenol oxidase from Haas avocado. J. Agric. Food Chem., 45, 1091-1096 (1997) [97] Pialis, P.; Saville, B.A.: Production of l-DOPA from tyrosinase immobilized on nylon 6,6: enzyme stability and scaleup. Enzyme Microb. Technol., 22, 261-268 (1998) [98] Hata, S.; Azumi, K.; Yokosawa, H.: Ascidian phenoloxidase: its release from hemocytes, isolation, characterization and physiological roles. Comp. Biochem. Physiol. B, 119, 769-776 (1998) [99] Kong, K.H.; Lee, J.L.; Park, H.J.; Cho, S.H.: Purification and characterization of the tyrosinase isozymes of pine needles. Biochem. Mol. Biol. Int., 45, 717-724 (1998) [100] Rescigno, A.; Sanjust, E.; Soddu, G.; Rinaldi, A.C.; Sollai, F.; Curreli, N.; Rinaldi, A.: Effect of 3-hydroxyanthranilic acid on mushroom tyrosinase activity. Biochim. Biophys. Acta, 1384, 268-276 (1998) [101] Espin, J.C.; Jolivet, S.; Wichers, H.J.: Kinetic study of the oxidation of g-lglutaminyl-4-hydroxybenzene catalyzed by mushroom (Agaricus bisporus) tyrosinase. J. Agric. Food Chem., 47, 3495-3502 (1999) [102] Chevalier, T.; de Rigal, D.; Mbeguie-A-Mbeguie, D.; Gauillard, F.; Richard.Forget, F.; Fils-Lycaon, B.R.: Molecular cloning and characterization of apricot fruit polyphenol oxidase. Plant Physiol., 119, 1261-1269 (1999) [103] Kong, K.H.; Park, S.Y.; Hong, M.P.; Cho, S.H.: Expression and characterization of human tyrosinase from a bacterial expression system. Comp. Biochem. Physiol. B, 125, 563-569 (2000) [104] Espin, J.C.; Varon, R.; Fenoll, L.G.; Gilabert, M.A.; Garcia-Ruiz, P.A.; Tudela, J.; Garcia-Canovas, F.: Kinetic characterization of the substrate specificity and mechanism of mushroom tyrosinase. Eur. J. Biochem., 267, 12701279 (2000) [105] Sugumaran, M.; Nellaiappan, K.; Valivittan, K.: A new mechanism for the control of phenoloxidase activity: inhibition and complex formation with quinone isomerase. Arch. Biochem. Biophys., 379, 252-260 (2000) [106] Ito, M.; Oda, K.: An organic solvent resistant tyrosinase from Streptomyces sp. REN-21: purification and characterization. Biosci. Biotechnol. Biochem., 64, 261-267 (2000) 192

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Monophenol monooxygenase

[107] Perez-Gilabert, M.; Garcia Carmona, F.: Characterization of catecholase and cresolase activities of eggplant polyphenol oxidase. J. Agric. Food Chem., 48, 695-700 (2000) [108] Kong, K.H.; Hong, M.P.; Choi, S.S.; Kim, Y.T.; Cho, S.H.: Purification and characterization of a highly stable tyrosinase from Thermomicrobium roseum. Biotechnol. Appl. Biochem., 31, 113-118 (2000) [109] Branza-Nichita, N.; Negroiu, G.; Petrescu, A.J.; Garman, E.F.; Platt, F.M.; Wormald, M.R.; Dwek, R.A.; Petrescu, S.M.: Mutations at critical N-glycosylation sites reduce tyrosinase activity by altering folding and quality control. J. Biol. Chem., 275, 8169-8175 (2000) [110] Chazarra, S.; Garcia-Cormona, F.; Cabanes, J.: Evidence for a tetrameric form of iceberg lettuce (Lactuca sativa L.) polyphenol oxidase: purification and characterization. J. Agric. Food Chem., 49, 4870-4875 (2001) [111] Housseau, F.; Moorthy, A.; Langer, D.A.; Robbins, P.F.; Gonzales, M.I.; Topalian, S.L.: N-linked carbohydrates in tyrosinase are required for its recognition by human MHC class II-restricted CD4+ T cells. Eur. J. Immunol., 31, 2690-2701 (2001) [112] Malkin, B.D.; Thickman, K.R.; Markworth, C.J.; Wilcox, D.E.: Kull, F.J.: Inhibition of potato polyphenol oxidase by anions and activity in various carboxylate buffers (pH 4.8) at constant ionic strength. J. Enzyme Inhib., 16, 135-145 (2001) [113] Gerdemann, C.; Eicken, C.; Krebs, B.: The crystal structure of catechol oxidase: New insight into the function of type-3 copper proteins. Acc. Chem. Res., 35, 183-191 (2002) [114] Shi, Y.L.; Benzie, I.F.F.; Buswell, J.A.: Role of tyrosinase in the genoprotective effect of the edible mushroom, Agaricus bisporus. Life Sci., 70, 15951608 (2002) [115] Debowska, R.; Podstolski, A.: Properties of diphenolase from Vanilla planifolia (Andr.) shoot primordia cultured in vitro. J. Agric. Food Chem., 49, 3432-3437 (2001)

193

 
 : 

:

   
 1.14.19.1    stearoyl-CoA,hydrogen-donor:oxygen oxidoreductase    stearoyl-CoA 9-desaturase    D9 terminal desaturase [4, 11] D9 -acyl CoA desaturase D9 -desaturase EC 1.14.99.5 (formerly) acyl coenzyme A desaturase acyl-CoA desaturase eicosatrienoyl-CoA desaturase fatty acid 9-desaturase fatty acid D9 -desaturase fatty acid desaturase fatty acyl CoA D9 -desaturase fatty acyl D9 -desaturase fatty acyl-CoA desaturase long-chain fatty acid D9 -desaturase palmitoyl CoA desaturase palmitoyl-CoA desaturase stearoyl coenzyme A desaturase stearoyl-CoA (D8 ) desaturase stearoyl-CoA desaturase stearyl coenzyme A desaturase stearyl-CoA desaturase
    9014-34-0

     Mycobacterium phlei ( ATCC 356 [1]) [1] Rattus norvegicus (Sprague-Dawley, male [2, 5]; Long-Evans [3, 6]; Wister, male [7]; Colworth-Wistar, male [13]) [2, 3, 5-7, 12, 13, 15, 16, 19, 20, 22] 194

1.14.19.1











Stearoyl-CoA 9-desaturase

Gallus gallus (embryo [15]) [4, 11, 15, 16] Micrococcus crysophilus (ATCC 15174 [8, 14]) [8, 14] Fusarium oxysporum (f. sp. lycopersici [9]) [9] Sus scrofa [17] Neurospora crassa [10] Bos taurus (cow [18]) [18] Cyprinus carpio (L. [20, 27]) [20, 26, 27] Helianthus annuus (L., sunflower [21]) [21] hamster [22] Homo sapiens [23] Mortierella alpina (strain CBS 528.72, ATCC 32222 [24]) [24] Picea glauca (white pruce [25]) [25] Spinacia oleracea [28] Mus musculus [29] Arabidopsis sp. [30]

!    "
 # 

stearoyl-CoA + AH2 + O2 = oleoyl-CoA + A + 2 H2 O (An iron protein. The rat liver enzyme is an enzyme system involving cytochrome b5 and EC 1.6.2.2, cytochrome-b5 reductase. Formerly EC 1.14.99.5; the reducing equivalents required for the desaturation site seem to be supplied from NADPH via cytochrome b5 as electron carrier [2]; arginyl and tyrosyl residues expected in the active site of enzyme [4]; multienzyme, insertion of a double bound in 9,10 position [5]; cytochrome b5, cytochrome b5 reductase and lipid and/or detergent are needed for all assays. A cis-hydrogen abstraction mechanism is involved in desaturation [6]; electron flow from NADH to cytochrome b5, via cytochrome b5 reductase. Different types of acyl-CoA desaturation show immunological differences [7]; incorporation of fatty acid into phospholipid. Enzyme may contain an essential thiol group. Endogenous saturated phospholipid is desaturated rapidly in all experiments in contrast with exogenous saturated phospholipid, which generally is not desaturated [8]; presence of an unusual microsomal electron transport system associated with the desaturase [9]; significant amounts of D8 -isomers are present [14]; introduction of double bonds at fixed positions from the carboxyl group of the fatty acid. d-hydrogen atoms are removed during desaturation. Mixed-function oxidase, acting via hydroxy acid intermediates. Acetyl-CoA desaturase, carboxylase and fatty acid synthetase are all controlled together as a unit and influenced by dietary components [15]; introduction of double bond at position 9 [16]; presence of two desaturase genes, isomers CDS1 and CDS2. Cooling treatment of carp causes significant changes to the activity of the D9 -desaturase. Modest cooling of carp shows an increase in acticity without any increase in the amount of desaturase protein, suggesting the activation of the pre-existing inactive latent enzyme, possibly by a post-translational mechanism [20]; the 195

Stearoyl-CoA 9-desaturase

1.14.19.1

synthesis is transcriptionally regulated by fat-free diet and sterols. Cholesterol-supplemented media as well as the transient transfection induces the expression of stearoyl-CoA desaturase RNA [22]; it is suggested that in HepG2 cells the trans-10,cis-12 conjugated linoleic acid isomer regulates human desaturase activity mainly by a posttranslational mechanism [23]; isolation of a third fatty acid D9 -desaturase. Higher activity with longer chain fatty acid substrates. Desaturase expression is induced significantly by increasing the ratio of carbon to nitrogen [24]; regioselectivity, substrate specificity, probably extra-plastidal [25]; increase of activity induced by cold is preceded by the activation of latent desaturase, probably by a posttranslational mechanism. Amounts of desaturase transcript are increased as a result of cold-induced gene transcription [26]; cold exposure leads to an induced activity, in vivo cooling causes an activity induction of up to 15 to 30fold. Increase of desaturase synthesis by means of activated gene transcription. Transfer of cells to 10 C causes a smaller increase in activity compared to cells maintained at 30 C [27]; the possible mechanism would be that dioxygen has to bind to both irons for effective two-electron transfer to the half-occupied antibonding pi orbitals of dioxygen to give a peroxide level intermediate [28]; existance of three highly homologous isoforms: SCD1, SCD2 and SCD3 which show different substrate specificities, here SCD1 is mainly analysed [29]; SSI2 gene encodes a member of a family of soluble fatty acid desaturases [30])    oxidation redox reaction reduction       palmitoyl-CoA + AH2 + O2 (Reversibility: ? [29]) [29] $ palmitoleoyl-CoA + A + H2 O [29]  stearate + AH2 + O2 ( biosynthesis of unsaturated fatty acid, synthesis of triacylglycerol from oleate [15]) (Reversibility: ? [14, 15]) [14, 15] $ oleate + A + H2 O [14, 15]  stearoyl-CoA + AH2 + O2 ( also other fatty acyl-CoA substrates [5]; oleic acid biosynthesis [9]) (Reversibility: ? [1-6, 9, 11, 12, 29]) [1-6, 9, 11, 12, 29] $ oleoyl-CoA + A + H2 O [1-6, 9, 11, 12, 29]  stearoyl-acyl carrier protein + AH2 + O2 ( insertion of a cis double bond between the 9 and 10 position, essential step in fatty acid biosynthesis [28]; key regulator of fatty acid desaturation of carbons 9 and 10 [30]) (Reversibility: ? [28, 30]) [28, 30] $ oleoyl-acyl carrier protein + A + H2 O [28, 30]  Additional information ( biosynthesis of unsaturated fatty acids. Possible substrates: acyl-CoA, acyl-acyl-carrier and acyl chains of phospholipids. The substrate in vivo is saturated phospholipid [8]; key enzyme in the synthesis of unsaturated fatty acyl-CoAs 196

1.14.19.1

Stearoyl-CoA 9-desaturase

[22]; rate limiting-step in the cellular synthesis of monounsaturated fatty acids mainly oleate and palmitoleate [23]; long-chain polyunsaturated fatty acid biosynthetic pathway [24]; incorporation of the first unsaturation bond into saturated fatty acids [26]; rate-limiting enzyme in the biosynthesis of monounsaturated fatty acids [29]) [8, 22-24, 26, 29] $ ?      ammonium stearate + NADH + O2 (Reversibility: ? [12]) [12] $ ?  heptadecanoate + AH2 + O2 ( low activity [15]) (Reversibility: ? [15]) [15] $ ?  myristoyl-CoA + AH2 + O2 ( 59% activity of the rate with stearoyl-CoA [4]) (Reversibility: ? [4, 13, 14]) [4, 13, 14] $ ?  myristoyl-CoA + NADH + O2 ( 59% activity of stearoyl-CoA [16]) (Reversibility: ? [16]) [16] $ ?  nonadecanoate + AH2 + O2 ( low activity [15]) (Reversibility: ? [15]) [15] $ ?  nonadecanoyl-CoA + NADH + O2 (Reversibility: ? [13]) [13] $ ?  palmitic acid + AH2 + O2 (Reversibility: ? [24, 25]) [24, 25] $ palmitoleic acid + A + H2 O [24, 25]  palmitoyl-CoA + AH2 + O2 ( 62% activity of the rate with stearoyl-CoA [4]) (Reversibility: ? [4, 14, 20]) [4, 14, 20] $ palmitoleoyl-CoA + A [20]  palmitoyl-CoA + NAD(P)H + O2 (Reversibility: ? [18]) [18] $ palmitoleate + NAD(P)+ + H2 O [18]  palmitoyl-CoA + NADH + O2 ( 62% activity of stearoylCoA [16]) (Reversibility: ? [13, 16, 17, 29]) [13, 16, 17, 29] $ palmitoleoyl-CoA + NAD+ + H2 O [29]  palmitoyl-CoA + NADPH + O2 (Reversibility: ? [1]) [1] $ ?  stearate + AH2 + O2 (Reversibility: ? [8]) [8] $ oleoyl-phospholipid + A + H2 O [8]  stearate + AH2 + O2 ( maximum activity [15]) (Reversibility: ? [14, 15, 24, 25, 27]) [14, 15, 24, 25, 27] $ oleate + A + H2 O [14, 15, 24, 25, 27]  stearoyl-CoA + AH2 + O2 (Reversibility: ? [10, 20, 30]) [10, 20, 30] $ oleoyl-CoA + A + H2 O [10, 20, 30]

197

Stearoyl-CoA 9-desaturase

1.14.19.1

 stearoyl-CoA + NAD(P)H + O2 ( fatty acid desaturation [2]) (Reversibility: ? [2, 9]) [2, 9] $ oleoyl-CoA + NAD(P)+ + H2 O [2, 9]  stearoyl-CoA + NADH + O2 ( terminal component in the enzyme system [3,4]; maximal activity [13]; preferred substrate for SCD1 [29]) (Reversibility: ? [3-6, 11-13, 16-18, 29]) [3-6, 11-13, 16-18, 29] $ oleoyl-CoA + NAD+ + H2 O [3-6, 11-13, 16, 17, 29]  stearoyl-CoA + NADPH + O2 (Reversibility: ? [1, 18]) [1, 18] $ oleoyl-CoA + NADP+ + H2 O [1, 18]  stearoyl-acyl carrier protein + AH2 + O2 (Reversibility: ? [30]) [30] $ oleoyl-acyl carrier protein + A + H2 O [30]  Additional information ( 12-19 carbon fatty acyl chains undergo 9,10 desaturation. Dephospho-, deamino- and (1-N6 -etheno)-CoA analogs of stearoyl-CoA are poorer substrates than stearoyl-CoA [6]; not acyl-CoA as substrate but phospholipid [8]; specific for C14 -C19 fatty acyl-CoA substrates [13]; temperature-dependent chain length change is not mediated by a temperature-dependent change in desaturase substrate specificity [14]; stearic and palmitic acids are no substrates [17]; glycerolipid such as phosphatidylcholine is involved as an intermediate substrate [25]; SCD2 and SCD3 preferentially utilize palmitoyl-CoA [29]; preference for 18-carbon versus 16-carbon chain length fatty acids [30]) [6, 8, 13, 14, 17, 25, 29, 30] $ ? 2  1,2 diglycerides ( only slight depression [18]) [18] 2,3-butanedione ( reversible [4]) [4] 2-mercaptoethanol [1] 5,5-dimethyl-1-pyrroline-N-oxide [11] CN- ( complete and reversible inhibition at 0.5 mM [3]; at high concentrations [6,8]; 44% inhibition at 1 mM [9]; sensitive [21]) [1-4, 6, 8, 9, 16, 17, 21] Cu+ ( strong, complete inhibition at 0.1 mM [1]) [1] Cu2+ ( and its acid chelates [4]; and its complexes of tyrosine, histidine and lysine, act as superoxide scavengers, inhibition at low concentrations [11]) [4, 11] EDTA ( 46% inhibition at 10 mM [9]; more than 95% inhibition [18]) [1, 9, 18] Fe2+ ( 76% inhibition at 10 mM [9]) [9] HgCl2 [2] l-cysteine ( not marked [8]) [8] NADH [1] adrenaline [11] azide ( at high concentrations [8]) [2, 4, 8, 16]

198

1.14.19.1

Stearoyl-CoA 9-desaturase

bis(salicylidene)-1,3-propanediamine [21] cycloheximide [15] cytochrome c ( 32% inhibition at 0.01 mM [9]) [9] decanoyl-CoA ( competitive inhibitor [4]; also copper complexes of tyrosine, histidine and lysine [11]; competitive inhibition of enzyme activity greater than 95% at 0.002 mM [16]) [4, 11, 16] dihydrolipoic acid [1] dodecanoyl-CoA ( competitive inhibitor [4]; competitive inhibition of enzyme activity greater than 90% at 0.002 mM [16]) [4, 16] ethanol ( depression [18]) [18] ethyl linoleate [15] ethyl palmitate [15] glutathione ( reduced, not marked [8]) [8] lysophosphatidylcholine [18] menadione ( 85% inhibition at 0.1 mM [1]; 66% inhibition at 0.1 mM [9]) [1, 9] methylene blue [1] o-phenanthroline ( 80% inhibition at 2 mM [1]; partially inhibitory [8]) [1, 8] oleoyl-CoA ( not competitively [16]; depression [18]) [16, 18] p-chloromercuribenzoate ( strong, complete inhibition at 0.02 mM [1]; a sulfonate, reversed by b-mercaptoethanol [4]; prevention by stearoyl-CoA [16]) [1, 4, 16] p-chloromercuribenzoate ( reversed by b-mercaptoethanol [16]) [16] p-hydroxymercuribenzoate [16] palmitoyl-CoA ( depression [18]) [18] phenazine methosulfate ( nearly complete inhibition at 0.3 mM [1]; 100% inhibition at 0.4 mM [9]) [1, 9] phenyllactate ( partial inhibition, maximal at 0.08 mM [12]) [12] phenylpyruvate ( partial inhibition [12]) [12] riboflavin ( 25-30% inhibition at 0.04 mM [1]) [1] stearoyl-CoA ( substrate inhibition above 0.05 mM [18]) [18] sterculic acid ( potent inhibitor [14]) [14] tetranitromethane [4] thenoyltrifluoroacetone ( i.e. 4,4,4-trifluoro-1-(2-thienyl)-1,3butanedione [9, 10]; 59% inhibition at 6 mM [9]) [9, 10] trifluoperazine [21] Additional information ( sulfhydryl compounds [1]; phenylalanine, indolepyruvic acid and 2-amino-1-phenylethanol show no inhibitory effect [12]; N-ethylmaleimide and iodoacetamide have no inhibitory effect [16]) [1, 12, 16]
"%  FAD ( required [1]) [1] FMN ( required, almost as active as FAD [1]) [1] NAD+ ( supports desaturation [18]) [18]

199

Stearoyl-CoA 9-desaturase

1.14.19.1

NADH ( dependent [3]; preferred electron donor [18]) [2-6, 9, 11, 12, 13, 15-18, 20, 25, 27, 29] NADP+ ( supports desaturation [18]) [18] NADPH ( specifically required, seems not to be directly involved in the desaturation reaction but serves to keep another cofactor in the reduced state, maybe FADH [1]; absolute requirement, preferred [9]) [1, 2, 9, 18, 21, 28] ascorbate [2, 11] cytochrome b5 ( required [2, 3, 13, 16]; hemoprotein [4]; dependent [17]; not substituent for ferredoxin [21]; fusion, C-terminal [24]; no evidence for [25]) [2-4, 13, 16, 17, 20, 21, 24, 25] flavin ( flavin-containing NADH-cytochrome b5 reductase (EC 1.6.2.2) [15]) [15] heme ( b-type, nonheme iron-sulfur component [9]; heme-containing cytochrome b5 [15]; non-heme iron-containing [21]) [9, 15, 21] Additional information ( phospholipid is required [3]; requires some other form of reduced cofactor than a reduced nicotinamide nucleotide, no special cofactor requirement [8]) [3, 8] &

  1,10-phenanthroline ( 26% stimulation [9]) [9] bovine serum albumin ( stimulates activity in the microsomal fraction [13, 15]) [13, 15] calf serum [27] catalase ( stimulation [11]) [11] cytochrome b5 reductase ( NADH-specific, required, EC 1.6.2.2 [2, 3, 13, 16]; flavoprotein [4]; NADH-dependent [20]) [2-4, 13, 16, 17, 20] ferredoxin ( in absence loss of desaturase activity [21]) [21] ferredoxin oxidoreductase [21] insulin ( causes 5-fold increace of activity [15]) [15] p-cresol ( as well as other phenols, interact with desaturase system causing a stimulation of oxidation of reduced cytochrome b5 [11]) [11] protein ( non-substrate soluble cytosolic protein stimulates [5, 15]; basic cytoplasmic protein from rat liver stimulates activity in crude extract, no activity of partially purified enzyme [13]) [5, 13, 15] stearoyl-CoA-binding protein ( stimulates activity in the crude microsomal fraction [13, 15]) [13, 15] thyroxine [15] triiodothyronine [15] Additional information ( desaturase expression is sensitive to hormonal manipulation [27]) [27]

200

1.14.19.1

Stearoyl-CoA 9-desaturase

'(   Cu2+ [4] Fe2+ ( specifically required [1]; contains one atom of non-heme iron per enzyme molecule, catalytic function [3, 4, 6, 15]; iron-sulfur protein(s) may act as electron carrier(s) in place of the cytchrome b5 and NADH cytochrome b5 reductase system associated with the mammalian desaturase systems [9]; contains a non-heme iron [16, 21]; each subunit of homodimer contains one binuclear non-heme iron active site. The reduced D9 -desaturase has two approximately equivalent 5-coordinate irons in a distorted square pyramidal geometry, a two-in-two-out equatorial distortion, the irons are rhombic, weakly antiferromagnetically coupled. Addition of substrate causes dramatic changes, first ion remains 5coordinate but is distorted toward a trigonal bipyramidal structure, second iron changes to 4-coordinate [28]; 20-25% stimulation [8]) [1, 3, 4, 6, 8, 9, 15, 16, 21, 28] Zn2+ ( 20-25% stimulation [8]) [4, 8] superoxid dismutase ( 8% stimulation [9]) [9] Additional information ( no special ion requirement [8]; enzyme contains active sulfhydryl groups [16]) [8, 16] ) &  * +, 21 (stearoyl-CoA, 25 C [3]; fatty acyl-CoA substrates with chain length of 14-19 carbon atoms show similar turnover number of about 22 [6]) [3, 6] 30 (stearoyl-acyl carrier protein) [28] " & *-% , 0.0002375 [25] 0.0004 [18] 0.00044 ( enzyme of cells grown at 37 C [9]) [9] 0.0009 [17] 0.0023 ( fed microsomes [2]) [2] 0.0079 ( induced microsomes [2]) [2] 0.0315 [13] 0.1 [16] 0.11 [4] 0.24-0.36 [3] 0.325 [6] 0.8 [30] Additional information ( enzyme activity is highest in microsomes from cells grown at 37 C and lowest in cells grown at 15 C [9]; maximal activity at a concentration of 0.03 mM stearoyl-CoA [12]; cold induces an 8 to 10fold increase in specific activity [26]) [9, 12, 26] . / *', 0.001 (FAD) [1] 0.0025 (FMN) [1] 0.003 (NADH) [18]

201

Stearoyl-CoA 9-desaturase

1.14.19.1

0.0033 (stearoyl-CoA) [17] 0.025 (stearoyl-CoA) [18] 0.038 (NADPH) [9] 0.089 (NADH) [9] 0.8 (NADPH) [1] Additional information ( substrates with 14-19 carbon fatty acyl chains show similar Km values of 0.0045-0.005 mM [6]) [6] 0  6.8 ( range from pH 6.5 to 7.5 [18]) [18] 7 ( with NADH and NADPH [2]) [2] 7.2 ( assay at [1, 2, 4, 29]) [1, 2, 4, 16, 29] 7.35 ( assay at [12]) [12] 7.4 ( assay at [17, 18]) [17, 18] 7.5 ( assay at [13]) [13] 7.6 ( assay at [27]) [27] 7.8 ( with ascorbate [2]) [2] 8.2-8.3 ( assay at pH 8.4 [9]) [9] 8.7 ( but assay at pH 7.4, to standardize the experiments in vitro with those carried out with intact bacteria [8]) [8] 0 

 5.5-9 ( pH 5.5: about 80% of maximum activity, pH 9.0: about 45% of maximum activity [18]) [18] )  *
, 21 ( assay at [8]) [8] 23 ( assay at [29]) [29] 25 ( assay at [9, 25]) [3, 6, 9, 25] 30 ( assay at [1, 2, 13, 18, 27]) [1, 2, 13, 18, 27] 37 ( assay at [5, 17]) [5, 17] ) 

 *
, 8-50 ( rate of desaturation of stearoyl-CoA increases [18]) [18]

  #   ' 1  26500 ( gel filtration [5]) [5] 33000 [20]   ? ( x * 53000, disc-gel electrophoresis [3]; x * 33600, SDS-PAGE [4, 16]; x * 53000 [15]; x * 55000 [16]; x * 35000, SDS-PAGE [21]; x * 43700, calculated [25]; x * 33650, calculated [26]; x * 33800 for cold treated carp, SDS-PAGE [27]) [3, 4, 15, 16, 21, 25, 26, 27] dimer ( homodimer, 2 * 40000 [28]) [28]

202

1.14.19.1

Stearoyl-CoA 9-desaturase

monomer ( 1 * 27000, SDS-PAGE [5]) [5] $   "

phospholipoprotein ( tightly bound phospholipid is removed during purification [3]; 4-12 mol of phospholipid per mol of polypeptide [6]) [3, 6] Additional information ( no detectable carbohydrate [6]) [6]

 2 %$ %' %

% Hep-G2 cell [23] adipose ( epididymal [2]; contains high amounts [29]) [2, 29] brain [29] eye ( ball [29]) [29] gland ( contains high amounts [29]) [29] leaf [30] liver [2-7, 11-13, 15-17, 20, 22, 26, 27, 29] mammary gland ( lactating [18]) [18] ovary [22] seed ( cotyledons [21]) [21, 25] spleen [29] stomach [17] testis [29] Additional information ( no activity in liver and perirenal adipose microsomes [18]) [18] 3#

endoplasmic reticulum ( bound [13, 15]; cytoplasmic surface of [16]) [13, 15, 16, 20, 24] membrane ( bound [8, 24, 25, 28]) [8, 24, 25, 28] microsome ( membrane bound [4, 15, 16, 18]; of hepatocytes [27]) [2-7, 9, 11-13, 15-18, 20, 26, 27, 29] $"

(preparation of 11 fractions, ion-exchange, gel filtration [3]; ammonium sulfate precipitation, gel filtration, ultra filtration, ion-exchange [5]; different fractions by sonication and centrifugation, ion-exchange [6]; partially [7, 13, 15]; ion-exchange [16]) [3, 5-7, 13, 15, 16] (several extractions besides ion-exchange and gel filtration [4]; anionic and nonionic detergents, solubilization and ion-exchange [16]) [4, 16] (sucrose gradient, filtration [17]) [17] [20] [28] [30]

203

Stearoyl-CoA 9-desaturase

1.14.19.1


 

(identified of positive clones by screening with hybridization of cDNA of carp with rat D9 -desaturase cDNA [26]) [26] (expression of D9 -3 gene in Saccharomyces cerevisiae [24]) [24] (heterologous gene expression in yeast [25]) [25] (coexpression of acyl carrier protein isoform I gene and holo-acyl carrier protein synthase of Escherichia coli [28]) [28] (expression in Escherichia coli, transfection of Arabidopsis by Agrobacterium tumefaciens clones [30]) [30] 

 

L146F ( point mutation in ssi2 gene, causes reduced desaturase activity [30]) [30] Additional information ( yeast expression strain carries a mutation in the stearoyl-CoA desaturase gene [25]) [25] 

agriculture ( disease resistance [30]) [30] medicine ( potential use of SCD1 as a target in the treatment of some eye diseases [29]) [29]

4  )  30 ( 30 min in distilled water or 0.1 M phosphate buffer, pH 7.2 causes in almost complete loss of activity [1]) [1] 32 ( 30 min, 70-80% of activity [3]) [3] Additional information ( repeated freezing causes loss of activity [9]; 50% loss of activity after repeated freezing and thawing [16]) [9, 16] 5    "

, rapid isolation of enzyme and storage at -70 C is required [3] , relatively labil [6] , 50% loss of activity caused by repeated freezing and thawing [4]    , -20 C, sedimented enzyme, 0.25 M sucrose, 2 weeks, without significant loss of activity [1] , -20 C, supernatant fraction, over 1 year, without loss of activity [1] , 4 C, 1 mM or 10 mM potassium phosphate buffer, pH 7.2, 24 hours, activity irreversibly lost [1] , -75 C, >95% pure last fraction, 3 months, no loss of activity [6] , -75 C, fractions during purification, overnight, 20-50% loss of activity [6] , 0 C, above 95% pure last fraction, 15-20 hours, 30-50% loss of activity [6] , 4 C, microsomes, 24 hours, inhibition of desaturase by the cytosolic fraction rather than stimulation [5] 204

1.14.19.1

Stearoyl-CoA 9-desaturase

, enzyme cannot be stored overnight without loss of 20-50% of activity before fraction 5 [3] , -60 C, 0.12 M sodium phosphate buffer, pH 8.0, 0.5% Triton X-100, stable for several months [16] , -70 C, stable at least 6 months [4] , -20 C, argon, microsomal fraction, stable for up to 4 weeks [9] , -30 C, freeze-dried microsomal preparation, 12 months stable, activity is retained [18] , -80 C, saline solution, 150 mM NaCl, 0.1 mM EDTA, 20 mM HEPES, pH 7.4 [20] , reduced enzyme is stable under O2 for hours [28]

"  [1] Fulco, A.J.; Bloch, K.: Cofactor requirements for the formation of D9 -unsaturated fatty acids in Mycobacterium phlei. J. Biol. Chem., 239, 993-997 (1964) [2] Oshino, N.; Imai, Y.; Sato, R.: A function of cytochrome b5 in fatty acid desaturation by rat liver microsomes. J. Biochem., 69, 155-167 (1971) [3] Strittmatter, P.; Spatz, L.; Corcoran, D.; Rogers, M. J.; Setlow, B.; Redline, R.: Purification and properties of rat liver microsomal stearyl coenzyme A desaturase. Proc. Natl. Acad. Sci. USA, 71, 4565-4569 (1974) [4] Joshi, V.C.; Prasad, M.R.; Sreekrishna, K.: Terminal enzyme of stearoyl-CoA desaturation from chicken liver. Methods Enzymol., 71, 252-258 (1981) [5] Jones, D.P.; Gaylor, J.L.: A non-substrate-binding protein that stimulates microsomal stearyl-CoA desaturase. Methods Enzymol., 71, 258-263 (1981) [6] Strittmatter, P.; Enoch, H.G.: Purification of stearyl-CoA desaturase from liver. Methods Enzymol., 52, 188-193 (1978) [7] Fujiwara, Y.; Okayasu, T.; Ishibashi, T.; Imai, Y.: Immunochemical evidence for the enzymatic difference of D6 -desaturase from D9 - and D5 -desaturase in rat liver microsomes. Biochem. Biophys. Res. Commun., 110, 36-41 (1983) [8] Foot, M.; Jeffcoat, R.; Russell, N.: Some properties, including the substrate in vivo, of the D9 -desaturase in Micrococcus cryophilus. Biochem. J., 209, 345-353 (1983) [9] Wilson, A.C.; Miller, R.W.: Growth temperature-dependent stearoyl coenzyme A desaturase activity of Fusarium oxysporum microsomes. Can. J. Biochem., 56, 1109-1114 (1978) [10] Baker, N.; Lynen, F.: Factors involved in fatty acyl CoA desaturation by fungal microsomes. The relative roles of acyl CoA and phospholipids as substrates. Eur. J. Biochem., 19, 200-210 (1971) [11] Sreekrishna, K.; Joshi, V.C.: Inhibition of the microsomal stearoyl coenzyme A desaturation by divalent copper and its chelates. Biochim. Biophys. Acta, 619, 267-273 (1980)

205

Stearoyl-CoA 9-desaturase

1.14.19.1

[12] Scott, W.; Foote, J.L.: The inhibition of stearoyl-coenzyme A desaturase by phenyllactate and phenylpyruvate. Biochim. Biophys. Acta, 573, 197-200 (1979) [13] Jeffcoat, R.; Brawn, P.R.; Safford, R.; James, A.T.: Properties of rat liver microsomal stearoyl-coenzyme A desaturase. Biochem. J., 161, 431-437 (1977) [14] Russell, N.J.: The positional specificity of a desaturase in the psychrophilic bacterium Micrococcus cryophilus (ATCC 15174). Biochim. Biophys. Acta, 531, 179-186 (1978) [15] Jeffcoat, R.: The physiological role and control of mammalian fatty acylcoenzyme A desaturases. Biochem. Soc. Trans., 5, 811-818 (1977) [16] Prasad, M.R.; Joshi, V.C.: Purification and properties of hen liver microsomal terminal enzyme involved in stearoyl coenzyme A desaturation and its quantitation in neonatal chicks. J. Biol. Chem., 254, 6362-6369 (1979) [17] Ghesquier, D.; Carreau, J.P.; Robert, J.C.; Abastado, M.; Cheret, A.M.; Lewin, M.J.M.: Cytochrome b5 -dependent D9 desaturation of fatty acids in gastric microsomes. Biochim. Biophys. Acta, 751, 349-354 (1983) [18] McDonald, T.M.; Kinsella, J.E.: Stearyl-CoA desaturase of bovine mammary microsomes. Arch. Biochem. Biophys., 156, 223-231 (1973) [19] Oshino, N.; Imai, Y.; Sato, R.: Electron-transfer mechanism associated with fatty acid desaturation catalyzed by liver microsomes. Biochim. Biophys. Acta, 128, 13-27 (1966) [20] Trueman, R.J.; Tiku, P.E.; Caddick, M.X.; Cossins, A.R.: Thermal thresholds of lipid restructuring and D9 -desaturase expression in the liver of carp (Cyprinus carpio L.). J. Exp. Biol., 203, 641-650 (2000) [21] Griffiths, G.; Griffiths, W.T.; Stobart, A.K.: D-9 desaturase activity in developing cotyledons of sunflower (Helianthus annuus. L). Phytochemistry, 48, 261-267 (1998) [22] Mziaut, H.; Korza, G.; Elkahloun, A.G.; Ozols, J.: Induction of stearoyl CoA desaturase is associated with high-level induction of emerin RNA. Biochem. Biophys. Res. Commun., 282, 910-915 (2001) [23] Choi, Y.; Park, Y.; Pariza, M.W.; Ntambi, J.M.: Regulation of stearoyl-CoA desaturase activity by the trans-10,cis-12 isomer of conjugated linoleic acid in HepG2 cells. Biochem. Biophys. Res. Commun., 284, 689-693 (2001) [24] MacKenzie, D.A.; Carter, A.T.; Wongwathanarat, P.; Eagles, J.; Salt, J.; Archer, D.B.: A third fatty acid D9 -desaturase from Mortierella alpina with a different substrate specificity to ole1p and ole2p. Microbiology, 148, 17251735 (2002) [25] Marillia, E.F.; Giblin, E.M.; Covello, P.S.; Taylor, D.C.: A desaturase-like protein from white spruce is a D9 desaturase. FEBS Lett., 526, 49-52 (2002) [26] Tiku, P.E.; Gracey, A.Y.; Macartney, A.I.; Beynon, R.J.; Cossins, A.R.: Coldinduced expression of D9 -desaturase in carp by transcriptional and posttranslational mechanisms. Science, 271, 815-818 (1996) [27] Macartney, A.I.; Tiku, P.E.; Cossins, A.R.: An isothermal induction of D9 desaturase in cultured carp hepatocytes. Biochim. Biophys. Acta, 1302, 207216 (1996) [28] Yang, Y.S.; Broadwater, J.A.; Pulver, S.C.; Fox, B.G.; Solomon, E.I.: Circular dichroism and magnetic circular dichroism studies of the reduced binuc206

1.14.19.1

Stearoyl-CoA 9-desaturase

lear non-heme iron site of stearoyl-ACP D9 -desaturase: substrate binding and comparison to ribonucleotide reductase. J. Am. Chem. Soc., 121, 2770-2783 (1999) [29] Miyazaki, M.; Kim, H.J.; Man, W.C.; Ntambi, J.M.: Oleoyl-CoA is the major de novo product of stearoyl-CoA desaturase 1 gene isoform and substrate for the biosynthesis of the Harderian gland 1-alkyl-2,3-diacylglycerol. J. Biol. Chem., 276, 39455-39461 (2001) [30] Kachroo, P.; Shanklin, J.; Shah, J.; Whittle, E.J.; Klessig, D.F.: A fatty acid desaturase modulates the activation of defense signaling pathways in plants. Proc. Natl. Acad. Sci. USA, 98, 9448-9453 (2001)

207

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:

   
 1.14.19.2    acyl-[acyl-carrier-protein],hydrogen-donor:oxygen oxidoreductase    acyl-[acyl-carrier-protein] desaturase    EC 1.14.99.6 (formely) desaturase, acyl-[acyl carrier protein] stearoyl-ACP desaturase stearoyl-[acyl carrier protein] desaturase stearyl acyl carrier protein desaturase stearyl-ACP desaturase stearyl-acyl carrier protein desaturase
    37256-86-3

    











208

Carthamus tinctorius (safflower [1]) [1, 5, 6, 8] Euglena gracilis [2, 9] Ricinus communis (castor bean [3]) [3, 4, 7, 11, 13, 15, 16, 17, 22, 23] Cucumis sativus [4] Spinacia oleracea (spinach [9]) [9, 20] Glycine max (soybean [10]) [10] Persea americana (avocado [4]) [4] Pelargonium xhortorum (geranium [12]) [12, 20] Thunbergia alata (black-eyed Susan vine [13]) [13, 14, 20] Asclepias syriaca (milkweed [14]) [14] Bignonia unguis-cati (cat's claw [15]) [15] Macadamia integrifolia [18] Nerium oleander [18] Elaeis guineensis (oil palm, clone 1 [19]) [19] Coriandrum sativum (coriander [20]) [20] Kochia scoparia [21] Elaeis guineensis (oil palm, clone 2 [19]) [19]

1.14.19.2

Acyl-[acyl-carrier-protein] desaturase

!    "
 # 

stearoyl-[acyl-carrier protein] + reduced acceptor + O2 = oleoyl-[acyl-carrier protein] + acceptor + 2 H2 O ( specific for stearoyl-CoA [1]; enzyme acts on derivatives of a number of long-chain fatty acids, requires ferredoxin, formerly EC 1.14.99.6 [2]; peroxodiferric intermediate is suggested [16])    oxidation redox reaction reduction       stearoyl-[acyl-carrier protein] + electron donor + O2 ( enzyme catalyzes the principal conversion of saturated fatty acids to unsaturated fatty acids in synthesis of vegetable oils [5]; involved in oleic acid biosynthesis [6, 8]) (Reversibility: ? [5]) [5, 6, 8] $ oleoyl-[acyl-carrier protein] + acceptor + H2 O [5, 6, 8]      10-(heptyloxy)-decanoyl-[acyl-carrier protein] + reduced acceptor + O2 (Reversibility: ? [23]) [23] $ 10-(heptyloxy)-9-decenoyl-[acyl-carrier protein] + acceptor + H2 O [23]  7-(decyloxy)-heptanoyl-[acyl-carrier protein] + reduced acceptor + O2 (Reversibility: ? [23]) [23] $ 7-(decyloxy)-9-heptenoyl-[acyl-carrier protein] + acceptor + H2 O [23]  8-(nonyloxy)-octanoyl-[acyl-carrier protein] + reduced acceptor + O2 (Reversibility: ? [23]) [23] $ 8-hydroxyoctanoyl-[acyl-carrier protein] + 1-nonanal + acceptor + H2 O [23]  9-(octyloxy)-nonanoyl-[acyl-carrier protein] + reduced acceptor + O2 (Reversibility: ? [23]) [23] $ 9-hydroxynonanoyl-[acyl-carrier protein] + 1-octanal + acceptor + H2 O [23]  heptaadecanoyl-[acyl-carrier protein] + reduced ferredoxin + O2 ( very low activity, vegetative ferredoxin from Anabaena [17]) (Reversibility: ? [17]) [17] $ 9-heptadecenoyl-[acyl-carrier protein] + oxidized ferredoxin + H2 O [17]  nonadecanoyl-[acyl-carrier protein] + reduced ferredoxin + O2 ( low activity, vegetative ferredoxin from Anabaena [17]) (Reversibility: ? [17]) [17] $ 9-nonadecenoyl-[acyl-carrier protein] + oxidized ferredoxin + H2 O [17]

209

Acyl-[acyl-carrier-protein] desaturase

1.14.19.2

 palmitoyl-[acyl-carrier protein] + reduced acceptor + O2 (Reversibility: ? [13]) [13] $ 6-hexadecenoyl-[acyl-carrier protein] + acceptor + H2 O [13]  palmitoyl-[acyl-carrier protein] + reduced acceptor + O2 ( 1% of activity with stearoyl-[acyl-carrier-protein] [6]; 8% of activity with stearoyl-[acyl-carrier-protein], 7fold higher activity than the enzyme from castor [14]; 4fold lower activity with stearoyl-[acyl-carrier protein] and myristoyl-[acyl-carrier protein] [15]) (Reversibility: ? [6, 12, 14, 15, 18]) [6, 8, 12, 14, 15, 18] $ 9-hexadecenoyl-[acyl-carrier protein] + acceptor + H2 O [6, 8, 12, 14, 15, 18]  pentadecanoyl-[acyl-carrier protein] + reduced ferredoxin + O2 ( very low activity, vegetative ferredoxin from Anabaena [17]) (Reversibility: ? [17]) [17] $ 9-pentadecenoyl-[acyl-carrier protein] + oxidized ferredoxin + H2 O [17]  stearoyl-CoA + electron donor + O2 ( 5% of activity with stearoyl-[acyl-carrier-protein] [6]; purified reconstituted enzyme shows same activity as with stearoyl-[acyl-carrier-protein], no activity in crude extracts [2]) (Reversibility: ? [2, 6]) [1, 2, 6, 8] $ 9-octadecenoyl + acceptor + H2 O [1, 2, 6, 8]  stearoyl-[acyl-carrier protein] + reduced acceptor + O2 (Reversibility: ? [13]) [13] $ 6-octadecenoyl-[acyl-carrier protein] + 9-octadecenoyl-[acyl-carrier protein]acceptor + H2 O ( ratio of activity: 2/1 [13]) [13]  stearoyl-[acyl-carrier protein] + reduced acceptor + O2 ( electron donor NADPH [1, 2, 4, 9]; electron donor NADPH-ferredoxin(II) [8]; no activity with NADH [1, 2]; purified enzyme is not active with NADH [2]; enzyme is specific for stearoyl-CoA [1, 6]; enzyme acts on derivatives of a number of long chain fatty acids [2]; a system composed of ferredoxin, grana lamellae, ascorbic acid, dichlorophenolindophenol and light is the most effective reductant [1]) (Reversibility: ? [1, 2, 4, 9, 14, 18]) [1, 2, 4, 6, 8, 9, 14, 18] $ oleoyl-[acyl-carrier protein] + acceptor + H2 O [1, 2, 4, 6, 8, 9, 14, 18]  tetradecanoyl-[acyl-carrier protein] + reduced acceptor + O2 ( 3fold more active as with palmitoyl-[acyl-carrier protein], very low activity with dodecanoyl-[acyl-carrier protein] and stearoyl-[acyl-carrier protein] [12]; 3% of activity with stearoyl-[acyl-carrier-protein], 30fold higher activity than castor enzyme [14]) (Reversibility: ? [12, 14]) [12, 14] $ 9-tetradecenoyl-[acyl-carrier protein] + acceptor + H2 O [12, 14] 2  FAD ( 0.005 mM, 74% inhibition [2]) [1, 2] FMN ( 0.006 mM, 73% inhibition [2]) [1, 2]

210

1.14.19.2

Acyl-[acyl-carrier-protein] desaturase

KCN ( 1 mM, strong inhibition [1]; 0.01 mM, 50% inhibition, 0.1 mM, 80% inhibition [2]) [1, 2] b-mercaptoethanol [1] cytochrome c3 [1] cytochrome c553 [1] Additional information ( not inhibited by CO [1, 2, 9]) [1, 2, 9]
"%  NADPH [1, 2, 4, 9, 10] ferredoxin ( required for activity [1,3,5]; stearyl acyl carrier protein desaturase system consists of 3 components: a reduced NADPHoxidoreductase, a desaturase and ferredoxin [2,9]; highest activity with Anabaena ferredoxin [20]; highest activity with Anabaena or impatiens ferredoxin [20]) [1-5, 9-23] Additional information ( NADH is ineffective as electron donor [1,2]) [1, 2] &

  catalase ( 5-10fold stimulation [4]; 3fold stimulation of purified enzyme, 4-5fold increase of activity in crude extracts [6]) [4, 6, 8] dithiothreitol ( enhances activity [1]) [1] reduced glutathione ( enhances activity [1]) [1] '(   iron ( 3.85 mol of iron per mol of holoenzyme, dimeric enzyme may contain a pair of identical diiron-oxo clusters [11]) [11, 16, 17] ) &  * +, 0.39 (tetradecanoyl-[acyl-carrier protein]) [22] 0.43 (tetradecanoyl-[acyl-carrier protein]) [17] 1.2 (9-(octyloxy)-nonanoyl-[acyl-carrier protein]) [23] 1.64 (tetradecanoyl-[acyl-carrier protein], T117R/G188L mutant enzyme [22]) [22] 1.9 (pentadecanoyl-[acyl-carrier protein]) [17] 2.4 (8-(nonyloxy)-octanoyl-[acyl-carrier protein]) [23] 2.8 (hexadecanoyl-[acyl-carrier protein]) [22] 4.9 (octadecanoyl-[acyl-carrier protein], T117R/G188L mutant enzyme [22]) [22] 5.7 (hexadecanoyl-[acyl-carrier protein]) [17] 12 (7-(decyloxy)-heptanoyl-[acyl-carrier protein]) [23] 12 (nonadecanoyl-[acyl-carrier protein]) [17] 14 (heptadecanoyl-[acyl-carrier protein]) [17] 20-30 (stearoyl-[acyl-carrier protein]) [16] 25.3 (hexadecanoyl-[acyl-carrier protein], T117R/G188L mutant enzyme [22]) [22] 30 (10-(heptyloxy)-decanoyl-[acyl-carrier protein]) [23] 33 (octadecanoyl-[acyl-carrier protein]) [17] 42.3 (octadecanoyl-[acyl-carrier protein]) [22]

211

Acyl-[acyl-carrier-protein] desaturase

1.14.19.2

49 (octadecanoyl-[acyl-carrier protein]) [23] 49 (tetradecanoyl-[acyl-carrier protein], spinach acyl-carrier protein [17]) [17] " & *-% , 0.065 [4] 0.1 ( substrate palmitoyl-[acyl-carrier protein] [13]) [13] 0.11 [6, 8] Additional information ( assay procedure [8]) [8] . / *', 0.00038 (stearoyl-[acyl-carrier-protein]) [6] 0.00046 (octadecanoyl-[acyl-carrier protein]) [22] 0.00051 (palmitoyl-[acyl-carrier-protein]) [6] 0.00055 (hexadecanoyl-[acyl-carrier protein], T117R/G188L mutant enzyme [22]) [22] 0.00098 (octadecanoyl-[acyl-carrier protein], T117R/G188L mutant enzyme [22]) [22] 0.001 (tetradecanoyl-[acyl-carrier protein], T117R/G188L mutant enzyme [22]) [22] 0.0013 (nonadecanoyl-[acyl-carrier protein]) [17] 0.002 (NADPH) [1] 0.0033 (octadecanoyl-[acyl-carrier protein]) [17] 0.0039 (octadecanoyl-[acyl-carrier protein]) [23] 0.0039 (tetradecanoyl-[acyl-carrier protein], spinach acyl-carrier protein [17]) [17] 0.00464 (tetradecanoyl-[acyl-carrier protein]) [22] 0.005 (hexadecanoyl-[acyl-carrier protein]) [22] 0.0058 (tetradecanoyl-[acyl-carrier protein]) [17] 0.0059 (heptadecanoyl-[acyl-carrier protein]) [17] 0.0066 (7-(decyloxy)-heptanoyl-[acyl-carrier protein]) [23] 0.0074 (10-(heptyloxy)-decanoyl-[acyl-carrier protein]) [23] 0.0083 (hexadecanoyl-[acyl-carrier protein]) [17] 0.0083 (stearoyl-CoA) [6] 0.06 (O2 ) [8] 0.069 (pentadecanoyl-[acyl-carrier protein]) [17] 0  6 [10] 0 

 5.5-7.7 ( pH 5.5: about 50% activity, pH 7.7: about 50% activity [10]) [10]

212

1.14.19.2

Acyl-[acyl-carrier-protein] desaturase

  #   ' 1  68000 ( gel filtration [6]) [6] 75000 ( recombinant enzyme lacking a putative transit peptide [11]) [11]   ? ( x * 39000, recombinant enzyme, SDS-PAGE, predicted from nucleotide sequence [12]; x * 45000, isoform 1, SDS-PAGE [21]; x * 49000, isoform 2, SDS-PAGE [21]) [12, 21] dimer ( 2 * 36000, SDS-PAGE [6]; a2 , 2 * 37000, recombinant enzyme lacking a putative transit peptide, SDS-PAGE [11]) [6, 11]

 2 %$ %' %

% cotyledon [1, 10] embryo [5] leaf ( 49000 Da isoform [21]) [21] mesocarp [19] seed ( found in developing seeds, 15-50 days after flowering, not in germinated seeds [10]; 2 isoforms [21]) [1, 5-8, 10, 14, 15, 18, 21] 3#

chloroplast [9] cytoplasm [1] cytosol [10] soluble [2] $"

(aceton powder, anion-exchange, affinity chromatography on acyl-carrier protein-Sepharose [5]) [5, 6, 8] (recombinant enzyme lacking a putative transit peptide [11]) [11, 16] (recombinant enzyme [12]) [12] (recombinant enzyme [13, 14]) [13, 14] (recombinant enzyme [15]) [15]
 #

(hanging-drop-vapor-diffusion, 12-18 mg/ml enzyme solution, reservoir solution contains 100 mM cacodylate buffer, pH 5.4, 200 mM magnesium acetate and 12-18% polyethyleneglycol 8000, needle shaped crystals too thin for X-ray study [7]) [7]
 

(expression of cDNA in Escherichia coli [5]) [5] (full-length cDNA clone [4]) [4]

213

Acyl-[acyl-carrier-protein] desaturase

1.14.19.2

(expression of cDNA in Escherichia coli [12]) [12] (expression of wild-type, A181T/A188G/Y189F/S205N/L206T/G207A, A188G/Y189F, A181T/A200F and A181T/A200F/S205N/L206T/G207A mutant enzyme in Escherichia coli [13]; expression of N-terminal truncated enzyme in Escherichia coli [14]) [13, 14] (expression in Escherichia coli [14]) [14] (expression in Escherichia coli [15]) [15] (expression of cDNA in Escherichia coli [18]) [18] (expression of cDNA in Escherichia coli [18]) [18] (cloning of isoform 1 [19]) [19] (expression of 2 isoforms in Escherichia coli [21]) [21] (cloning of isoform 2 [19]) [19] 

 

A181T/A188G/Y189F/S205N/L206T/G207A ( compared to wild-type: reduced D6 -desaturase activity with palmitoyl-[acyl-carrier protein] as substrate, strong D9 -desaturase activity with stearoyl-[acyl-carrier protein] as substrate, exhibits D9-desaturase activity with palmitoyl-[acyl-carrier protein] as substrate [13]) [13] A181T/A200F ( increase in D6 -desaturase activity with palmitoyl[acyl-carrier protein] as substrate, strong D9 -desaturase activity with stearoyl-[acyl-carrier protein] as substrate [13]) [13] A181T/A200F/S205N/L206T/G207A ( reduced D6 desaturase activity with palmitoyl-[acyl-carrier protein] as substrate, very low D9 desaturase activity, no D6 -desaturase activity with stearoyl-[acyl-carrier protein] as substrate [13]) [13] A188G/Y189F ( reduced D6 -desaturase activity with palmitoyl-[acylcarrier protein] as substrate [13]) [13] L118F/P179I ( 15fold higher activity with palmitoyl-[acyl-carrier protein] than wild-type enzyme, low D10 -desaturase activity [13]) [13] L118W ( 100% activity with palmitoyl-[acyl-carrier protein], 89% activity with stearoyl-[acyl-carrier protein] and 8.2% activity with myristoyl-[acyl-carrier protein], wild-type is only active with stearoyl-[acyl-carrier protein] [15]) [15] T117R/G188L ( 82fold higher specificity for palmitoyl-[acyl-carrier protein] with respect to wild-type [22]) [22]

4  )  50 ( 1 min, inactivation [8]) [8] 5    "

, unstable to dialysis [8]

214

1.14.19.2

Acyl-[acyl-carrier-protein] desaturase

   , -70 C, 50% glycerol, 0.1% bovine serum albumin, 100 mM potassium phosphate buffer, pH 6.8, at least 2 months, no loss of activity [6] , 4 C, overnight, 30% loss of activity [6] , 0 C, 10 h, 50% loss of activity [2] , frozen state, 1 month, complete loss of activity [9]

"  [1] Jaworski, J.G.; Stumpf, P.K.: Fat metabolism in higher plants. Properties of a soluble stearyl-acyl carrier protein desaturase from maturing Carthamus tinctorius. Arch. Biochem. Biophys., 162, 158-165 (1974) [2] Nagai, J.; Bloch, K.: Enzymatic desaturation of stearyl acyl carrier protein. J. Biol. Chem., 243, 4626-4633 (1968) [3] Knutzon, D.S.; Scherer, D.E.; Schreckengost, W.E.: Nucleotide sequence of a complementary DNA clone encoding stearoyl-acyl carrier protein desaturase from castor bean, Ricinus communis. Plant Physiol., 96, 344-345 (1991) [4] Shanklin, J.; Somerville, C.: Stearoyl-acyl-carrier-protein desaturase from higher plants is structurally unrelated to the animal and fungal homologs. Proc. Natl. Acad. Sci. USA, 88, 2510-2514 (1991) [5] Thompson, G.A.; Scherer, D.E.; Foxall-van Aken, S.; Kenny, J.W.; Young, H.L.; Shitani, D.K.; Kridl, J.C.; Knauf, V.C.: Primary structures of the precursor and mature forms of stearoyl-acyl carrier protein desaturase from safflower embryos and requirement of ferredoxin for enzyme activity. Proc. Natl. Acad. Sci. USA, 88, 2578-2582 (1991) [6] McKeon, T.A.; Stumpf, P.K.: Purification and characterization of the stearoyl-acyl carrier protein desaturase and the acyl-acyl carrier protein thioesterase from maturing seeds of safflower. J. Biol. Chem., 257, 12141-12147 (1982) [7] Schneider, G.; Lindquist, Y.; Shanklin, J.; Somerville, C.: Preliminary crystallographic data for stearoyl-acyl carrier protein desaturase from castor seed. J. Mol. Biol., 225, 561-564 (1992) [8] McKeon, T.A.; Stumpf, P.K.: Stearoyl-acyl carrier protein desaturase from safflower seeds. Methods Enzymol., 71, 275-281 (1981) [9] Nagai, J.; Bloch, K.: Enzymatic desaturation of stearyl acyl carrier protein. J. Biol. Chem., 241, 1925-1927 (1966) [10] Stumpf, P.K.; Porra, R.J.: Lipid biosynthesis in developing and germinating soybean cotyledons. The formation of oleate by a soluble stearyl acyl carrier protein desaturase. Arch. Biochem. Biophys., 176, 63-70 (1976) [11] Fox, B.G.; Shanklin, J.; Somerville, C.; Munck, E.: Stearoyl-acyl carrier protein D9 desaturase from Ricinus communis is a diiron-oxo protein. Proc. Natl. Acad. Sci. USA, 90, 2486-2490 (1993) [12] Schultz, D.; Cahoon, E.B.; Shanklin, J.; Craig, R.; Cox-Foster, D.L.; Mumma, R.O.; Medford, J.I.: Expression of a D9 14:0-acyl carrier protein fatty acid desaturase gene is necessary for the production of w5 anacardic acids

215

Acyl-[acyl-carrier-protein] desaturase

[13] [14] [15] [16]

[17] [18] [19] [20] [21] [22]

[23]

216

1.14.19.2

found in pest-resistant geranium (Pelargonium xhortorum). Proc. Natl. Acad. Sci. USA, 93, 8771-8775 (1996) Cahoon, E.B.; Lindqvist, Y.; Schneider, G.; Shanklin, J.: Redesign of soluble fatty acid desaturases from plants from altered substrate specificity and double bond position. Proc. Natl. Acad. Sci. USA, 94, 4872-4877 (1997) Cahoon, E.B.; Coughlan, S.J.; Shanklin, J.: Characterization of a structurally and functionally diverged acyl-acyl carrier protein desaturase from milkweed seed. Plant Mol. Biol., 33, 1105-1110 (1997) Cahoon, E.B.; Shah, S.; Shanklin, J.; Browse, J.: A determinant of substrate specificity predicted from the acyl-acyl carrier protein desaturase of developing cat's claw seed. Plant Physiol., 117, 593-598 (1998) Broadwater, J.A.; Ai, J.; Loehr, T.M.; Sanders-Loehr, J.; Fox, B.G.: Peroxodiferric intermediate of stearoyl-acyl carrier protein D9 desaturase: oxidase reactivity during single turnover and implications for the mechanism of desaturation. Biochemistry, 37, 14664-14671 (1998) Haas, J.A.; Fox, B.G.: Role of hydrophobic partitioning in substrate selectivity and turnover of the Ricinus communis stearoyl acyl carrier protein D9 desaturase. Biochemistry, 38, 12833-12840 (1999) Gummeson, P.O.; Lenman, M.; Lee, M.; Singh, S.; Stymne, S.: Characterization of acyl-ACP desaturases from Macadamia integrifolia Maiden & Betche and Nerium oleander L. Plant Sci., 154, 53-60 (2000) Shah, F.H.; Rashid, O.; San, C.T.: Temporal regulation of two isoforms of cDNA clones encoding D9 -stearoyl-ACP desaturase from oil palm (Elaeis guineensis). Plant Sci., 152, 27-33 (2000) Schultz, D.J.; Suh, M.C.; Ohlrogge, J.B.: Stearoyl-acyl carrier protein and unusual acyl-acyl carrier protein desaturase activities are differentially influenced by ferredoxin. Plant Physiol., 124, 681-692 (2000) Whitney, H.; Sayanova, O.; Lewis, M.J.; Pickett, J.; Napier, J.A.: Isolation of two putative acyl-acyl carrier protein desaturase enzymes from Kochia scoparia. Biochem. Soc. Trans., 28, 623-624 (2000) Whittle, E.; Shanklin, J.: Engineering D9 -16:0-acyl carrier protein (ACP) desaturase specificity based on combinatorial saturation mutagenesis and logical redesign of the castor D9 -18:0-ACP desaturase. J. Biol. Chem., 276, 21500-21505 (2001) Rogge, C.E.; Fox, B.G.: Desaturation, chain scission, and register-shift of oxygen-substituted fatty acids during reaction with stearoyl-ACP desaturase. Biochemistry, 41, 10141-10148 (2002)

3  
 

:!

   
 1.14.19.3    linoleoyl-CoA,hydrogen-donor:oxygen oxidoreductase    linoleoyl-CoA desaturase    D6 -acyl CoA desaturase D6 -desaturase D6 -fatty acyl-CoA desaturase EC 1.4.99.25 (formerly) desaturase, fatty acid D6 desaturase, linoleate fatty acid 6-desaturase fatty acid D6 -desaturase linoleate desaturase linoleic acid desaturase linoleic desaturase linoleoyl CoA desaturase linoleoyl-coenzyme A desaturase long-chain fatty acid D6 -desaturase
    9014-34-0

    





Arabidopsis thaliana (Heynh [7]) [7] Borago officinalis (common borage [6]) [6] Glycine max (soybean, cv Tracy [7]) [7] Homo sapiens (human [8, 9, 11-13]) [8, 9, 11-13] Linum usitatissimum (linseed, cultivar Punjab [2]) [2] Mortierella alpina [10] Mus musculus [11] Rattus norvegicus (strain Wistar [4, 5]; Wistar BB/E n=12, Wistar BB/E n=6 [9]; INS-1 b-cells [13]; strain Sprague-Dawley [12]) [1, 3-5, 8, 9, 11-13] Spinacia oleracea [2, 7] 217

Linoleoyl-CoA desaturase

1.14.19.3

!    "
 # 

linoleoyl-CoA + AH2 + O2 = g-linolenoyl-CoA + A + 2 H2 O    oxidation redox reaction reduction       linoleic acid + AH2 + O2 ( key enzyme localized in the endoplasmic reticulum [8]; D6 -desaturase reaction is the rate-limiting step in the conversion of linoleic acid and a linoleic acids to the longer, more highly unsaturated members of the n-6 and n-3 polyunsaturated fatty acids, metabolic pathway in mammalian cells [11]; important for the generation of unsaturated fatty acids, D6 -desaturase required for the conversion of dietary linoleic acid to arachidonic acid [13]) (Reversibility: ? [1-13]) [1-13] $ g-linolenic acid + A + H2 O      18-carbon fatty acid + AH2 + O2 (Reversibility: ? [12]) [12] $ ? + A + H2 O  24-carbon fatty acid + AH2 + O2 (Reversibility: ? [12]) [12] $ ? + A + H2 O  a-linolenic acid + AH2 + O2 (Reversibility: ? [8, 12]) [8, 12] $ stearidonic acid + A + H2 O [8, 12]  linolenic acid + AH2 + O2 (Reversibility: ? [13]) [13] $ arachidonic acid + A + H2 O [13]  linoleoyl phosphatidylcholine + AH2 + O2 (Reversibility: ? [6]) [6] $ g-linolenoylphosphatidylcholine + A + H2 O  monogalactosydiacylglycerol + AH2 + O2 (Reversibility: ? [7]) [7] $ ? + A + H2 O  octadeca-9,12-dienoic acid + AH2 + O2 (Reversibility: ? [1-13]) [1-13] $ g-linolenic acid + A + H2 O [1-13] 2  4-chloro-5-dimethylamino-2-phenyl-3(2H)-pyridazinone ( BASF 13-338, San 9785, herbicide known to reduce levels of linolenic acid, inhibits in vitro desaturation of fatty acids [7]) [7] KCN [4] N-ethylmaleimide ( only little effect [4]; n-3 D6 -desaturase is not inhibited, it increases, NEM specifically inhibits D6 -desaturase activity in the n-6 series, but not in the n-3-series [8]) [4, 8]

218

1.14.19.3

Linoleoyl-CoA desaturase

Tiron [4] bathophenanthroline sulfonate ( mild inhibition [4]) [4] b-mercaptoethanol ( mild inhibition [4]) [4] conjugated linoleic acid ( all CLA isomers inhibits significantly the activity of D6 -desaturase, the cis 9, trans 11 isomer is the most potent inhibitor [10]) [10] dithiothreitol ( mildly inhibition [4]) [4] iron chelators [4] p-chloromercuribenzene sulfonate [4]
"%  NADH ( absolutely essential for D6 -desaturation [4]) [4] NADPH ( absolutely essential for D6 -desaturation [4]) [4] cytochrome b5 ( absolutely essential for D6 -desaturation [4]) [4] linoleoyl-CoA ( absolutely essential for D6 -desaturation [4]) [4] &

  NADH-cytochrome b5 reductase ( EC 1.6.2.2, absolutely essential for D6 -desaturation [4]) [4] NADPH-cytochrome b5 reductase ( EC 1.6.2.2, absolutely essential for D6 -desaturation [4]) [4] O2 ( absolutely essential for D6 -desaturation [4]) [4] bovine serum albumin ( 1-10 mg/ml, stimulates enzyme activity of unwashed microsomes by 50% [1]) [1] catalase ( bovine catalase [1]; EC 1.11.1.6 [2]; activity stabilized by addition [7]) [1, 2, 7] '(   Fe2+ ( D6 -desaturase contains 15.1 nmol iron/mg protein [4]) [4] " & *-% , 0.0344 [4] . / *', 0.045 (linoleoyl-CoA) [4] 0  7 [4] 0 

 6-9 [4]

  #   ' 1  45000 ( recombinant D6 -desaturase expressed in COS-7 cells, analysed by Western blot [12]) [12] 65000-68000 ( gel filtration [4]) [4] 66000 ( SDS-PAGE [4]) [4]

219

Linoleoyl-CoA desaturase

1.14.19.3

  monomer ( 1 * 66000, SDS-PAGE [4]) [4] $   "

phospholipoprotein ( 20-40 M phospholipid per mol of protein [4]) [4]

 2 %$ %' %

% brain [11, 12] cerebellum ( neurons [11]) [11] cotyledon [2, 6] fetus [8] fibroblast ( skin fibroblast [11]) [11] leaf [2, 7] liver ( fetal liver [8]) [1, 3-5, 8, 9, 12] retina [11] seed [2, 6] 3#

chloroplast [2, 7] cytoplasm [1] endoplasmic reticulum [8] microsome ( membrane [4-6, 8]; membrane-bound [1]) [1-6, 8, 9, 12] $"

[7] [7] [4]
 

(human sequence determined [13]; D6 -desaturase gene identified in human chromosome 11 [12]) [12, 13] (Saccharomyces cerevisiae transformed with fungal D6 -desaturase gene [10]) [10] (RT-PCR analysis of INS-1 b-cell content of mRNA, primer pairs basedon known rat sequence [13]; plasmid coding for rat D6 -desaturase constructed using pCMV for expression in mammalian cells, rat D6 -desaturase sequence, GenBank accession number AB021980 PCR amplified, expressed by transiently transforming COS-7 cells [12]) [12, 13]

220

1.14.19.3

Linoleoyl-CoA desaturase

4     , -70 C, stored under nitrogen the enzyme is unstable, loses 80% of its activity after repeated freezing and thawing [4]

"  [1] Jeffcoat, R.; Dunton, A.P.; James, A.T.: Evidence for the different responses of D9 -, D6 - and D5 -fatty acyl-CoA desaturases to cytoplasmic proteins. Biochim. Biophys. Acta, 528, 28-35 (1978) [2] Browse, J.A.; Slack, C.R.: Catalase stimulates linoleate desaturase activity in microsomes from developing linseed cotyledons. FEBS Lett., 131, 111-114 (1981) [3] Mahfouz, M.; Johnson, S.; Holman, R.T.: Inhibition of desaturation of palmitic, linoleic and eicosa-8,11,14-trienoic acids in vitro by isomeric cis-octadecenoic acids. Biochim. Biophys. Acta, 663, 58-68 (1981) [4] Okayasu, T.; Nagao, M.; Ishibashi, T.; Imai, Y.: Purification and partial characterization of linoleoyl-CoA desaturase from rat liver microsomes. Arch. Biochem. Biophys., 206, 21-28 (1981) [5] Garda, H.A.; Brenner, R.R.: Short chain aliphatic alcohols increase rat-liver microsomal membrane fluidity and affect the activities of some microsomal membrane-bound enzymes. Biochim. Biophys. Acta, 769, 160-170 (1984) [6] Griffiths, G.; Stobart, A.K.; Stymne, S.: D6 - and D12 -desaturase activities and phosphatidic acid formation in microsomal preparations from the developing cotyledons of common borage (Borago officinalis). Biochem. J., 252, 641-647 (1988) [7] Norman, H.A.; Pillai, P.; St.John, J.B.: In vitro desaturation of monogalactosyldiacylglycerol and phosphatidylcholine molecular species by chloroplast homogenates. Phytochemistry, 30, 2217-2222 (1991) [8] Rodriguez, A.; Sarda, P.; Boulot, P.; Leger, C.L.; Descomps, B.: Differential effect of N-ethyl maleimide on D6 -desaturase activity in human fetal liver toward fatty acids of the n-6 and n-3 series. Lipids, 34, 23-30 (1999) [9] Brown, J.E.; Lindsay, R.M.; Riemersma, R.A.: Linoleic acid metabolism in the spontaneously diabetic rat: D6 -desaturase activity vs. product/precursor ratios. Lipids, 35, 1319-1323 (2000) [10] Chuang, L.T.; Thurmond, J.M.; Liu, J.W.; Kirchner, S.J.; Mukerji, P.; Bray, T.M.; Huang, Y.S.: Effect of conjugated linoleic acid on fungal D6 -desaturase activity in a transformed yeast system. Lipids, 36, 139-143 (2001) [11] Williard, D.E.; Nwankwo, J.O.; Kaduce, T.L.; Harmon, S.D.; Irons, M.; Moser, H.W.; Raymond, G.V.; Spector, A.A.: Identification of a fatty acid D6 -desaturase deficiency in human skin fibroblasts. J. Lipid Res., 42, 501-508 (2001)

221

Linoleoyl-CoA desaturase

1.14.19.3

[12] D'Andrea, S.; Guillou, H.; Jan, S.; Catheline, D.; Thibault, J.N.; Bouriel, M.; Rioux, V.; Legrand, P.: The same rat D6 -desaturase not only acts on 18- but also on 24-carbon fatty acids in very-long-chain polyunsaturated fatty acid biosynthesis. Biochem. J., 364, 49-55 (2002) [13] Ramanadham, S.; Zhang, S.; Ma, Z.; Wohltmann, M.; Bohrer, A.; Hsu, F.F.; Turk, J.: D6 -, stearoyl CoA-, and D5 -desaturase enzymes are expressed in bcells and are altered by increases in exogenous PUFA concentrations. Biochim. Biophys. Acta, 1580, 40-56 (2002)

222

9 
 

=

   
 1.14.20.1    penicillin-N,2-oxoglutarate:oxygen oxidoreductase (ring-expanding)    deacetoxycephalosporin-C synthase    DAOCS deacetoxycephalosporin-C synthetase penicillin N expandase
    85746-10-7

     Penicillium chrysogenum [1] Streptomyces clavuligerus (recombinant enzyme expressed in Escherichia coli [5]) [2, 3, 4, 5, 6, 7, 8, 11, 13, 14, 16, 17, 18] Streptomyces lactamdurans [12] Streptomyces jumonjinensis [8] Acremonium chrysogenum [8] Cephalosporium acremonium (bifunctional enzyme deacetoxycephalosporin C sythetase/hydroxylase [4, 9]) [4, 9, 10, 15]

!    "
 # 

penicillin N + 2-oxoglutarate + O2 = deacetoxycephalosporin C + succinate + CO2 + H2 O    oxidation redox reaction reduction

223

Deacetoxycephalosporin-C synthase

1.14.20.1

      penicillin N + 2-oxoglutarate + O2 (, the enzyme catalyzes the committed step in the biosynthesis of cephalosporin antibiotics [2]; , committed step in biosynthesis of cephamycin C [6]; , the enzyme is involved in catalyzing the biosynthesis of cephalosporins and cephamycin [13]) (Reversibility: ? [2]) [2, 6, 13] $ deacetoxycephalosporin C + succinate + CO2 + H2 O      3-exomethylenecephalosporin C + 2-oxoglutarate + O2 (, 8% of the activity with cephalosporin N, recombinant enzyme [14]) (Reversibility: ? [14]) [14] $ deacetylcephalosporin C + succinate + CO2 + H2 O [14]  adipoyl-6-aminopenicillanic acid + 2-oxoglutarate + O2 (, poor substrate [7]) (Reversibility: ? [7]) [7] $ 7-aminodeacetoxycephalosporanic acid + succinate + CO2 + H2 O [7]  amoxicillin + 2-oxoglutarate + O2 (Reversibility: ? [8]) [8] $ ? + succinate + CO2 + H2 O  ampicillin + 2-oxo-4-methylpentanoate + O2 (, wild-type enzyme has a requirement for 2-oxoglutarate and cannot efficiently use hydrophobic 2-oxoacids, the mutants R258A, R258L, R258H and R258F have broadened cosubstrate selectivity and are able to utilize hydrophobic 2-oxoacids [5]) (Reversibility: ? [5]) [5] $ cephalexin + succinate + CO2 + H2 O [5]  ampicillin + 2-oxoglutarate + O2 (, no activity [14]) (Reversibility: ? [5, 8]) [5, 8] $ cephalexin + succinate + CO2 + H2 O [5]  ampicillin + 2-oxohexanoate + O2 (, wild-type enzyme has a requirement for 2-oxoglutarate and cannot efficiently use hydrophobic 2oxoacids, the mutants R258A, R258L, R258H and R258F have broadened cosubstrate selectivity and are able to utilize hydrophobic 2-oxoacids [5]) (Reversibility: ? [5]) [5] $ cephalexin + ?  carbenicillin + 2-oxoglutarate + O2 (Reversibility: ? [8]) [8] $ ? + succinate + CO2 + H2 O  metampicillin + 2-oxoglutarate + O2 (Reversibility: ? [8]) [8] $ ? + succinate + CO2 + H2 O  penicillin G + 2-oxo-4-methylpentanoate + O2 (, wild-type enzyme has a requirement for 2-oxoglutarate and cannot efficiently use hydrophobic 2-oxoacids, the mutants R258A, R258L, R258H and R258F have broadened cosubstrate selectivity and are able to utilize hydrophobic 2oxoacids [5]) (Reversibility: ? [2, 5]) [2, 5] $ phenylacetyl-7-aminodeacetoxy cephalosporanic acid + ?  penicillin G + 2-oxoglutarate + O2 (, no activity [12, 14]) (Reversibility: ? [2, 5, 6, 7, 8]) [2, 5, 6, 7, 8]

224

1.14.20.1

Deacetoxycephalosporin-C synthase

$ phenylacetyl-7-aminodeacetoxy cephalosporanic acid + succinate + CO2 + H2 O [5]  penicillin G + 2-oxohexanoate + O2 (, wild-type enzyme has a requirement for 2-oxoglutarate and cannot efficiently use hydrophobic 2oxoacids, the mutants R258A, R258L, R258H and R258F have broadened cosubstrate selectivity and are able to utilize hydrophobic 2-oxoacids [5]) (Reversibility: ? [2, 5]) [2, 5] $ phenylacetyl-7-aminodeacetoxy cephalosporanic acid + ?  penicillin N + 2-oxoglutarate + O2 (, bifunctional enzyme: penicillin N expandase/DAOC hydroxylase [4]; , separate genes encode penicillin N expandase and DAOC hydroxylase [4]; , wild-type enzyme has a requirement for 2-oxoglutarate and cannot efficiently use hydrophobic 2-oxoacids, the mutants R258A, R258L, R258H and R258F have broadened cosubstrate selectivity and are able to utilize hydrophobic 2-oxoacids [5]; , oxoglutarate analogs are not used as substrate [12]; , almost absolute requirement for 2-oxoglutarate [15]) (Reversibility: ? [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14]) [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14] $ deacetoxycephalosporin C + succinate + CO2 + H2 O [2, 14]  penicillin V + 2-oxoglutarate + O2 (, no activity [14]) (Reversibility: ? [8]) [8] $ ? + succinate + CO2 + H2 O  phenethicillin + 2-oxoglutarate + O2 (Reversibility: ? [8]) [8] $ ? + succinate + CO2 + H2 O  Additional information (, no activity with isopenicillin N or 6aminopenicillanic acid [12]) [12] $ ? 2  1,10-phenanthroline [14] 2-oxobutyrate (, 0.1 mM, complete inhibition [12]) [12] 3-exomethylenecephalosporin C (, 0.28 mM, 71% inhibition of the formation of deacetoxycephalosporin C [14]) [14] 3-oxoadipate (, 0.1 mM, 56% inhibition [12]) [12] Co2+ (, 1 mM, 79% inhibition [12]) [12] Cu2+ (, 1 mM, complete inhibition [12]) [12] DTNB [14] EDTA [14] NEM [14] Zn2+ (, 1 mM, 84% inhibition [12]) [12] ampicillin (, 2.8 mM, 13% inhibition of the formation of deacetoxycephalosporin C [14]) [14] iodoacetic acid [14] p-hydroxymercuribenzoate [14] penicillin G (, 2.8 mM, 57% inhibition of the formation of deacetoxycephalosporin C [14]) [14]

225

Deacetoxycephalosporin-C synthase

1.14.20.1

penicillin V (, 2.8 mM, 47% inhibition of the formation of deacetoxycephalosporin C [14]) [14] phosphate [12] Additional information (, when 2-oxoglutarate is added to the enzyme before the mixture of ATP, Fe2+ and ascorbate activity is only about 40% of the reaction [15]) [15] &

  ascorbate (, stimulates activity of native and recombinant enzyme [14]) [14] bovine serum albumin (, stimulates [16]) [16] catalase (, stimulates [16]) [16] dithiothreitol (, stimulates activity of native and recombinant enzyme [14]; , stimulates, even in presence of ascorbate [16]) [14, 16] Additional information (, best activity is obtained by adding to the enzyme a mixture of ATP, Fe2+ , and ascorbate, followed by addition of 2-oxoglutarate, and then penicillin N. Simultaneous addition of ATP, Fe2+ , and ascorbate is preferable to sequential addition [15]) [15] '(   Fe2+ (, required [12, 14]; , Km : 0.071 mM [12]) [12, 14] Fe3+ (, can replace Fe2+ under reducing conditions [12, 14]) [12, 14] iron (, non-heme iron-binding protein [13]) [13] ) &  * +, 0.84 (ampicillin, , wild-type enzyme [5]) [5] 1.2 (penicillin N, , wild-type enzyme [6]; , mutant enzyme DI305-310, DR306-310 and mutant DR307-310 [2]) [2, 6] 1.26 (penicillin G, , wild-type enzyme [5]) [5] 3 (penicillin G, , wild-type enzyme [2, 6]) [2, 6] 3 (penicillin N, , wild-type enzyme [2]) [2] 3.6 (penicillin G, , mutant enzyme DK310-314 [2]) [2] 3.72 (penicillin G) [7] 4.38 (adipoyl-6-aminopenicillanic acid) [7] 4.8 (penicillin G, , mutant enzyme DR307-310 [2]) [2] 4.8 (penicillin N, , mutant enzyme DI310-314 [2]) [2] 25.2 (penicillin N) [7] " & *-% , 0.166 (, native enzyme [14]) [14] 0.264 [9] 0.432 (, recombinant enzyme [14]) [14] 66 (, recombinant enzyme [7]) [7] Additional information [10, 12] . / *', 0.003 (oxoglutarate) [12] 0.0066 (penicillin N) [7]

226

1.14.20.1

Deacetoxycephalosporin-C synthase

0.018 (2-oxoglutarate, , recombinant enzyme [14]) [14] 0.022 (2-oxoglutarate, , native enzyme [14]) [14] 0.025 (penicillin N, , mutant enzyme DK310-314 [2]) [2] 0.026 (penicillin N, , mutant enzyme DR306-310 [2]) [2] 0.028 (deacetoxycephalosporin C) [14] 0.029 (penicillin N, , recombinant enzyme [14]) [14] 0.033 (penicillin N, , wild-type enzyme [6]) [6] 0.034 (penicillin N, , mutant enzyme DI305-310 [2]) [2] 0.035 (penicillin N, , native enzyme [14]) [14] 0.036 (penicillin N, , wild-type enzyme [2]) [2] 0.044 (penicillin N, , mutant enzyme DR307-310 [2]) [2] 0.052 (penicillin N) [12] 0.5 (penicillin G, , mutant enzyme DK310-314 [2]) [2] 0.7 (penicillin G, , wild-type enzyme [2,6]) [2, 6] 1.1 (penicillin G, , wild-type enzyme [5]) [5] 1.3 (adipoyl-6-aminopenicillanic acid) [7] 1.6 (penicillin G, , mutant enzyme DR307-310 [2]) [2] 2.03 (penicillin G) [7] 2.6 (ampicillin, , wild-type enzyme [5]) [5] Additional information (, KM -values for mutant enzymes with penicillin G or ampicillin as prime substrate and 2-oxo-4-methylpentanoate, 2oxoglutarate or 2-oxohexanoate as cosubstrate [5]; , Km -values for mutant enzymes with penicillin N and penicillin G as substrate [6]) [5, 6] . / *', 0.034 (3-exomethylenecephalosporin C) [14] 0  5-11 [12] 7 (, in piperazineethanesulfonic acid buffer, recombinant enzyme [14]) [14] 7.4 (, native enzyme [14]) [14] )  *
, 25-30 [12] 36 (, native and recombinant enzyme [14]) [14] ) 

 *
, 5-40 (, 20 C: 15% of maximal activity, 35 C: 15% of maximal activity [12]) [12] 20-35 (, 20 C: 90% of maximal activity, 35 C: 90% of maximal activity [12]) [12]

  #   ' 1  27000 (, gel filtration [12]) [12]

227

Deacetoxycephalosporin-C synthase

1.14.20.1

  ? (, x * 34519, calculation from nucleotide sequence [11]; , x * 34554, in solution the monomeric apoenzyme is in equilibrium with atrimeric form, electrospray mass spectrometry [7]; , x * 36000, SDS-PAGE [11]; , x * 60000, SDS-PAGE [8]) [7, 8, 11] monomer (, 1 * 28000, SDS-PAGE [12]) [12]

 2 %$ %' %

3#

soluble [7, 10] $"

(recombinant enzyme expressed in Escherichia coli [2, 7, 14]; recombinant wild-type enzyme and mutant enzymes R258K, R258H, R258A, R258L and R258F [5]; wild-type and mutant enzymes R74I, R74Q, R75I/D270G, R75Q, R306L and R307Q [6]; efficient large-scale purification [7]) [2, 5, 6, 7, 14] [12] (purification of partially folded recombinant expandase/hydroxylase from insoluble granules of recombinant Escherichia coli [4]; purification of refolding-competent recombinant enzyme [9]) [4, 9, 10, 15]
 #

(crystallization of wild-type enzyme and of the DR306 mutant complexed with iron(II) and 2-oxoglutarate to 2.1 A and the DR306A mutant complexed with iron(II), succinate and unhydrated carbon dioxide to 1.96 A [2]; recombinant enzyme expressed in E. coli, high-resolution structures for apoenzyme, the enzyme complexed with Fe(II), and with Fe(II) and 2-oxoglutarate [3]; hanging drop method, recombinant enzyme expressed in E. coli [7]) [2, 3, 7]
 

(expression in Escherichia coli [7, 11]; expression of wild-type and mutant enzymes at high levels in Escherichia coli [13]) [7, 11, 13] (overexpression as an insoluble and inactive enzyme in granules of recombinant Escherichia coli [9]; expression at high levels in Escherichia coli under control of the trc promoter [10]) [9, 10] 

 

D185L (, no detectable ring expansion activity [13]) [13] DR307-310 (, mutant in which residues 307-310 are excised, reduced turnover number for penicillin N compared to wild-type enzyme, higher KM value for penicillin N compared to wild-type enzyme, increased KM -value for penicillin G compared to wild-type enzyme, increased turnover-number for penicillin G compared to wild-type enzyme [2]) [2]

228

1.14.20.1

Deacetoxycephalosporin-C synthase

DI305-310 (, mutant truncated by six residues, reduced turnover number for penicillin N compared to wild-type enzyme, lower KM -value for penicillin N compared to wild-type enzyme [2]) [2] DK310-314 (, mutant truncated by five residues, increased turnover number for penicillin N compared to wild-type enzyme, lower KM -value for penicillin N compared to wild-type enzyme, lower Km -value for penicillin G compared to wild-type enzyme, slightly higher turnover number for penicillin G compared to wild-type enzyme [2]) [2] DR306-310 (, mutant truncated by five residues, reduced turnover number for penicillin N compared to wild-type enzyme, lower KM -value for penicillin N compared to wild-type enzyme [2]) [2] H183L (, no detectable ring expansion activity [13]) [13] H243L (, no detectable ring expansion activity [13]) [13] N304L (, mutant enzyme with improved efficiency in penicillin conversion [17]; , mutant enzyme shows increased penicillin analog conversion [18]) [17, 18] R160Q (, more than 95% loss of activity [6]) [6] R258A (, wild-type enzyme has a requirement for 2-oxoglutarate and cannot efficiently use hydrophobic 2-oxoacids, the mutant enzyme has broadened cosubstrate selectivity and is able to utilize hydrophobic 2-oxoacids. The efficiency of 2-oxoglutarate utilization is decreased as compared to the wild-type enzyme [5]) [5] R258F (, wild-type enzyme has a requirement for 2-oxoglutarate and cannot efficiently use hydrophobic 2-oxoacids, the mutant enzyme has broadened cosubstrate selectivity and is able to utilize hydrophobic 2-oxoacids. The efficiency of 2-oxoglutarate utilization is decreased as compared to the wild-type enzyme [5]) [5] R258H (, wild-type enzyme has a requirement for 2-oxoglutarate and cannot efficiently use hydrophobic 2-oxoacids, the mutant enzyme has broadened cosubstrate selectivity and is able to utilize hydrophobic 2-oxoacids. The efficiency of 2-oxoglutarate utilization is decreased as compared to the wild-type enzyme [5]) [5] R258L (, wild-type enzyme has a requirement for 2-oxoglutarate and cannot efficiently use hydrophobic 2-oxoacids, the mutant enzyme has broadened cosubstrate selectivity and is able to utilize hydrophobic 2-oxoacids. The efficiency of 2-oxoglutarate utilization is decreased as compared to the wild-type enzyme [5]) [5] R266L (, 2-oxoglutarate conversion is very low and the same whether penicillin N, penicillin G or no penicillin substrate is present [6]) [6] R266Q (, 2-oxoglutarate conversion is very low and the same whether penicillin N, penicillin G or no penicillin substrate is present [6]) [6] R306L (, mutant enzyme with improved efficiency in penicillin conversion [17]) [17] R306L (, mutation enhances penicillin N conversion compared to the level of wild-type enzyme, turnover number for penicillin N is increased, no enhancement in activity with penicillin G as substrate, little effect on kinetic values using penicillin G as substrate [6]) [6] 229

Deacetoxycephalosporin-C synthase

1.14.20.1

R307L (, mutant enzyme with improved efficiency in penicillin conversion [17]) [17] R307Q (, mutation enhances penicillin N conversion compared to the level of wild-type enzyme, turnover number for penicillin N is increased, no enhancement in activity with penicillin G as substrate. Mutation increase the Km -value by 10fold, but has little effect on the turnover number for penicillin G [6]) [6] R74I (, turnover number and Km -values are similar to that for wildtype enzyme. 2-Oxoglutarate conversion is significantly stimulated in presence of penicillin N and penicillin G compared to wild type enzyme. Penicillin oxidation is reduced relative to the wild type enzyme [6]) [6] R74Q (, turnover number and Km -values are similar to that for wildtype enzyme. 2-Oxoglutarate conversion is significantly stimulated in presence of penicillin N and penicillin G compared to wild type enzyme. Penicillin oxidation is reduced relative to the wild type enzyme [6]) [6] R74Q/R266I (, 2-oxoglutarate conversion is not stimulated by penicillin substrates [6]) [6] R74Q/R266Q (, 2-oxoglutarate conversion is not stimulated by penicillin substrates [6]) [6] R75I/D270G (, 2-oxoglutarate conversion is not stimulated by penicillin substrates [6]) [6] R75Q (, turnover number and Km -values are similar to that for wildtype enzyme. 2-Oxoglutarate conversion is significantly stimulated in presence of penicillin N compared to wild type enzyme [6]) [6] Additional information (, the truncation of the C-terminus at position 310 in the wild-type enzyme results in reduction of indiscriminate conversion of penicillin analog but this defect is compensated by the replacement of asparagine with leucine at position 304 [17]) [17]

4  0  6.5-9 (, 4 C, 1 h, stable, native enzyme [14]) [14] 7 (, 4 C, 120 h, 80% loss of activity [12]) [12] 8 (, 4 C, 120 h, 70% loss of activity [12]) [12] 9 (, 4 C, 120 h, 35% loss of activity [12]) [12] 10 (, enzyme has similar stability at 4 C and at 25 C [9]) [9] 11 (, enzyme is more stable at 4 C than at 25 C [9]) [9] )  25 (, 60 min, stable [12]) [12] 30 (, 60 min, 25% loss of activity [12]) [12] 40 (, 10 min, 50% loss of activity [14]) [14] 65 (, 60 min, complete inactivation [12]) [12] Additional information (, at pH 10 enzyme has similar stability at 4 C and at 25 C. At pH 11 the enzyme is more stable at 4 C than at 25 C [9]) [9] 230

1.14.20.1

Deacetoxycephalosporin-C synthase

   &   urea (, purification is achieved by an anion-exchange-chromatographic step in the presence of denaturing concentrations of urea. The main obstacle to converting the homogenous unfolded protein into the active enzyme is a urea-dependent aggregation during refolding that led to irreversible enzyme inactivation [9]) [9] 5    "

, increasing ionic strength to 100 decreases stability of the enzyme [12] , the enzyme is very unstable and can be partially stabilized in 25 mM Tris-HCl, pH 9.0, in the presence of 0.1 mM DTT [12] , purified enzyme is stable when flash-frozen and stored at -80 C [10]    , purified enzyme is stable when flash-frozen and stored at -80 C [10]

"  [1] Cantwell, C.; Beckmann, R.; Whiteman, P.; Queener, S.W.; Abraham, E.P.: Isolation of deacetoxycephalosporin-c from fermentation broths of Penicillium chrysogenum transformants - construction of a new fungal biosynthetic-pathway. Proc. R. Soc. Lond. B Biol. Sci., 248, 283-289 (1992) [2] Lee, H.J.; Lloyd, M.D.; Harlos, K.; Clifton, I.J.; Baldwin, J.E.; Schofield, C.J.: Kinetic and crystallographic studies on deacetoxycephalosporin C synthase (DAOCS). J. Mol. Biol., 308, 937-948 (2001) [3] Valegard, K.; van Scheltinga, A.C.T.; Lloyd, M.D.; Hara, T.; Ramaswamy, S.; Perrakis, A.; Thompson, A.; Lee, H.J.; Baldwin, J.E.; Schofield, C.J.; Hajdu, J.; Andersson, I.: Structure of a cephalosporin synthase. Nature, 394, 805809 (1998) [4] Yeh, W.K.; Ghag, S.K.; Queener, S.W.: Enzymes for epimerization of isopenicillin N, ring expansion of penicillin N, and 3'-hydroxylation of deacetoxycephalosporin C. Function, evolution, refolding, and enzyme engineering. Ann. N.Y. Acad. Sci., 672, 396-408 (1992) [5] Lee, H.J.; Dai, Y.F.; Shiau, C.Y.; Schofield, C.J.; Lloyd, M.D.: The kinetic properties of various R258 mutants of deacetoxycephalosporin C synthase. Eur. J. Biochem., 270, 1301-1307 (2003) [6] Lipscomb, S.J.; Lee, H.J.; Mukherji, M.; Baldwin, J.E.; Schofield, C.J.; Lloyd, M.D.: The role of arginine residues in substrate binding and catalysis by deacetoxycephalosporin C synthase. Eur. J. Biochem., 269, 2735-2739 (2002) [7] Lloyd, M.D.; Lee, H.J.; Harlos, K.; Zhang, Z.H.; Baldwin, J.E.; Schofield, C.J.; Charnock, J.M.; Garner, C.D.; Hara, T.; Terwisscha van Scheltinga, A.C.; Valeg.Ang.rd, K.; Viklund, J.A.C.; Hajdu, J.; Andersson, I.; Danielsson, A.; Bhikhabhai, R.: Studies on the active site of deacetoxycephalosporin C synthase. J. Mol. Biol., 287, 943-960 (1999) [8] Chin, H.S.; Sim, J.; Seah, K.I.; Sim, T.S.: Deacetoxycephalosporin C synthase isozymes exhibit diverse catalytic activity and substrate specificity. FEMS Microbiol. Lett., 218, 251-257 (2003) 231

Deacetoxycephalosporin-C synthase

1.14.20.1

[9] Ghag, S.K.; Brems, D.N.; Hassell, T.C.; Yeh, W.K.: Refolding and purification of Cephalosporium acremonium deacetoxycephalosporin C synthetase/hydroxylase from granules of recombinant Escherichia coli. Biotechnol. Appl. Biochem., 24, 109-119 (1996) [10] Baldwin, J.E.; Blackburn, J.M.; Heath, R.J.; Sutherland, J.D.: High-level soluble expression and purification of deacetoxycephalosporin C/deacetylcephalosporin C synthase. Bioorg. Med. Chem. Lett., 2, 663-668 (1992) [11] Kovacevic, S.; Weigel, B.J.; Tobin, M.B.; Ingolia, T.D.; Miller, J.R.: Cloning, characterization, and expression in Escherichia coli of the Streptomyces clavuligerus gene encoding deacetoxycephalosporin C synthetase. J. Bacteriol., 171, 754-760 (1989) [12] Cortes, J.; Martin, J.F.; Castro, J.M.; Laiz, L.; Liras, P.: Purification and characterization of a 2-oxoglutarate-linked ATP-independent deacetoxycephalosporin C synthase of Streptomyces lactamdurans. J. Gen. Microbiol., 133, 3165-3174 (1987) [13] Sim, J.; Sim, T.S.: Mutational evidence supporting the involvement of tripartite residues His183, Asp185, and His243 in Streptomyces clavuligerus deacetoxycephalosporin C synthase for catalysis. Biosci. Biotechnol. Biochem., 64, 828-832 (2000) [14] Dotzlaf, J.E.; Yeh, W.K.: Purification and properties of deacetoxycephalosporin C synthase from recombinant Escherichia coli and its comparison with the native enzyme purified from Streptomyces clavuligerus. J. Biol. Chem., 264, 10219-10227 (1989) [15] Shen, Y.Q.; Wolfe, S.; Demain, A.L.: Desacetoxycephalosporin C synthetase: importance of order of cofactor/reactant addition. Enzyme Microb. Technol., 6, 402-404 (1984) [16] Lubbe, C.; Wolfe, S.; Demain, A.L.: Dithiothreitol reactivates desacetoxycephalosporin C synthetase after inactivation. Enzyme Microb. Technol., 7, 353-356 (1985) [17] Chin, H.S.; Sim, T.S.: C-terminus modification of Streptomyces clavuligerus deacetoxycephalosporin C synthase improves catalysis with an expanded substrate specificity. Biochem. Biophys. Res. Commun., 295, 55-61 (2002) [18] Chin, H.S.; Sim, J.; Sim, T.S.: Mutation of N304 to leucine in Streptomyces clavuligerus deacetoxycephalosporin C synthase creates an enzyme with increased penicillin analog conversion. Biochem. Biophys. Res. Commun., 287, 507-513 (2001)

232

*,     



   
 1.14.21.1    (S)-cheilanthifoline,NADPH:oxygen oxidoreductase (methylenedioxy-bridgeforming)    (S)-stylopine synthase    (S)-cheilanthifoline oxidase (methylenedioxy-bridge-forming) EC 1.1.3.32 (formerly) synthase, (S)-stylopine
    138791-29-4

     Eschscholzia californica [1]

!    "
 # 

(S)-cheilanthifoline + NADPH + H+ + O2 = (S)-stylopine + NADP+ + 2 H2 O    oxidation redox reaction reduction       (S)-cheilanthifoline + NADPH + O2 (, enzyme is induced 20 h after challenging the cell suspension culture with elicitor [1]) [1] $ (S)-stylopine + NADP+ [1]      (S)-cheilanthifoline + NADPH + O2 (, NADPH is essential for activity, NADH displays only 0.5% turnover of that of NADPH [1]) [1] $ (S)-stylopine + NADP+ [1] 233

(S)-Stylopine synthase

1.14.21.1


"%  FAD (, 0.004 mM, together with the optimal concentration of NADPH, 0.2 mM, enhances activity by 50%) [1] FMN (, 0.004 mM, together with the optimal concentration of NADPH, 0.2 mM, enhances activity by 50%) [1] NADPH (, essential for activity, NADH displays only 0.5% turnover of that of NADPH) [1] cytochrome p450 (, the enzyme is a cytochrome P450 dependent monooxygenase [1]) [1] . / *', 0.0004 ((S)-cheilanthifoline, ) [1] 0  8 [1] 0 

 7-9 (, about 40% of maximal activity at pH 7 and pH 9) [1] )  *
, 30 [1]

 2 %$ %' %

% cell suspension culture [1] 3#

microsome [1]

4  )  4 (, half-life: 27 h) [1] 25 (, half-life: 2.3 h) [1]    , -20 C, 15% loss of activity after 4 months [1]

"  [1] Bauer, W.; Zenk, M.H.: Two methylenedioxy bridge forming cytochrome P450 dependent enzymes are involved in (S)-stylopine biosynthesis. Phytochemistry, 30, 2953-2961 (1991)

234

*,
 "   



   
 1.14.21.2    (S)-scoulerine,NADPH:oxygen oxidoreductase (methylenedioxy-bridge-forming)    (S)-cheilanthifoline synthase    (S)-scoulerine oxidase (methylenedioxy-bridge-forming) EC 1.1.3.33 (formerly) S-cheilanthifoline synthase synthase, (S)-cheilanthifoline
    138791-27-2

     Eschscholzia californica [1]

!    "
 # 

(S)-scoulerine + NADPH + H+ + O2 = (S)-cheilanthifoline + NADP+ + 2 H2 O    oxidation redox reaction reduction       (S)-scoulerine + NADPH + O2 (, enzyme is induced 20 h after challenging the cell suspension cultur with elicitor [1]) [1] $ (S)-cheilanthifoline + NADP+ [1]

235

(S)-Cheilanthifoline synthase

1.14.21.2

     (S)-scoulerine + NADPH + O2 (, NADPH is essential for activity, NADH displays only 0.5% turnover of that of NADPH [1]) [1] $ (S)-cheilanthifoline + NADP+ [1]
"%  FAD (, 0.004 mM, together with the optimal concentration of NADPH, 0.2 mM, enhances activity by 50%) [1] FMN (, 0.004 mM, together with the optimal concentration of NADPH, 0.2 mM, enhances activity by 50%) [1] NADPH (, essential for activity, NADH displays only 0.5% turnover of that of NADPH) [1] cytochrome p450 (, the enzyme is a cytochrome P450 dependent monooxygenase [1]) [1] . / *', 0.0009 ((S)-scoulerine, ) [1] 0  8 [1] 0 

 7-9 (, about 50% of maximal activity at pH 7 and pH 9) [1] )  *
, 30 [1]

 2 %$ %' %

% cell suspension culture [1] 3#

microsome [1]

4  )  4 (, half-life: 30 h) [1] 25 (, half-life: 2.5 h) [1]    , -20 C, 15% loss of activity after 4 months [1]

"  [1] Bauer, W.; Zenk, M.H.: Two methylenedioxy bridge forming cytochrome P450 dependent enzymes are involved in (S)-stylopine biosynthesis. Phytochemistry, 30, 2953-2961 (1991)

236

>    

!

   
 1.14.21.3    (S)-N-methylcoclaurine,NADPH:oxygen oxidoreductase (C-O phenol-coupling)    berbamunine synthase    (S)-N-methylcoclaurine oxidase (C-O phenol-couling) (S)-N-methylcoclaurine oxidase [C-O phenol-coupling] CYP80 CYPLXXX cytochrome P450 80 EC 1.1.3.34 (formerly) benzyltetrahydroisoquinoline oxidase oxidase, benzyltetrahydroisoquinoline
    144941-42-4

     Berberis stolonifera [1-3]

!    "
 # 

(S)-N-methylcoclaurine + (R)-N-methylcoclaurine + NADPH + H+ + O2 = berbamunine + NADP+ + 2 H2 O (, regioselective and stereoselective formation of a C-O phenol couple in bisbenzylisoquinoline alkaloid biosynthesis without concomitant incorporation of activated oxygen into the product [1])    oxidation redox reaction reduction

237

Berbamunine synthase

1.14.21.3

      (S)-N-methylcoclaurine + (R)-N-methylcoclaurine + NADPH + O2 (, enzyme mediates highly regiospecific and stereospecific oxidative phenol coupling to afford natural bisbenzylisoquinoline alkaloids [1]) [1] $ berbamunine + guattegaumerine + NADP+ [1]      (S)-N-methylcoclaurine + (R)-N-methylcoclaurine + (S)-coclaurine + NADPH + O2 [1, 3] $ (R,S)-berbamunine + (R,R)-guattegaumerine + (R,S)-2'-norberbamunine + NADP+ [1, 3]  (S)-N-methylcoclaurine + (R)-N-methylcoclaurine + NADPH + O2 [1, 2] $ berbamunine + guattegaumerine + NADP+ (, incubation with equimolar amounts of both (R)-N-methylcoclaurine and (S)-N-methylcoclaurine leads to the formation of berbamunine and guattegaumerine [1]; , two molecules of (R)-N-methylcoclaurine for the (R,R) dimer guattegaumerine or one molecule each of (R)-N-methylcoclaurine and (S)-Nmethylcoclaurine form the (R,S) dimer berbamunine. The ratio of the two bisbenzylisoquinolines formed can be altered by the reductase source or by varying the enantiomer composition of the substrates [2]) [1, 2]  Additional information (, incubation with (S)-N-methylcoclaurine and (R)-coclaurine does not lead to a dimeric product, as well as incubations with (S)-N-methylcoclaurine alone or with either (S)-coclaurine or (R)-coclaurine together or individually [1]) [1] $ ?
"%  cytochrome p450 (, enzyme contains 18.2 nmol of cytochrome P450 per mg of enzyme [1]) [1] ) &  * +, Additional information [1, 2] " & *-% , Additional information [1, 3] . / *', Additional information (, normal Michaelis-Menten kinetics are not observed for the formation of guattegaumerine or berbamunine) [2] 0  7.5 (, formation of berbamunine or guattegaumerine) [2] 8-8.5 [1] )  *
, 35 (, formation of berbamunine) [2] 35-38 [1] 38 (, formation of guattegaumerine) [2]

238

1.14.21.3

Berbamunine synthase

) 

 *
, 7-49 (, 50% of maximal activity at 7 C and 49 C, formation of berbamunine) [2] 18-46 (, 50% of maximal activity at 18 C and 46 C, formation of guattegaumerine) [2] 22-46 (, 50% of maximal activity at 22 C and 46 C) [1]

  #     ? (, x * 46000, SDS-PAGE [1,2]; , x * 55474, calculation from nucleotide sequence [2]) [1, 2]

 2 %$ %' %

% cell suspension culture [1, 2] $"

(heterologously expressed enzyme from Sf9 cell culture [2]) [1-3]
 

(expression in functional form in insect cell culture using a baculovirusbased expression system [2]; expression in Sf9 cells [2]) [2, 3]

"  [1] Stadler, R.; Zenk, M.H.: The purification and characterization of a unique cytochrome P-450 enzyme from Berberis stolonifera plant cell cultures. J. Biol. Chem., 268, 823-831 (1993) [2] Kraus, P.F.X.; Kutchan, T.M.: Molecular cloning and heterologous expression of a cDNA encoding berbamunine synthase, a C-O phenol-coupling cytochrome P450 from the higher plant Berberis stolonifera. Proc. Natl. Acad. Sci. USA, 92, 2071-2075 (1995) [3] Kutchan, T.M.: Heterologous expression of alkaloid biosynthetic genes - a review. Gene, 179, 73-81 (1996)

239

   



   
 1.14.21.4    (R)-reticuline,NADPH:oxygen oxidoreductase (C-C phenol-coupling)    salutaridine synthase    (R)-reticuline dehydrogenase (R)-reticuline oxidase (C-C phenol-coupling) EC 1.1.3.35 (formerly) dehydrogenase, (R)-reticuline
    149433-84-1

    





Papaver somniferum [1] Sus scrofa [2] Bos taurus [2] Mus musculus [2] Ovis aries [2] Rattus norvegicus [2] Homo sapiens (very low activity) [2]

!    "
 # 

(R)-reticuline + NADPH + H+ + O2 = salutaridine + NADP+ + 2 H2 O (, regioselective and stereoselective para-ortho oxidative coupling [1])    oxidation redox reaction reduction

240

1.14.21.4

Salutaridine synthase

      (R)-reticuline + NADPH + O2 (, formation of salutaridine, a key intermediate in morphine biosynthesis [1]; , critical step in morphine biosynthesis [2]) [1, 2] $ salutaridine + NADP+ [1, 2]      (R)-reticuline + NADPH + O2 (, strictly dependent on NADPH as reducing cofactor and on (R)-configurated reticuline [1]; , highly regioselective and stereoselective enzyme [2]) [1, 2] $ salutaridine + NADP+ [1, 2]  (R/S)-orientaline [2] $ ? 2  CO (, in darkness but not in light) [1] ancymidole [1] prochloraz [1]
"%  cytochrome p450 (, enzyme contains cytochrome P450 [1,2]; , 20 nmol cytochrome P450 per mg of enzyme [2]) [1, 2] . / *', 0.003 ((R)-reticuline, ) [2] 0.017 ((R)-reticuline, ) [1] 0.15 (NADPH, ) [1] 0  7.5 [1] 0 

 6.5-8.5 (, about 50% of maximal activity at pH 6.5 and pH 8.5) [1] )  *
, 20-25 [1] ) 

 *
, 10-30 (, 10 C: about 30% of maximal activity, 30 C: about 65% of maximal activity) [1]

  #     ? (, x * 50000, SDS-PAGE [2]) [2]

241

Salutaridine synthase

1.14.21.4

 2 %$ %' %

% capsule [1] cell suspension culture (thebaine-producing) [1] liver [2] root [1] shoot [1] Additional information (, no enzyme activity in latex [1]) [1] 3#

lysosome [2] microsome (, membrane-bound [1]) [1, 2] mitochondrion ( light mitochondrial fraction [2]) [1, 2] $"

[2]

4     , -20 C, microsome-bound enzyme, freezing causes a total loss of activity [1] , -70 C, microsome-bound enzyme, 20% loss of activity after 1 month [1] , 22 C, microsome-bound enzyme, half-life: 2 h [1] , in ice water, microsome-bound enzyme, half-life: 20 h [1]

"  [1] Gerardy R.; Zenk, M.H.: Formation of salutaridine from (R)-reticuline by a membrane-bound cytochrome P-450 enzyme from Papaver somniferum. Phytochemistry, 32, 79-86 (1993) [2] Amann, T.; Roos, P.H.; Huh, H.; Zenk, M.H.: Purification and characterization of a cytochrome p450 enzyme from pig liver, catalyzing the phenol oxidative coupling of (R)-reticuline to salutaridine, the critical step in morphine biosynthesis. Heterocycles, 1, 425-440 (1995)

242

*,
    



   
 1.14.21.5    (S)-tetrahydrocolumbamine,NADPH:oxygen oxidoreductase (methylenedioxybridge-forming)    (S)-canadine synthase    (S)-tetrahydroberberine synthase (S)-tetrahydrocolumbamine oxidase (methylenedioxy-bridge-forming) EC 1.1.3.36 (formerly) canadine synthase
    114308-22-4

    









Thalictrum tuberosum [1] Berberis aristata [1] Berberis crataegina [1] Berberis taliensis [1] Berberis stolonifera [1] Berberis henryana [1] Coptis japonica [1, 3] Thalictrum glaucum [1] Thalictrum macrocarpum [1] Berberidaceae [2] Ranunculaceae [2] Thalictrum glabrum [1]

!    "
 # 

(S)-tetrahydrocolumbamine + NADPH + H+ + O2 = (S)-canadine + NADP+ + 2 H2 O (, formation of a methylenedioxy bridge in ring A [3]) 243

(S)-Canadine synthase

1.14.21.5

   oxidation redox reaction reduction       (S)-tetrahydrocolumbamine + NADPH + O2 (, penultimate step in the biosynthesis of the protoberberine alkaloid, berberine [1]; , reaction in protoberberine biosynthetic pathway [2]) [1, 2] $ (S)-canadine + NADP+ + H2 O (, (S)-canadine is identical with (S)-tetrahydroberberine [1]) [1, 2]      (S)-tetrahydrocolumbamine + NADH + O2 (, 23% of the activity compared to NADPH [2]) [2] $ ?  (S)-tetrahydrocolumbamine + NADPH + O2 (, high substrate specificity [1]) [1, 2] $ (S)-canadine + NADP+ + H2 O (, (S)-canadine is identical with (S)-tetrahydroberberine [1]) [1, 2] 2  CO (, inhibition can be partly reverted by blue light [2]) [2] cytochrome c (, microsomal-bound enzyme) [1] juglone (, microsomal-bound enzyme) [1] ketoconazole (, microsomal-bound enzyme) [1] menadione (, microsomal-bound enzyme) [1] metyrapone (, microsomal-bound enzyme) [1] plumbagin (, microsomal-bound enzyme) [1] prochloraz (, microsomal-bound enzyme) [1] propiconazole (, microsomal-bound enzyme) [1] tetcyclacis (, microsomal-bound enzyme) [1] triadimefone (, microsomal-bound enzyme) [1] tropolone (, microsomal-bound enzyme) [1]
"%  cytochrome p450 (, the enzyme forms a complex with a cytochrome P-450 reductase [1]; , cytochrome P-450 enzyme [2]) [1, 2] . / *', 0.0115 (tetrahydrocolumbamine, canadine synthase in complex with a cytochrome P-450 reductase, ) [1] 0.033 (NADPH, canadine synthase in complex with a cytochrome P-450 reductase, ) [1] 0  8.5 (, canadine synthase in complex with a cytochrome P-450 reductase) [1]

244

1.14.21.5

(S)-Canadine synthase

)  *
, 40 (, canadine synthase in complex with a cytochrome P-450 reductase) [1]

 2 %$ %' %

% cell culture (, protoberberine producing cell line [1]) [1, 3] 3#

microsome [1]

"  [1] Rueffer, M.; Zenk, M.H.: Canadine synthase from Thalictrum tuberosum cell cultures catalyses the formation of the methylenedioxy bridge in berberine synthesis. Phytochemistry, 36, 1219-1223 (1994) [2] Rueffer, M.: New reactions in the protoberberine biosynthetic pathway. Chem. Listy, 87, 215-217 (1993) [3] Galneder, E.; Rueffer, M.; Wanner, G.; Tabata, M.; Zenk, M.H.: Alternative final steps in berberine biosynthesis in Coptis japonica cell cultures. Plant Cell Rep., 7, 1-4 (1988)

245

$      

::

   
 1.14.99.1    (5Z,8Z,11Z,14Z)-icosa-5,8,11,14-tetraenoate,hydrogen-donor:oxygen oxidoreductase    prostaglandin-endoperoxide synthase    (PG)H synthase PG synthetase cyclooxygenase fatty acid cyclooxygenase prostaglandin G/H synthase prostaglandin endoperoxide synthetase prostaglandin synthase prostaglandin synthase-2 prostaglandin synthetase synthase, prostaglandin
    9055-65-6

     Homo sapiens (overview [14]; recombinant enzyme, product of COX-2 [28-31]; recombinant isozymes 1 and 2 [34, 41]) [14, 25, 28-31, 34, 35, 37, 41] Cavia porcellus [23] Sus scrofa (overview [14]) [14] Ovis aries (recombinant enzyme [32]) [1, 3, 6, 8, 10, 11, 13, 15-17, 19, 20, 32, 39] Bos taurus (overview [14]) [2, 4, 7, 12, 14, 18, 22, 26, 27] Rattus norvegicus [5, 24, 35] Oryctolagus cuniculus [9, 21] Mus musculus (recombinant enzyme [38, 40]) [33, 38, 40] Canis canis (prostatic epithelial cell line [36]) [35, 36]

246

1.14.99.1

Prostaglandin-endoperoxide synthase

Saimiri sciureus (squirrel monkey [35]) [35] Macaca mulatta (rhesus monkey [35]) [35] Salvelinus fontinalis (brook trout, isozymes 1 and 2 [42]) [42]

!    "
 # 

arachidonate + AH2 + 2 O2 = prostaglandin H2 + A + H2 O ( mechanism, enzyme acts both as dioxygenase and as peroxidase [8, 19])    oxidation redox reaction reduction       Additional information ( enzyme has a central position in prostanoic metabolism: first step in formation of prostaglandins and thromboxanes, the conversion of arachidonic acid to prostaglandin endoperoxides G and H [1]; first step in prostaglandin synthesis [15, 16]) [1, 15, 16] $ ?      8,11,14-eicosatrienoic acid + O2 ( bis-dioxygenase activity, cyclooxygenase activity, presence of hematin [7]) (Reversibility: ? [7, 18]) [7, 18] $ prostaglandin G1 + ? ( 9a,11a-epidioxy-15(S)-hydroperoxy-13trans-prostenoic acid [7]) [7, 18]  8,11,14-eicosatrienoic acid + O2 ( hydroperoxidase activity, presence of hematin and tryptophan [7]) (Reversibility: ? [7]) [7, 18] $ prostaglandin H1 + ? ( 9a,11a-epidioxy-15(S)-hydroxy-13-trans-prostenoic acid [7])  arachidonate + electron donor + O2 (Reversibility: ? [4, 19, 24, 28, 29, 38]) [4, 19, 24, 28, 29, 38] $ prostaglandin + H2 + oxidized electron donor + H2 O  arachidonic acid + ? (Reversibility: ? [22]) [22] $ prostaglandin E2 + ? [22]  arachidonic acid + ? (Reversibility: ? [30, 40]) [30, 40] $ 15(R)-hydroxy-eicosatetraenoic acid ( product of aspirin acetylated enzyme or S516M mutant [30]; product of aspirin treated enzyme [40,41]; no S-isomer found [41]) [30, 40, 41]  arachidonic acid + ? (Reversibility: ? [6]) [6] $ 15-hydroperoxy-9a,11a-peroxiprosta-5,13-dienoic acid + ? [6]  Additional information ( also catalyzed: transformation of arachidonic acid into prostaglandin E2 , prostaglandin F2 and 12-hydroxy-5,8,10-heptadecatrienoic acid [19]; formation of prostaglandin E2 ,

247

Prostaglandin-endoperoxide synthase

1.14.99.1

prostaglandin F2a and prostaglandin D2 from arachidonic acid [21]; electron donors used by hydroperoxidase: phenylbutazone, sulindac [8-10]; cooxidation of: 4-chloroaniline to yield N-(4-chlorophenyl)-hydroxylamine and 1-chloro-4-nitrosobenzene [20]; xenobiotics such as benzo(a)pyrene cannot act as electron donor, but undergo cooxydation during hydroperoxidase reaction [8]; functional differentiation of cyclooxygenase and peroxidase activities by trypsin treatment [16]; relative activities of isozymes 1,2 depend on source of arachidonic acid - exogenous versus endogenous [38]; major products of arachidonic acids are prostaglandins D2 and E2 , minor products prostaglandin F2a and 6-keto-prostaglandin F1a [41]) [1, 8-10, 16, 19-21, 38, 41] $ ? 2  1,10-phenanthroline ( weak [21]) [21] 1-mercapto-9,11,15-trihydroxyprosta-5,13-diene ( inhibition of prostaglandin G1 synthesis [18]) [18] 1-mercapto-9-oxo-11,15-dihydroxyprosta-5,13-dione ( inhibition of prostaglandin G1 synthesis [18]) [18] 2,2'-bipyridyl ( weak [21]) [21] 2,3-dimercaptopropanol ( inhibition of prostaglandin G1 synthesis [18]) [18] 2-hydroxybutyric acid ( weak [5]) [5] 5-bromo-2-[4-fluorophenyl]-3-[4-methylsulfonylphenyl]-thiophene ( DuP-697, selective for isozyme 2 [28]; 50% inhibition at 8.7 nM [29]) [28, 29] 6-[2,4-difluorophenoxy]-5-methyl-sulfonylamino-1-indanone ( CGP28238, an isozyme-2 specific inhibitor, 65% inhibition at 100 nM [27]) [27] 6-methoxy-2-naphthyl acetic acid ( active metabolite of nabumetone, isozyme 1, 50% inhibition at 0.2-0.8 mM, isozyme 2, 50% inhibition at 0.015-0.55 mM [40]) [40] 6-methylnaphthylacetic acid ( recombinant protein, 50% inhibition at 0.08-0.1 mM [25]) [25] 8-hydroxyquinoline [21] 9,11-dihydroxy-15S-mercaptoprosta-5,13-dienoic acid ( or 15R-isomer, inhibition of prostaglandin G1 synthesis [18]) [18] BW 755C ( recombinant protein, 50% inhibition at 0.01-0.02 mM [25]) [25] dl-propanolol [24] EDTA ( weak [21]) [21] EGTA ( weak [21]) [21] ETYA ( recombinant protein, 50% inhibition at 0.015-0.025 mM [25]) [25] L-745 ( isozyme 1, 50% inhibition at 0.369 mM, isozyme 2, 50% inhibition at 0.002 mM [37]) [37]

248

1.14.99.1

Prostaglandin-endoperoxide synthase

N-[2-cyclohexyloxy-4-nitrophenyl]methanesulfonamide ( NS-398, selective for isozyme 2 [28]; 50% inhibition at 81 nM [29]; isozyme 1, 50% inhibition at 0.075 mM, isozyme 2, 50% inhibition at 0.002 mM [34]; little inhibition in gastrointestinal tissues [35]; isozyme 1, 50% inhibition at 0.017 mM, isozyme 2, 50% inhibition at 0.0001 mM [37]) [28, 29, 34, 35, 37] SC58125 ( isozyme 1, 50% inhibition at 0.039 mM, isozyme 2, 50% inhibition at 0.0003 mM [37]) [37] acetoacetic acid ( weak [5]) [5] acetylsalicylic acid ( inhibition of prostaglandin G1 synthesis [7]; inhibition of oxygenase activity by acetylating a serine residue of the enzyme [15]; isozyme 1, complete inhibition, isozyme 2, change in reaction, main product from arachidonate is 15-hydroxyeicosatetraenoic acid [40]) [7, 15, 40] albumin ( bovine serum albumin inhibits by binding of arachidonic acid [21]) [4, 21] anilorac ( isozyme 1, 50% inhibition at 0.0007 mM, isozyme 2, 50% inhibition at 0.009 mM [34]) [34] azide ( weak [21]) [21] butyric acid [5] crotonic acid [5] cyanide [21] diclofenac ( recombinant protein, 50% inhibition at 0.04 mM [25]; 50% inhibition at 9.4 nM [29]; isozyme 1, 50% inhibition at 0.0009 mM, isozyme 2, 50% inhibition at 0.0015 mM [34]; isozyme 1, 50% inhibition at 0.0003 mM, isozyme 2, 50% inhibition at 18 nM [37]) [25, 29, 34, 37] diethyldithiocarbamate [21] dihydrolipoic acid ( inhibition of prostaglandin G1 synthesis [18]) [18] dithiothreitol ( inhibition of prostaglandin G1 synthesis [18]) [18] docosahexaenoic acid ( isozyme 1, 50% inhibition at 0.011 mM, isozyme 2, 50% inhibition at 0.015 mM [40]) [40] eicosa-5,8,11,14-tetraynoic acid [22] ellagic acid ( at high concentration and in presence of cofactors inhibition, at low concentrations stimulation [2]) [2] etodalac ( recombinant protein, 50% inhibition at 0.06-0.07 mM [25]) [25] fatty acid ( of low molecular mass [5]) [5] fenclofenac ( isozyme 1, 50% inhibition at 0.007 mM, isozyme 2, 50% inhibition at 0.004 mM [34]) [34] flosulide ( selective for isozyme 2, 50% inhibition at 130 nM [29] [29]) [29] flufenamic acid ( 50% inhibition at 0.02 mM [29]) [29] flurbiprofen ( isozyme 1, 50% inhibition at 40 nM, isozyme 2, 50% inhibition at 500 nM [34]; isozyme 1, 50% inhibition at 0.0009 mM, iso-

249

Prostaglandin-endoperoxide synthase

1.14.99.1

zyme 2, 50% inhibition at 0.0009 mM [37]; isozyme 1, 50% inhibition at 0.0005 mM, isozyme 2, 50% inhibition at 0.003 mM [40]) [34, 37, 40] haptoglobin [4] human serum [4] ibuprofen ( recombinant protein, 50% inhibition at 0.04 mM [25]; reversible [28]; 50% inhibition at 0.253 mM [29]; isozyme 1, 50% inhibition at 0.0026 mM, isozyme 2, 50% inhibition at 0.0015 mM [34]; isozyme 1, 50% inhibition at 0.009 mM, isozyme 2, 50% inhibition at 0.018 mM [37]; isozyme 1, 50% inhibition at 0.09 mM, isozyme 2, 50% inhibition at 0.008 mM [40]) [25, 28, 29, 34, 37, 40] indomethacin ( inhibition of prostaglandin G1 synthesis [7]; acts on isozyme 1 and 2, 85% inhibition at 100 nM [27]; reversible and time-dependent inhibition [28]; 50% inhibition at 100 nM [29]; isozyme 1, 50% inhibition at 0.0017 mM, isozyme 2, 50% inhibition at 0.025 mM [34]; inhibition in gastrointestinal tissues [35]; isozyme 1, 50% inhibition at 0.0005 mM, isozyme 2, 50% inhibition at 0.0003 mM [37]; isozyme 1, 50% inhibition at 0.005 mM, isozyme 2, 50% inhibition at 0.130-0.160 mM [40]) [7, 21, 22, 23, 27, 28, 29, 34, 35, 37, 40] ketoprofen ( isozyme 1, 50% inhibition at 0.0005 mM, isozyme 2, 50% inhibition at 0.0025 mM [34]; isozyme 1, 50% inhibition at 0.011 mM, isozyme 2, 50% inhibition at 0.018 mM [37]) [34, 37] meclofenamic acid ( isozyme 1, 50% inhibition at 0.002 mM, isozyme 2, 50% inhibition at 0.015 mM [40]) [21, 40] mefenamic acid ( isozyme 1, 50% inhibition at 0.01 mM, isozyme 2, 50% inhibition at 0.0003 mM [34]) [34] meloxicam ( isozyme 1, 50% inhibition at 0.005 mM, isozyme 2, 50% inhibition at 0.0004 mM [37]) [37] naproxen ( recombinant protein, 50% inhibition at 0.05-0.06 mM [25]; isozyme 1, 50% inhibition at 0.0006 mM, isozyme 2, 50% inhibition at 0.002 mM [34]) [25, 34] nemesulide ( isozyme 1, 50% inhibition at 0.07 mM, isozyme 2, 50% inhibition at 0.0013 mM [34]; isozyme 1, 50% inhibition at 0.009 mM, isozyme 2, 50% inhibition at 0.0005 mM [37]) [34, 37] niflumic acid ( isozyme 1, 50% inhibition at 0.016 mM, isozyme 2, 50% inhibition at 0.0001 mM [34]) [34] non-steroidal anti-inflammatory agents (inhibition of cyclooxygenase activity) [11, 12] p-aminophenol [21] piroxicam ( isozyme 1, 50% inhibition at 0.009-0.024 mM, isozyme 2, 50% inhibition at 0.070-0.240 mM [37]; isozyme 1, 50% inhibition at 0.075 mM, isozyme 2, 50% inhibition at 0.002 mM [40]) [37, 40] propionic acid [5] sulindac sulfide ( isozyme 1, 50% inhibition at 0.0004 mM, isozyme 2, 50% inhibition at 0.012 mM [40]) [40] suprofen ( isozyme 1, 50% inhibition at 0.0005 mM, isozyme 2, 50% inhibition at 0.002mM [34]) [34] 250

1.14.99.1

Prostaglandin-endoperoxide synthase

tannic acid ( at high concentration and in presence of cofactors inhibition, at low concentrations stimulation [2]) [2] Additional information ( effect of cofactor, enzyme and substrate concentration on inhibition by human serum, haptoglobin and albumin [4]; mechanism of selective inhibition [28]) [4, 28]
"%  heme ( enzyme contains a heme group [6]; either free or protein-bound heme is required for cyclooxygenase- and hydroperoxidase activity [7]) [6, 7] &

  5-hydroxytryptamine ( stimulates [21]) [21] benzoquinone ( stimulates conversion of prostaglandin G1 to H1 [7]) [7] cysteine ( stimulates [21]) [21] dopamine ( 3,4-dihydroxyphenylethylamine, stimulates [21]) [21, 24] ellagic acid ( at high concentration and in presence of cofactors inhibition, at low concentrations stimulation [2]) [2] epinephrine ( stimulates conversion of prostaglandin G1 to H1 [7]) [7, 23, 24] hemin ( activates [6]) [6] hydroperoxyeicosatetraenoic acid [3] hydroquinone ( stimulates conversion of prostaglandin G1 to H1 [7]) [7, 23] indole ( stimulates conversion of prostaglandin G1 to H1 [7]) [7] kynurenine ( stimulates conversion of prostaglandin G1 to H1 [7]) [7] melatonin ( stimulates [7]) [7, 21] norepinephrine [24] oestrogens ( weak stimulation [21]) [21] phenylalanine ( stimulates conversion of prostaglandin G1 to H1 [7]) [7] quinol ( stimulates [21]) [21] reduced glutathione ( stimulates [21]) [21] serotonin ( stimulates conversion of prostaglandin G1 to H1 [7]) [7, 23] tannic acid ( at high concentration and in presence of cofactors inhibition, at low concentrations stimulation [2]) [2] thyroid hormones ( weak stimulation [21]) [21] thyrotropin ( tissue-specific stimulation in thyroid [22]) [22] tryptophan ( stimulates conversion of prostaglandin G1 to H1 [7]) [7] tyrosine ( stimulates conversion of prostaglandin G1 to H1 [7]) [7] Additional information ( stimulation of enzyme in crude extract by some amines [24]) [24]

251

Prostaglandin-endoperoxide synthase

1.14.99.1

'(   iron ( possibly contains heme and non-heme iron [6]) [6] " & *-% , 2.4 ( prostaglandin H1 synthesis [7]) [7] 43 [15] . / *', 0.001 (arachidonic acid, isozyme 1 and 2 [28]) [28] 0.0045 (arachidonic acid, isozyme 1 [34]) [34] 0.005 (arachidonic acid, isozyme 2 [34]) [34] 0.006 (arachidonic acid) [29] 0.0083 (arachidonic acid) [21] 0.015 (arachidonic acid) [31] 0.16 (arachidonic acid) [22] . / *', 0.005 (indomethacin) [23] 0  7-7.3 ( formation of prostaglandin E2 from arachidonic acid [22]) [22] 7.5-8 ( formation of prostaglandin F2a [21]) [21] 8 ( synthesis of prostaglandin G1 , conversion to prostaglandin H1 [7]) [7] 8-8.5 ( formation of prostaglandin E2 and D2 [21]) [21] 0 

 6.5-9 ( about 70% of maximum activity at pH 6.0 and 9.0 of prostaglandin E2 formation, about 50% of maximum activity at pH 6.0 and 9.0 of prostaglandin F2a formation [21]) [21] 7.2-9 ( about 60% of maximum activity at pH 7.2 and 9.0 of prostaglandin D2 formation [21]) [21] )  *
, 37 [22]

  #   ' 1  65620 ( amino acid sequence deduced from nucleotide sequence of cDNA, MW of unglycosylated enzyme [1]) [1] 300000-350000 ( gel filtration [7]) [7]   ? ( x * 72000, SDS-PAGE, oxygenase and peroxidase activity are present in a single polypeptide chain, in nonionic detergent the enzyme is a dimer of 2 identical subunits [15]; x * 70000, SDS-PAGE in absence and presence of 2-mercaptoethanol [6]; x * 72000, SDS-PAGE [26]; x * 68000, SDS-PAGE [31]; x * 75000, SDS-PAGE, isozyme 1 and 2 [34]) [6, 15, 26, 31, 34]

252

1.14.99.1

Prostaglandin-endoperoxide synthase

$   "

glycoprotein ( both isozyme 1 and 2 [34, 41]) [1, 34, 41]

 2 %$ %' %

% fibroblast [25] kidney ( medulla [21]) [21] lung [23] monocyte ( isozyme 2 [37]) [37] myometrium [27] ovary [42] platelet ( isozyme 1 [37]) [37] prostate gland [36] seminal vesicle [2-4, 16, 17, 20] spleen [5] stomach ( fundus [24]) [5, 24] thyroid gland [22] vesicular gland [1, 6, 7, 11, 13, 15, 18, 19] Additional information ( enzyme can be associated with endoplasmic reticulum, nuclear envelope and plasma membrane even within the same cell [13, 14]; distribution of isozymes 1 and 2 in gastrointestinal tissues [35]; distribution in different organs [42]) [13, 14, 35, 42] 3#

cytoplasmic membrane ( probably associated with [22]) [22] microsome [7, 15-17, 19-23] Additional information ( enzyme can be associated with endoplasmic reticulum, nuclear envelope and plasma membrane even within the same cell [13, 14]) [13, 14] $"

(recombinant enzyme, apoenzyme [31]) [31] [6, 15, 17] [7]
 

[1] [26] (codes for protein of 604 amino acids, 89% identity to human protein [36]) [36] (isozymes 1 and 2 [42]) [42] 

 

Arg120Glu ( Arg120 important for interaction with substrate and with inhibitors containing a free carboxylic acid moiety [39]) [39]

253

Prostaglandin-endoperoxide synthase

1.14.99.1

Cys313Ser ( cyclooxygenase and peroxidase activity reduced by 8090%, no significant effect on inhibition, dimer formation, glycosylation [32]) [32] Cys540Ser ( cyclooxygenase and peroxidase activity reduced by 8090%, no significant effect on inhibition, dimer formation, glycosylation [32]) [32] S516M ( mutation mimics acetylation of Ser516, mutant still sensitive to most inhibitors, not: diclofenac, meclofenamic acid [30]) [30] 

medicine ( use of peroxidase activity for luminol assay of inflammation [29]; study of aspirin acetylated enzyme in order to inhibit prostaglandin synthesis [30]; inhibition of prostaglandin synthesis by suppression of enzyme expression using isomallotochromanol [33]) [29, 30, 33]

4  0  6-8 ( 24 C, 5 min, stable [7]) [7] )  30 ( pH 8.0, 5 min, stable [6]) [6] 50 ( pH 8.0, 5 min, complete loss of activity [7]) [7] 5    "

, diethyldithiocarbamate stabilizes [6] , ethylene glycol stabilizes [6] , flufenamate stabilizes [6] , glycerol stabilizes [6]

"  [1] DeWitt, D.L.; Smith, W.L.: Primary structure of prostaglandin G/H synthase from sheep vesicular gland determined from the complementary DNA sequence [published erratum appears in Proc Natl Acad Sci U S A 1988 Jul;85(14):5056]. Proc. Natl. Acad. Sci. USA, 85, 1412-1416 (1988) [2] Saeed, S.A.; Butt, N.M.; McDonald-Gibson, W.J.: The effect of ellagic acid and tannic acid on prostaglandin synthase activity in bovine seminal-vesicle homogenates. Biochem. Soc. Trans., 8, 443 (1981) [3] Narayanan, R.; Harrington, M.G.: Hydroperoxyeicosatetraenoic acid: an activator of prostaglandin synthase activity. Biochem. Soc. Trans., 8, 449-450 (1980) [4] Denning-Kendall, P.A.; Saeed, S.A.: Effect of cofactor, enzyme and substrate concentration on inhibition of prostaglandin synthase of bull seminal vesicles by human serum, haptoglobin and albumin. Biochem. Soc. Trans., 9, 379-380 (1981)

254

1.14.99.1

Prostaglandin-endoperoxide synthase

[5] Ryan, J.; Davis, G.: Inhibition of prostaglandin synthase by some low molecular mass fatty acids. Biochem. Soc. Trans., 16, 398-399 (1988) [6] Hemler, M.; Lands, W.E.M.: Purification of the cyclooxygenase that forms prostaglandins. Demonstration of two forms of iron in the holoenzyme. J. Biol. Chem., 251, 5575-5579 (1976) [7] Miyamoto, T.; Ogino, N.; Yamamoto, S.; Hayaishi, O.: Purification of prostaglandin endoperoxide synthetase from bovine vesicular gland microsomes. J. Biol. Chem., 251, 2629-2636 (1976) [8] Marnett, L.J.; Dix, T.A.; Sachs, R.J.; Siedlik, P.H.: Oxidations by fatty acid hydroperoxides and prostaglandin synthase. Adv. Prostaglandin Thromboxane Leukot. Res., 11, 79-86 (1983) [9] Zenser, T.V.; Mattammal, M.B.; Armbrecht, H.J.; Davis, B.B.: Benzidine binding to nucleic acids mediated by the peroxidative activity of prostaglandin endoperoxide synthetase. Cancer Res., 40, 2839-2845 (1980) [10] Egan, R.W.; Gale, P.O.H.; Baptista, E.M.; Kennicott, K.L.; VandenHeuvel, W.J.A.; Walker, R.W.; Fagerness, P.W.; Kuehl, F.A.: Oxidation reactions by prostaglandin cyclooxygenase-hydroperoxidase. J. Biol. Chem., 256, 73527361 (1981) [11] Van der Ouderaa, F.J.; Buytenhek, M.; Nugteren, D.H.; Van Dorp, D.A.: Acetylation of prostaglandin endoperoxide synthetase with acetylsalicylic acid. Eur. J. Biochem., 109, 1-8 (1980) [12] Mizuno, K.; Yamamoto, S.; Lands, W.E.M.: Effects of non-steroidal anti-inflammatory drugs on fatty acid cyclooxygenase and prostaglandin hydroperoxidase activities. Prostaglandins, 23, 743-757 (1982) [13] DeWitt, D.L.; Rollins, T.E.; Day, J.S.; Gauger, J.A.; Smith, W.L.: Orientation of the active site and antigenic determinants of prostaglandin endoperoxide synthase in the endoplasmic reticulum. J. Biol. Chem., 256, 10375-10382 (1981) [14] Smith, W.L.: Prostaglandin biosynthesis and its compartmentation in vascular smooth muscle and endothelial cells. Annu. Rev. Physiol., 48, 251-262 (1986) [15] Roth, G.J.; Siok, C.J.; Ozols, J.: Structural characteristics of prostaglandin synthetase from sheep vesicular gland. J. Biol. Chem., 255, 1301-1304 (1980) [16] Marnett, L.J.; Chen, Y.N.P.; Maddipati, K.R.; Ple, P.; Labeque, R.: Functional differentiation of cyclooxygenase and peroxidase activities of prostaglandin synthase by trypsin treatment. Possible location of a prosthetic heme binding site. J. Biol. Chem., 263, 16532-16535 (1988) [17] Marnett, L.J.; Siedlick, P.H.; Ochs, R.C.; Pagels, W. R.; Das, M.; Honn, K.V.; Warnock, R.H.; Tainer, B.E.; Eling, T.E.: Mechanism of the stimulation of prostaglandin H synthase and prostacyclin synthase by the antithrombotic and antimetastatic agent, nafazatrom. Mol. Pharmacol., 26, 328-335 (1984) [18] Ohki, S.; Ogino, N.; Yamamoto, S.; Hayaishi, O.; Yamamoto, H.; Miyake, H.; Hayashi, M.: Inhibition of prostaglandin endoperoxide synthetase by thiol analogues of prostaglandin. Proc. Natl. Acad. Sci. USA, 74, 144-148 (1977) [19] Wlodawer, P.; Samuelsson, B.: On the organization and mechanism of prostaglandin synthetase. J. Biol. Chem., 248, 5673-5678 (1973)

255

Prostaglandin-endoperoxide synthase

1.14.99.1

[20] Golly, I.; Hlavica, P.: N-Oxidation of 4-chloroaniline by prostaglandin synthase. Biochem. J., 260, 803-809 (1985) [21] Tai, H.H.; Tai, C.L.; Hollander, C.S.: Biosynthesis of prostaglandins in rabbit kidney medulla. Properties of prostaglandin synthase. Biochem. J., 154, 257-264 (1976) [22] Friedman, Y.; Lang, M.; Burke, G.: Further characterization of bovine thyroid prostaglandin synthase. Biochim. Biophys. Acta, 397, 331-341 (1975) [23] Parkes, D.G.; Eling, T.E.: Characterization of prostaglandin synthetase in guinea pig lung. Isolation of a new prostaglandin derivative from arachidonic acid. Biochemistry, 13, 2598-2604 (1974) [24] Pace-Asciak, C.: Prostaglandin synthetase activity in the rat stomach fundus. Activation by l-norepinephrine and related compounds. Biochim. Biophys. Acta, 280, 161-171 (1972) [25] Miller, D.B.; Munster, D.; Wasvary, J.S.; Simke, J.P.; Peppard, J.V.; Bowen, B.R.; Marshall, P.J.: The heterologous expression and characterization of human prostaglandin G/H synthase-2 (COX-2). Biochem. Biophys. Res. Commun., 201, 356-362 (1994) [26] Liu, J.; Antaya, M.; Goff, A.K.; Boerboom, D.; Silversides, D.W.; Lussier, J.G.; Sirois, J.: Molecular characterization of bovine prostaglandin G/H synthase2 and regulation in uterine stromal cells. Biol. Reprod., 64, 983-991 (2001) [27] Doualla-Bell, F.; Guay, J.M.; Bourgoin, S.; Fortier, M.A.: Prostaglandin G/H synthase (PGHS)-2 expression in bovine myometrium: influence of steroid hormones and PGHS inhibitors. Biol. Reprod., 59, 1433-1438 (1998) [28] Kargman, S.; Wong, E.; Greig, G.M.; Falgueyret, J.P.; Cromlish, W.; Ethier, D.; Yergey, J.A.; Riendeau, D.; Evans, J.F.; Kennedy, B.; Tagari, P.; Francis, D.A.; O'Neill, G.P.: Mechanism of selective inhibition of human prostaglandin G/H synthase-1 and -2 in intact cells. Biochem. Pharmacol., 52, 11131125 (1996) [29] Forghani, F.; Ouellet, M.; Keen, S.; Percival, M.D.; Tagari, P.: Analysis of prostaglandin G/H synthase-2 inhibition using peroxidase-induced luminol luminescence. Anal. Biochem., 264, 216-221 (1998) [30] Mancini, J.A.; Vickers, P.J.; O'Neill, G.P.; Boily, C.; Falgueyret, J.P.; Riendeau, D.: Altered sensitivity of aspirin-acetylated prostaglandin G/H synthase-2 to inhibition by nonsteroidal anti-inflammatory drugs. Mol. Pharmacol., 51, 52-60 (1997) [31] George, H.J.; Van Dyk, D.E.; Straney, R.A.; Trzaskos, J.M.; Copeland, R.A.: Expression purification and characterization of recombinant human inducible prostaglandin G/H synthase from baculovirus-infected insect cells. Protein Expr. Purif., 7, 19-26 (1996) [32] Smith, C.J.; Marnett, L.J.: Effects of cysteine-to-serine mutations on structural and functional properties of prostaglandin endoperoxide synthase. Arch. Biochem. Biophys., 335, 342-350 (1996) [33] Ishii, R.; Horie, M.; Saito, K.; Arisawa, M.; Kitanaka, S.: Prostaglandin E(2) production and induction of prostaglandin endoperoxide synthase-2 is inhibited in a murine macrophage-like cell line, RAW 264.7, by Mallotus japonicus phloroglucinol derivatives. Biochim. Biophys. Acta, 1571, 115-123 (2002) 256

1.14.99.1

Prostaglandin-endoperoxide synthase

[34] Barnett, J.; Chow, J.; Ives, D.; Chiou, M.; Mackenzie, R.; Osen, E.; Nguyen, B.; Tsing, S.; Bach, C.; Freire, J.; et al.: Purification, characterization and selective inhibition of human prostaglandin G/H synthase 1 and 2 expressed in the baculovirus system. Biochim. Biophys. Acta, 1209, 130-139 (1994) [35] Kargman, S.; Charleson, S.; Cartwright, M.; Frank, J.; Riendeau, D.; Mancini, J.; Evans, J.; O'Neill, G.: Characterization of prostaglandin G/H synthase 1 and 2 in rat, dog, monkey, and human gastrointestinal tracts. Gastroenterology, 111, 445-454 (1996) [36] Boutemmine, D.; Bouchard, N.; Boerboom, D.; Jones, H.E.; Goff, A.K.; Dore, M.; Sirois, J.: Molecular characterization of canine prostaglandin G/H synthase-2 and regulation in prostatic adenocarcinoma cells in vitro. Endocrinology, 143, 1134-1143 (2002) [37] Patrignani, P.; Panara, M.R.; Sciulli, M.G.; Santini, G.; Renda, G.; Patrono, C.: Differential inhibition of human prostaglandin endoperoxide synthase-1 and -2 by nonsteroidal anti-inflammatory drugs. J. Physiol. Pharmacol., 48, 623-631 (1997) [38] Chulada, P.C.; Loftin, C.D.; Winn, V.D.; Young, D.A.; Tiano, H.F.; Eling, T.E.; Langenbach, R.: Relative activities of retrovirally expressed murine prostaglandin synthase-1 and -2 depend on source of arachidonic acid. Arch. Biochem. Biophys., 330, 301-313 (1996) [39] Mancini, J.A.; Riendeau, D.; Falgueyret, J.P.; Vickers, P.J.; O'Neill, G.P.: Arginine 120 of prostaglandin G/H synthase-1 is required for the inhibition by nonsteroidal anti-inflammatory drugs containing a carboxylic acid moiety. J. Biol. Chem., 270, 29372-29377 (1995) [40] Meade, E.A.; Smith, W.L.; DeWitt, D.L.: Differential inhibition of prostaglandin endoperoxide synthase (cyclooxygenase) isozymes by aspirin and other non-steroidal anti-inflammatory drugs. J. Biol. Chem., 268, 6610-6614 (1993) [41] O'Neill, G.P.; Mancini, J.A.; Kargman, S.; Yergey, J.; Kwan, M.Y.; Falgueyret, J.P.; Abramovitz, M.; Kennedy, B.P.; Ouellet, M.; Cromlish, W.: Overexpression of human prostaglandin G/H synthase-1 and -2 by recombinant vaccinia virus: inhibition by nonsteroidal anti-inflammatory drugs and biosynthesis of 15-hydroxyeicosatetraenoic acid. Mol. Pharmacol., 45, 245-254 (1994) [42] Roberts, S.B.; Langenau, D.M.; Goetz, F.W.: Cloning and characterization of prostaglandin endoperoxide synthase-1 and -2 from the brook trout ovary. Mol. Cell. Endocrinol., 160, 89-97 (2000)

257

.    8(7   

::

   
 1.14.99.2    kynurenate,hydrogen-donor:oxygen oxidoreductase (hydroxylating)    kynurenine 7,8-hydroxylase    EC 1.14.1.4 (formerly) hydroxylase, kynurenate hydroxylase, kynurenate 7,8kynurenate 7,8-hydroxylase kynurenic acid hydroxylase kynurenic hydroxylase
    9029-63-4

     Pseudomonas fluorescens (ATCC 11299B) [1] Pseudomonas sp. (overview [2]) [2]

!    "
 # 

kynurenate + AH2 + O2 = 7,8-dihydro-7,8-dihydroxykynurenate + A    oxidation redox reaction reduction       kynurenate + NADH + O2 (Reversibility: ? [1, 2]) [1, 2] $ 7,8-dihydro-7,8-dihydroxykynurenate + NAD+  kynurenate + NADPH + O2 (Reversibility: ? [1, 2]) [1, 2] $ 7,8-dihydro-7,8-dihydroxykynurenate + NADP+ 258

1.14.99.2

Kynurenine 7,8-hydroxylase

     kynurenate + NADH + O2 (Reversibility: ? [1, 2]) [1, 2] $ 7,8-dihydro-7,8-dihydroxykynurenate + NAD+  kynurenate + NADPH + O2 (Reversibility: ? [1, 2]) [1, 2] $ 7,8-dihydro-7,8-dihydroxykynurenate + NADP+ 2  Ag+ [1] Cu2+ [1] Hg2+ [1] iodoacetate ( inhibition reversed by addition of GSH or cysteine [1]) [1] kynurenate ( competitive inhibition at 0.2 mM [1]) [1] p-chloromercuribenzoate ( inhibition reversed by addition of GSH or cysteine [1]) [1]
"%  NADH [1, 2] NADPH [1] . / *', 0.008 (kynurenate) [1] 0.04 (NADH) [1] 0  7 [1] )  *
, 23 ( assay at [1]) [1]

 2 %$ %' %

$"

(partial [1]) [1] [2]

4  0  9 ( stable in presence 10 mM cysteine [1]) [1] )  60 ( 5 min, complete inactivation [1]) [1] 5    "

, 2-mercaptoethanol stabilizes activity [1] , GSH stabilizes activity [1] , cysteine stabilizes activity [1]

259

Kynurenine 7,8-hydroxylase

1.14.99.2

, precipitation of supernatant with ammonium sulfate in the absence of cysteine, complete loss of activity [1] , thioglycolate stabilizes activity [1]    , -10 C, ammonium sulfate fractions, loss of activity after a few days [1] , -10 C, supernatant after high speed centrifugation of crude extract, several months, no loss in activity [1]

"  [1] Taniuchi, H.; Hayaishi, O.: Studies on the metabolism of kynurenic acid. J. Biol. Chem., 238, 283-293 (1963) [2] Gibson, D.T.: Assay of enzymes of aromatic metabolism. Methods Microbiol., 6A, 463-478 (1971)

260

0    * #

,

::!

   
 1.14.99.3    heme,hydrogen-donor:oxygen oxidoreductase (a-methene-oxidizing, hydroxylating)    heme oxygenase (decyclizing)    ORP33 proteins haem oxygenase oxygenase, heme (decyclizing) proteins, specific or class, ORP33 (oxygen-regulated protein 33,000-mol.-wt.)
    9059-22-7

     Rattus norvegicus (2 isoforms: heme oxygenase-1, inducible [12, 14, 16]; heme oxygenase-2, constitutive [12, 14, 16]; only minute amounts of heme oxygenase-1 in testis [12]; spleen heme oxygenase-1 is not induced by hematin [16]; heme oxygenase-1 is a heat shock protein that is inducible by numerous stimuli, including heavy metals, oxidative stress and injury, and cytokines, a 3 isomer, heme oxygenase-3, exhibits 90% homology to heme oxygenase-2, its mRNA has been detected in several tissues including kidney [32]) [1, 2, 5-8, 12, 14, 16-19, 28, 31, 32, 33, 34, 35, 36] Sus scrofa [2, 9] Gallus gallus [2, 3] Homo sapiens [2, 10, 21, 22, 23, 24, 27, 29, 30] Bos taurus [2, 4, 11, 15, 19] Oryctolagus cuniculus (2 isoforms: heme oxygenase-1, inducible, heme oxygenase-2, constitutive [14]) [2, 14] Cyanidium caldarium (heme oxygenase is induced by light and d-aminolevulinic acid, induction is inhibited by d-glucose [20]) [13, 20] Corynebacterium diphtheriae [25, 26, 29] Cavia porcellus (guinea pig [33]) [33]

261

Heme oxygenase (decyclizing)

1.14.99.3

mammalia (heme oxygenase-1 is identical to the major 32000 Da mammalian stress-protein inducible by heat shock, H2 O2, ultraviolet-A radiation, NaAsO2, pro-inflammatory cytokines, bacterial endotoxins, growth factors, NO, and tumor promotors, heme oxygenase-1 confers protection against oxidative stress [34]) [34] Neisseria meningitidis (pathogenic bacteria [37]) [37]

!    "
 # 

heme + 3 AH2 + 3 O2 = biliverdin + Fe2+ + CO + 3 A + 3 H2 O ( mechanism [7]; a ferric hydroperoxide species must be an active intermediate in the first oxygenation step [26]; determination of single turnover rate constants and reaction intermediates for heme oxygenase-1 [30]; hydroperoxoferri-heme oxygenase-1 is the reactive species directly forming the ameso-hydroxyheme product by attack of the distal OH of the hydroperoxo moiety at the heme a-carbon [36])    oxidation redox reaction reduction       heme + NADH + O2 ( NADH-dependent heme degradation system may have a biological role in regulating the concentration of respiratory hemoproteins and the disposition of the aberrant forms of the mitochondrial hemoproteins [15]) (Reversibility: ? [15]) [15] $ biliverdin + Fe2+ + CO + NAD+ [15]  heme + NADPH + O2 ( involved in heme metabolism [1,2]) (Reversibility: ? [1, 2]) [1, 2] $ biliverdin + Fe2+ + CO + NADP+ + H2 O [1, 2]      a-meso-formylmesoheme + NADPH ( exclusively oxidized at a non-formyl substituted meso-carbon [24]) (Reversibility: ? [24]) [24] $ meso-formylmesobiliverdin + NADP+ [24]  heme + electron donor + O2 ( electron donor NADH [1, 8, 15, 17]; NADH-dependent heme-degradation activity, 16% of NADH activity with 0.5 mM NADPH [15]; NADPH is more effective than NADH [1, 12]; electron donor NADPH [1, 3, 4, 8, 11, 15, 17, 18]; NADH can replace NADPH at concentrations higher than 5 mM in vitro, NADH is unlikely to be an electron donor in vivo [11]; algal heme oxygenase requires a second reductant in addition to reduced pyridine nucleotide [13]; oxidation of Co-heme [7]; overview, substrate specificity [5-7, 12]; enzyme oxidizes protoheme, hematoheme, hematoheme dimethyl ester, dicysteinyl hematoheme, and heme undeca-

262

1.14.99.3

Heme oxygenase (decyclizing)

peptide, conversion of hematoheme to hematobilirubin requires the presence of: NADPH, NADPH-cytochrome c reductase, biliverdin reductase and O2 [8]; testis heme oxygenase 2 oxidizes Fe-protopophyrin, ferric hematoporphyrin acetate and ferric hematoporphyrin [12]; synthetic hemins XIII and III and iron porphyrin are better substrates than the natural substrate hemin IX, 83 and 86% of hemin IX activity with mesohemin IX and hematohemin IX respectively [5,6]; enzyme catalyzes oxidative cleavage of both heme b and heme c [19]; iron-protoporphyrin IX is the most active substrate, lower activity with: iron-mesoporphyrin IX, iron-deuteroheme IX, iron-coproheme I, a and b chain of hemoglobin, poor substrates: oxyhemoglobin, carboxyhemoglobin, myoglobin [2]; a-meso-oxyprotoheme is an intermediate of heme degradation that is converted stereospecifically into biliverdin IXa via verdoheme IXa [4]; porphyrins without chelated iron and metalloporphyrins other than iron porphyrins are not oxidized [2]; cytochrome c and myoglobin are not oxidized [15]; Ni, Mn, and Sn protoporphyrin IX is not oxidized [7]; intact cytochrome c is not oxidized [8]; electron donor NADPH, reductase: human or E. coli NADPH-cytochrome P450 reductase or putidaredoxin/putidaredoxin reductase [25]; electron donor ascorbic acid [26]) (Reversibility: ? [1, 2, 3, 4, 8, 13, 18, 23, 25]) [1, 2, 3, 4-8, 12, 13, 15, 18, 19, 23, 25, 26, 31] $ biliverdin + Fe2+ + CO + oxidized eletron donor + H2 O ( biliverdin IXa is the sole biliverdin isomer formed [3]; regiospecific oxidation of a-meso position of heme to form the a-biliverdin isomer [31]) [1, 2, 3, 4-8, 12, 13, 15, 18, 19, 23, 25, 26, 31]  methemoglobin + electron donor + O2 ( 30% of activity with heme [1]) (Reversibility: ? [1, 2]) [1, 2] $ ? 2  1,10-phenanthroline ( weak [2]) [2] 2,2'-dipyridyl ( weak [2]) [2] 2-mercaptoethanol ( 0.1 mM, 81% inhibition [1]) [1] 3-morpholinosydnonimine ( NO-donor, 27% inhibition of recombinant heme oxygenase-2 [28]) [28] 4-hydroxymercuribenzoate ( 1 mM, complete inhibition [1]) [1, 2] CO ( strong inhibition [2]) [2, 8, 12] Co-protoporphyrin ( 0.005 mM, 82.5% inhibition of kidney heme oxygenase [10]) [10, 11] Co2+ ( 0.4 mM, significant inhibition [2]) [2] Cu2+ ( 0.2 mM, significant inhibition [2]) [2] EDTA ( weak [2]) [2] Fe-deuteroporphyrin IX 2,4-bisglycol ( 0.01 mM, 46.8% inhibition of kidney heme oxygenase [10]) [10] Hg2+ ( inhibition of NADPH-cytochrome c reductase or biliverdin reductase in reconstituted heme oxygenase system [11]; 0.3 mM, complete inhibition [1]) [1, 3, 11]

263

Heme oxygenase (decyclizing)

1.14.99.3

KCN ( strong inhibition [2]) [2, 8, 12] Mn-protoporphyrin [11] NaN3 ( strong inhibition [2]) [2, 8, 12] S-nitroso-N-acetyl-pennicillamine ( NO-donor, 23% inhibition of recombinant heme oxygenase-2 [28]) [28] Sn-protoporphyrin ( 0.005 mM, complete inhibition of kidney heme oxygenase [10]; 0.002 mM, 80% inhbition [13]) [10, 11, 13, 17] Zn-deuteroporphyrin IX 2,4-bisglycol ( 0.002 mM, complete inhibition of kidney heme oxygenase [10]) [10] Zn-protoporphyrin ( 0.005 mM, 93.4% inhibition of kidney heme oxygenase [10]) [10, 11] cysteine ( 1 mM, 67% inhibition [1]) [1, 2] dithiothreitol ( 0.01 mM, 88% inhibition [1]) [1] iodoacetamide ( 5 mM, 39% inhibition, 10 mM, 79% inhibition [1]) [1] p-chloromercuribenzoate ( inhibition of NADPH-cytochrome c reductase in reconstituted heme oxygenase system [11]) [1, 11] porphyrins ( overview [7]; protoporphyrin IX, Zn-protoporphyrin IX, 2,4-diacetyldeuteroporphyrin IX, deuteroporphyrin IX, coproporphyrin II, III and IV [5]; metalloporphyrins, decreasing order of inhibition potency: Sn-mesoporphyrin, Sn-protoporphyrin, Zn-protoporphyrin, Mn-protoporphyrin, Co-protoporphyrin [3]; Zn-deuteroporphyrin IX 2,4-bis glycol, synthetic metal porphyrins [10]; protoporphyrin IX, Zn-protoporphyrin IX, 2,4-diacetyldeuteroporphyrin IX, deuteroporphyrin IX, coproporphyrin II, III and IV [5]; metalloporphyrins [11]; b, g and d-oxyprotohaem IX [4]; Ni, Mn, and Sn-protoporphyrin IX [7]) [3, 4, 5, 7, 10, 11] sodium nitroprusside ( NO-donor, 58% inhibition of recombinant heme oxygenase-2 [28]) [28]
"%  NADH ( NADPH is more effective than NADH [1]) [1] NADPH ( 0.5 mM, 16% as effective as NADH [15]; NADPH is more effective than NADH [1]) [1, 15] heme ( hemoprotein, heme is both substrate and cofactor, heme oxygenase-2 binds heme at heme regulatory motifs with a conserved Cys-Pro pair [28]) [28] &

  Fe/S cluster ( heme oxygenase consists of 3 required protein components: a ferredoxin-like Fe-S cluster protein that can be replaced by ferredoxin, a protein that is inactivated by diethyldicarbonate, inactivation is blocked by heme, a protein with ferredoxin-linked cytochrome c reductase activity [13]) [13] cysteine ( 0.1 mM, slight stimulation, inhibition at 1 mM [1]) [1]

264

1.14.99.3

Heme oxygenase (decyclizing)

'(   iron ( heme oxygenase consists of 3 required protein components: a ferredoxin-like Fe-S cluster protein that can be replaced by ferredoxin, a protein that is inactivated by diethyldicarbonate, inactivation is blocked by heme, a protein with ferredoxin-linked cytochrome c reductase activity [13]) [13] ) &  * +, 3.5 (heme) [11] 19.2 (heme) [3] " & *-% , 0.00000625 ( activity in kidney microsomes, micromol bilirubin/ min/mg [10]) [10] 0.000175 ( spleen enzyme [9]) [9] 0.00046 ( NADH-dependent heme degradation in heart mitochondria [15]) [15] 0.0064 ( testis heme oxygenase-2, partialy purified [14]) [14] 0.017 ( 28000 Da recombinant tryptic fragment of heme oxygenase-2 [21]) [21] 0.024 ( recombinant C-terminal truncated heme oxygenase-1 [22]) [22] 0.05 ( hematoheme oxidation [8]) [8] 0.052 ( protoheme oxidation [8]) [8] 0.067 ( testicular heme oxygenase-2 [12]) [12] 0.075-0.1 ( liver heme oxygenase from Co-treated animals, 2 fractions during purification [18]) [18] 0.095 ( testis heme oxygenase-2 [14]) [14] 0.104 ( liver heme oxygenase-1 [14]) [14] 0.11 ( liver heme oxygenase-1 [12]) [12] 0.113 ( spleen enzyme [11]) [11] 0.138 ( liver heme oxygenase-1 [14]) [14] 0.433 [3] 12.7 [1] Additional information ( assay procedure [2]) [2, 8, 9, 18] . / *', 0.00024 (heme, heme oxygenase-1 [34]) [34] 0.00067 (heme, heme oxygenase-2 [34]) [34] 0.0009 (heme b, spleen heme oxygenase [19]) [19] 0.0009 (protoheme) [8] 0.00093 (heme) [11] 0.0018 (protoheme IX) [4] 0.003 (heme, heme oxygenase-1, wild-type [23]) [23] 0.003 (heme, recombinant C-terminal truncated heme oxygenase-1 [22]) [22] 0.0036 (a-meso-oxyprotoheme IX) [4] 0.0038 (heme, in the absence of bovine serum albumin [3]) [3]

265

Heme oxygenase (decyclizing)

1.14.99.3

0.005 (heme, in the presence of bovine serum albumin [3]) [3] 0.005 (protoheme IX, enzyme from spleen and liver [2]) [2] 0.006 (heme, recombinant heme oxygenase-1/cytochrome P450 reductase fusion protein [22]) [22] 0.0061 (NADPH) [3] 0.01 (hematoheme) [8] 0.01 (hemin, intestinal enzyme [17]) [17] 0.014 (heme, heme oxygenase-1, H132G and H132A mutants [23]) [23] 0.0164 (protoheme IX, enzyme from spleen [2]) [2] 0.017 (Fe-heme) [7] 0.018 (heme, heme oxygenase-1, H132S mutant [23]) [23] 0.023 (NADPH) [11] 0.029 (heme c, spleen heme oxygenase [19]) [19] 0.04 (heme) [1] 0.125 (Co-heme) [7] . / *', 0.000033 (Sn-protoporphyrin) [11] 0.0000825 (Co-protoporphyrin) [11] 0.00013 (Zn-protoporphyrin) [11] 0  7.2-7.5 [3] 7.4 ( liver heme oxygenase-1 and testis heme oxygenase-2 [12]) [2, 8, 12] 7.5 [1] 0 

 6.5-7.9 ( pH 6.5: 63% activity, pH 7.9: 50% activity [3]) [3] )  *
, 37 ( assay at [2,3]) [2, 3] ) 

 *
, Additional information ( no activity at 0 C [1]) [1]

  #   ' 1  150000 ( gel filtration [9]) [9] 180000 ( gel filtration [1]) [1] 200000 ( gel filtration [2, 18]) [2, 18] Additional information ( heme oxygenase consists of 3 components: a 22000 Da ferredoxin-like Fe/S cluster protein that can be replaced by ferredoxin, a 38000 Da protein that is inactivated by diethyldicarbonate, inactivation is blocked by heme, a 37000 Da protein with ferredoxin-linked cytochrome c reductase activity [13]) [13]

266

1.14.99.3

Heme oxygenase (decyclizing)

  ? ( x * 30000, liver heme oxygenase-1, SDS-PAGE [12, 14]; x * 30700, liver heme oxygenase-1, SDS-PAGE [14]; x * 31000, SDS-PAGE [11]; x * 32000, SDS-PAGE [9]; x * 32000, kidney heme oxygenase, immunoblot [10]; x * 33000, SDS-PAGE [3, 18]; x * 36000, testis heme oxygenase-2, SDS-PAGE [12,14]; x * 36000, heme oxygenase-2, SDS-PAGE [14]; x * 42000, heme oxygenase-2, SDS-PAGE [14]; x * 68000, possibly a trimer, SDS-PAGE [1]; x * 36000, recombinant heme oxygenase-2, SDS-PAGE [21]; x * 25000, recombinant enzyme, SDSPAGE [25]; x * 24000, SDS-PAGE, deduced from amino acid sequence [26]) [1, 3, 9-12, 14, 18, 21, 25, 26]

 2 %$ %' %

% brain [2] heart [2, 15] kidney ( heme oxygenase-3 [32]; high level of heme oxygenase-2 in the outer medulla followed by inner medulla/papilla and cortex, heme oxygenase-1 is barely detectable in tissue from untreated animals but increases strongly in SnCl2 treated animals [32]) [2, 10, 32] liver ( 24 h after a CoCl2 injection heme-oxygenase-1 activity increases 13fold, enzyme may play a role in the liver oxidative-stress defence [33]) [1-3, 5, 6, 8, 14, 16, 18, 19, 33] lung [2] macrophage ( peritoneal, alveolar [2]) [2] marrow [2] small intestine epithelium [17] small intestine mucosa [2] spleen ( heme oxygenase-1 and 2, relative ratio 5/1 [16]) [2, 4, 9, 11, 16, 19] testis [12, 14] 3#

membrane ( recombinant heme oxygenase-2 expressed in E. coli [21]) [21] microsome [1-3, 5, 6, 8-11, 17-19] mitochondrion ( 77% activity in inner membrane, specifically associated with complex I, NADH: ubiquinone oxidoreductase [15]; liver, 7% of microsomal activity [8]) [8, 15] soluble [13] $"

(DEAE-cellulose, Sephadex G-200 [1]; heme oxygenase-1 [16]; heme oxygenase-2 [12, 16]; 2 separate fractions after first DEAE-cellulose, hydroxyapatite, Sephadex G-150, DEAE-cellulose [18]) [1, 12, 16, 18]

267

Heme oxygenase (decyclizing)

1.14.99.3

(ammonium sulfate, DEAE-cellulose, hydroxyapatite, Sephadex G-200 [9]) [9] (DEAE-Sephacel, carboxymethyl-cellulose, hydroxyapatite, Superose 6/ 12 [3]) [3] (ammonium sulfate, Sephadex G-75, DEAE-cellulose, hydroxyapatite, tryptic 28000 Da fragment of recombinant heme oxygenase-2 [21]; recombinant C-terminal truncated heme oxygenase-1, ammonium sulfate, Mono Q, recombinant heme oxygenase-1/cytochrome P450 reductase fusion protein, ammonium sulfate, 2',5'-ADP-Sepharose [22]) [21, 22] (Triton X-100 + cholate, DEAE-cellulose, hydroxyapatite, Sepharose CL6B, hydroxyapatite [11]) [11] (partial [13]) [13] (recombinant wild-type and His-tagged enzyme, ion-exchange, gel filtration [25]) [25, 26]
 #

(truncated heme oxygenase-1, hanging-drop vapor diffusion, enzyme solution is mixed with an equal volume of each reservoir solution and equilibrated, crystals are obtained at 293 K, reservoir solution contains 4 M sodium formate, 18 mg/ml enzyme concentration in 50 mM potassium phosphate, pH 7.0, hexagonal rod-shaped crystals appear after 3 d, X-ray structure of heme oxygenase in complex with heme bound to azide at 1.9 A resolution [35]) [35] (heme oxygenase-1 [27]) [27] (crystal structure at 1.5 A resolution, comparison with heme oxygenase-1 from mammalian sources [37]) [37]
 

(expression of wild-type heme oxygenase-1 and 2 and heme oxygenase-2 C264A/C281A double mutant in Escherichia coli [28]; expression of truncated heme oxygenase-1 in Escherichia coli [36]) [28, 35, 36] (expression of heme oxygenase-2 in Escherichia coli [21]; expression of C-terminal truncated heme oxygenase-1 and of a heme oxygenase-1 cytochrome P450 reductase fusion protein [22]) [21, 22, 23] (expression of wild-type and His-tagged enzyme in Escherichia coli [25]) [25, 26] 

 

D140A ( mutant of truncated heme oxygenase-1 [36]) [36] D140F ( mutant of truncated heme oxygenase-1 [36]) [36] H132A ( heme oxygenase-1, 40-50% of wild-type activity [23]) [23] H132G ( heme oxygenase-1, 40-50% of wild-type activity [23]) [23] H132S ( heme oxygenase-1, 20% of wild-type activity [23]) [23] H20A ( capable of NADPH dependent hydroxylation of heme to a-mesohydroxyheme in contrast to human H25A heme oxygenase-1 mutant, ability to catalyze the conversion of verdoheme to biliverdin is rescued by imidazole titration [29]) [29]

268

1.14.99.3

Heme oxygenase (decyclizing)

R183D ( in contrast to wild-type heme oxygenase-1 which converts heme exclusively to a-biliverdin, the R183D mutant converts heme to 20% dbiliverdin in addition to a-biliverdin [31]) [31] R183E ( in contrast to wild-type heme oxygenase-1 which converts heme exclusively to a-biliverdin, the R183E mutant converts heme to 35% dbiliverdin and small amounts of b- and g-biliverdin in addition to a-biliverdin [31]) [31] R183N ( same a-regioselectivity as wild-type, only a-biliverdin is produced [31]) [31] R183Q ( same a-regioselectivity as wild-type, only a-biliverdin is produced [31]) [31] R183a ( same a-regioselectivity as wild-type, only a-biliverdin is produced [31]) [31]

4  )  0-4 ( several hours [2]) [2] 35 ( liver heme oxygenase-1: 65-70% activity after 10 min at 60 C, testis heme oxygenase-2: 20% activity after 10 min at 60 C [12]) [12] 50 ( 10 min, 80% loss of liver heme oxygenase-1 and testis heme oxygenase 2 activity [14]; complete loss of activity [8]) [8, 14] 60 ( 5 min, complete loss of activity [1]) [1] 65 ( 10 min, 30% loss of heme oxygenase-1 activity, 80% loss of heme oxygenase-2 activity [12]) [12] 90 ( 15 min, 95% loss of activity, crude extract [15]) [15] 5    "

, 50% loss of activity during one cycle of freezing and thawing [1] , 10-20% loss of activity upon thawing [11]    , -20 C, 2 weeks [1] , -30 C, several weeks, no loss of activity [8] , 4 C, 20 mM potassium phosphate, pH 7.5, 0.2% Triton X-100, 20% glycerol, 2 d, 50% loss of activity [11] , liquid nitrogen, 6 months, no loss of activity [11]

"  [1] Maines, M.D.; Ibrahim, N.G.; Kappas, A.: Solubilization and partial purification of heme oxygenase from rat liver. J. Biol. Chem., 252, 5900-5903 (1977) [2] Schacter, B.A.: Assay of microsomal heme oxygenase in liver and spleen. Methods Enzymol., 52, 367-372 (1978)

269

Heme oxygenase (decyclizing)

1.14.99.3

[3] Bonkovsky, H.L.; Healy, J.F.; Pohl, J.: Purification and characterization of heme oxygenase from chick liver. Comparison of the avian and mammalian enzymes. Eur. J. Biochem., 189, 155-160 (1990) [4] Yoshinaga, T.; Sudo, Y.; Sano, S.: Enzymic conversion of a-oxyprotohaem IX into biliverdin IX a by haem oxygenase. Biochem. J., 270, 659-664 (1990) [5] Frydman, R.B.; Tomaro, M.L.; Buldain, G.; Awruch, J.; Diaz, L.; Frydman, B.: Specificity of heme oxygenase: a study with synthetic hemins. Biochemistry, 20, 5177-5182 (1981) [6] Tomaro, M.L.; Frydman, R.B.; Frydman, B.; Pandey, R.K.; Smith, K.M.: The oxidation of hemins by microsomal heme oxygenase. Structural requirements for the retention of substrate activity. Biochim. Biophys. Acta, 791, 342-349 (1984) [7] Maines, M.D.; Kappas, A.: Enzymatic oxidation of cobalt protoporphyrin IX: observations on the mechanism of heme oxygenase action. Biochemistry, 16, 419-423 (1977) [8] Kutty, R.K.; Maines, M.D.: Oxidation of heme c derivatives by purified heme oxygenase. Evidence for the presence of one molecular species of heme oxygenase in the rat liver. J. Biol. Chem., 257, 9944-9952 (1982) [9] Yoshida, T.; Kikuchi, G.: Purification and properties of heme oxygenase from pig spleen microsomes. J. Biol. Chem., 253, 4224-4229 (1978) [10] Matasek, P.; Solangi, K.; Goodman, A.I.; Levere, R.D.; Chernick, R.J.; Abraham, N.G.: Properties of human kidney heme oxygenase: inhibition by synthetic heme analogues and metalloporphyrins. Biochem. Biophys. Res. Commun., 157, 480-487 (1988) [11] Yoshinaga, T.; Sassa, S.; Kappas, A.: Purification and properties of bovine spleen heme oxygenase. Amino acid composition and sites of action of inhibitors of heme oxidation. J. Biol. Chem., 257, 7778-7785 (1982) [12] Trakshel, G.M.; Kutty, R.K.; Maines, M.D.: Purification and characterization of the major constitutive form of testicular heme oxygenase. The noninducible isoform. J. Biol. Chem., 261, 11131-11137 (1986) [13] Cornejo, J.; Beale, S.I.: Algal heme oxygenase from Cyanidium caldarium. Partial purification and fractionation into three required protein components. J. Biol. Chem., 263, 11915-11921 (1988) [14] Trakshel, G.M.; Maines, M.D.: Multiplicity of heme oxygenase isozymes. HO-1 and HO-2 are different molecular species in rat and rabbit. J. Biol. Chem., 264, 1323-1328 (1989) [15] Kutty, R.K.; Maines, M.D.: Characterization of an NADH-dependent haemdegrading system in ox heart mitochondria. Biochem. J., 246, 467-474 (1987) [16] Braggins, P.E.; Trakshel, G.M.; Kutty, R.K.; Maines, M.D.: Characterization of two heme oxygenase isoforms in rat spleen: comparison with the hematin-induced and constitutive isoforms of the liver. Biochem. Biophys. Res. Commun., 141, 528-533 (1986) [17] Rosenberg, D.W.; Kappas, A.: Characterization of heme oxygenase in the small intestinal epithelium. Arch. Biochem. Biophys., 274, 471-480 (1989) [18] Yoshida, T.; Kikuchi, G.: Purification and properties of heme oxygenase from rat liver microsomes. J. Biol. Chem., 254, 4487-4491 (1979) 270

1.14.99.3

Heme oxygenase (decyclizing)

[19] Yoshinaga, T.; Sassa, S.; Kappas, A.: The oxidative degradation of heme c by the microsomal heme oxygenase system. J. Biol. Chem., 257, 7803-7807 (1982) [20] Rhie, G.E.; Beale, S.I.: Regulation of heme oxygenase activity in Cyanidium caldarium by light, glucose, and phycobilin precursors. J. Biol. Chem., 269, 9620-9626 (1994) [21] Ishikawa, K.; Takeuchi, N.; Takahashi, S.; Matera, K.M.; Sato, M.; Shibahara, S.; Rousseau, D.L.; Ikeda-Saito, M.; Yoshida, T.: Heme oxygenase-2. Properties of the heme complex of the purified tryptic fragment of recombinant human heme oxygenase-2. J. Biol. Chem., 270, 6345-6350 (1995) [22] Wilks, A.; Black, S.M.; Miller, W.L.; Ortiz de Montellano, P.R.: Expression and characterization of truncated human heme oxygenase (hHO-1) and a fusion protein of hHO-1 with human cytochrome P450 reductase. Biochemistry, 34, 4421-4427 (1995) [23] Wilks, A.; de Montellano, P.R.O.; Sun, J.; Loehr, T.M.: Heme oxygenase (HO1): His-132 stabilizes a distal water ligand and assists catalysis. Biochemistry, 35, 930-936 (1996) [24] Torpey, J.; Ortiz de Montellano, P.R.: Oxidation of a-meso-formylmesoheme by heme oxygenase. Electronic control of the reaction regiospecificity. J. Biol. Chem., 272, 22008-22014 (1997) [25] Wilks, A.; Schmitt, M.P.: Expression and characterization of a heme oxygenase (Hmu O) from Corynebacterium diphtheriae. Iron acquisition requires oxidative cleavage of the heme macrocyle. J. Biol. Chem., 273, 837841 (1998) [26] Chu, G.C.; Katakura, K.; Zhang, X.; Yoshida, T.; Ikeda-Saito, M.: Heme degradation as catalyzed by a recombinant bacterial heme oxygenase (Hmu O) from Corynebacterium diphtheriae. J. Biol. Chem., 274, 21319-21325 (1999) [27] Schuller, D.J.; Wilks, A.; De Montellano, P.R.O.; Poulos, T.L.: Crystal structure of human heme oxygenase-1. Nat. Struct. Biol., 6, 860-867 (1999) [28] Ding, Y.; McCoubrey, W.K., Jr.; Maines, M.D.: Interaction of heme oxygenase-2 with nitric oxide donors: is the oxygenase an intracellular sink for NO?. Eur. J. Biochem., 264, 854-861 (1999) [29] Wilks, A.; Moenne-Loccoz, P.: Identification of the proximal ligand His-20 in heme oxygenase (Hmu O) from Corynebacterium diphtheriae. Oxidative cleavage of the heme macrocycle does not require the proximal histidine. J. Biol. Chem., 275, 11686-11692 (2000) [30] Liu, Y.; Ortiz de Montellano, P.R.: Reaction intermediates and single turnover rate constants for the oxidation of heme by human heme oxygenase-1. J. Biol. Chem., 275, 5297-5307 (2000) [31] Zhou, H.; Migita, C.T.; Sato, M.; Sun, D.; Zhang, X.; Ikeda-Saito, M.; Fujii, H.; Yoshida, T.: Participation of carboxylate amino acid side chain in regiospecific oxidation of heme by heme oxygenase. J. Am. Chem. Soc., 122, 8311-8312 (2000) [32] Botros, F.T.; Laniado-Schwartzmann, M.; Abraham, N.G.: Regulation of cyclooxygenase- and cytochrome P450 -derived eicosanoids by heme oxygenase in the rat kidney. Hypertension, 39, 639-644 (2002) 271

Heme oxygenase (decyclizing)

1.14.99.3

[33] Christova, T.Y.; Duridanova, D.B.; Setchenska, M.S.: Enhanced heme oxygenase activity increases the antioxidant defense capacity of guinea pig liver upon acute cobalt chloride loading: comparison with rat liver. Comp. Biochem. Physiol. C, 131, 177-184 (2002) [34] Ryter, S.W.; Otterbein, L.E.; Morse, D.; Choi, A.M.K.: Heme oxygenase/carbon monoxide signaling pathways: Regulation and functional significance. Mol. Cell. Biochem., 234-235, 249-263 (2002) [35] Sugishima, M.; Sakamoto, H.; Higashimoto, Y.; Omata, Y.; Hayashi, S.; Noguchi, M.; Fukuyama, K.: Crystal structure of rat heme oxygenase-1 in complex with heme bound to azide. Implication for regiospecific hydroxylation of heme at the a-meso carbon. J. Biol. Chem., 277, 45086-45090 (2002) [36] Davydov, R.; Kofman, V.; Fujii, H.; Yoshida, T.; Ikeda-Saito, M.; Hoffman, B.M.: Catalytic mechanism of heme oxygenase through EPR and ENDOR of cryoreduced oxy-heme oxygenase and its Asp 140 mutants. J. Am. Chem. Soc., 124, 1798-1808 (2002) [37] Schuller, D.J.; Zhu, W.; Stojiljkovic, I.; Wilks, A.; Poulos, T.L.: Crystal structure of heme oxygenase from the gram-negative pathogen Neisseria meningitidis and a comparison with mammalian heme oxygenase-1. Biochemistry, 40, 11552-11558 (2001)

272

$      

::

   
 1.14.99.4    progesterone,hydrogen-donor:oxygen oxidoreductase (hydroxylating)    progesterone monooxygenase    CYP2G1 [6] FMO [2] cytochrome P-450/monooxygenase [5] progesterone hydroxylase
    37256-85-2

     Ceropithecus aethiops (african green monkey, COS1 cells, DSMZ NoACC63 [3, 4]) [3, 4] Cladosporium resinae [1] Cuniculus sp. (rabbit [2, 6]) [2, 6] Cylindrocarpon radicicola (ACC 11011 [1]) [1] Ictalurus punctatus (channel catfish [2]) [2] Mus musculus (mouse, strain C57BL/6 [6]) [6] Oncorhynchus mykiss (rainbow trout [2]) [2] Rattus norvegicus [6] Streptomyces roseochromogenes (ATCC 13400 [5]) [5] Sus scrofa (pig [2]) [2]

!    "
 # 

progesterone + AH2 + O2 = testosterone acetate + A + H2 O (Has a wide specificity. A single enzyme from Cylindrocarpon radicicola (EC1.14.13.54 ketosteroid monooxygenase) catalyses both this reaction and that catalysed by EC1.14.99.12 4-androstene-3,17-dione monooxygenase) 273

Progesterone monooxygenase

1.14.99.4

   oxidation redox reaction reduction       progesterone + NADPH + O2 (Reversibility: ? [1-6]) [1-6] $ testosterone acetate + NADP+ + H2 O [1-6]      16a,17a-oxidopregn-4-ene-3,20-dione + NADPH + O2 ( degradation of pregnane side chains [1]) (Reversibility: ? [1]) [1] $ 16a-hydroxyandrost-4-ene-3,17-dione [1]  17a-hydroxypregn-4-ene-3,20-dione + NADPH + O2 ( degradation of pregnane side chains, about 210% the rate of the reaction with progesterone [1]) (Reversibility: ? [1]) [1] $ androst-4-ene-3,17-dione [1]  17a-methylpregn-4-ene-3,20-dione + NADPH + O2 ( about 4.3% the rate of the reaction with progesterone [1]) (Reversibility: ? [1]) [1] $ ?  5a-dihydrotestosterone + NADPH + O2 (Reversibility: ? [6]) [6] $ ?  androstenedione + NADPH + O2 (Reversibility: ? [6]) [6] $ ?  deoxycorticosterone + NADPH + O2 ( degradation of pregnane side chains, about 64% the rate of the reaction with progesterone [1]) (Reversibility: ? [1]) [1] $ testosterone [1]  estradiol + NADPH + O2 (Reversibility: ? [6]) [6] $ ?  pregna-4,16-diene-3,20-dione + NADPH + O2 ( degradation of pregnane side chains, about 104% the rate of the reaction with progesterone [1]) (Reversibility: ? [1]) [1] $ androst-4-ene-3,17-dione [1]  progesterone + NADPH + O2 (Reversibility: ? [1-6]) [1-6] $ testosterone acetate + NADP+ + H2 O [1-6]  progesterone + NADPH + O2 (Reversibility: ? [5]) [5] $ 16a-hydroxyprogesterone + 2b,16a-dihydroxyprogesterone + NADP+ + H2 O [5]  testosterone + NADPH + O2 (Reversibility: ? [2, 6]) [2, 6] $ 6b-hydroxytestosterone + NADP+ + H2 O [2, 6]  Additional information ( cortisol, 20a-hydroxy-16a,17a-oxidopregn-4-ene-3,20-dione and 20b-hydroxypregn-4-ene-3-one are no substrates [1]) [1] $ ?

274

1.14.99.4

Progesterone monooxygenase

2  KCN [1] ketoconazole [5] methylene blue [1] p-hydroxymercuribenzoate [1] phenazine methosulfate [1]
"%  NADPH ( absolute requirement, FMN, FAD, NADH or 2-amino-6,7dimethyl-4-hydroxy-5,6,7,8-tetrahydropteridine cannot replace NADPH as hydrogen-donor [1]) [1] cytochrome P450 [2, 4, 5] &

  coumarin ( hydroxylase activity is amplifiable by coumarin, increases 50% above that found constitutively [5]) [5] . / *', 0.0051 (testosterone) [6] 0.0076 (progesterone) [6] 0  7.5 ( progesterone, deoxycorticosterone, 17a-hydroxypregn-4-ene3,20-dione as substrates [1]) [1]

  #   ' 1  65000 ( PAGE, gel filtration [5]) [5]

 2 %$ %' %

% liver [2, 6] lung [2] olfactory mucosa [6] 3#

microsome [2, 6] $"

[1] [2] [5]

275

Progesterone monooxygenase

1.14.99.4


 

(cDNA obtained by PCR, cloning into a baculoviral expression vector pVL1392, production of the enzyme in cultured insect cells and heterologous expression of CYP2G1 in rabbits and rats [6]) [6] 

nutrition ( multiple drugs, pesticides and therapeutic agents are used in aquaculturing of channel catfish, flavin-containing monooxygenase enzymatic systems can metabolize these chemicals in the fish [2]; enzyme plays a significant role in biotransformation of pesticides in rainbow trout [2]) [2]

4  0  5.4-9 ( irreversible inactivated below [1]) [1] 7-8 ( most stable [1]) [1] 5    "

, 1 mg/ml albumin stabilizes crude enzyme extract for 1 month when kept frozen [1] , 10 h dialysis of purified enzyme against 0.01 M Tris buffer, pH 7.5, 0.005 M EDTA, leads to 50% loss of activity [1] , EDTA affords some protection, especially for crude extracts [1] , preparation is highly unstable [1]    , 0-6 , 85% of the enzyme activity is lost in 24 h [1] , 0 C, 80% enzyme activity is lost in 7 days, half-life of a highly purified preparation is 12 h [1]

"  [1] Rahim, M.A.; Sih, C.J.: Mechanisms of steroid oxidation by microorganisms. XI. Enzymatic cleavage of the pregnane side chain. J. Biol. Chem., 241, 36153623 (1966) [2] Schlenk, D.; Ronis, M.J.J.; Miranda, C.L.; Buhler, D.R.: Channel catfish liver monooxygenases. Immunological characterization of constitutive cytochromes P450 and the absence of active flavin-containing monooxygenases. Biochem. Pharmacol., 45, 217-221 (1993) [3] Straub, P.; Johnson, E.F.; Kemper, B.: Hydrophobic side chain requirements for lauric acid and progesterone hydroxylation at amino acid 113 in cytochrome P450 2C2, a potential determinant of substrate specificity. Arch. Biochem. Biophys., 306, 521-527 (1993)

276

1.14.99.4

Progesterone monooxygenase

[4] Straub, P.; Lloyd, M.; Johnson, E.F.; Kemper, B.: Differential effects of mutations in substrate recognition site 1 of cytochrome P450 2C2 on lauric acid and progesterone hydroxylation. Biochemistry, 33, 8029-8034 (1994) [5] Berrie, J.R.; Williams, R.A.D.; Smith, K.E.: Purification and characterization of progesterone hydroxylase, cytochrome P-450 from Streptomyces roseochromogenes ATCC 13400. Biochem. Soc. Trans., 25, 18S (1997) [6] Hua, Z.; Zhang, Q.; Su, T.; Lipinskas, T.W.; Ding, X.: cDNA cloning, heterologous expression, and characterization of mouse CYP2G1, an olfactory-specific steroid hydroxylase. Arch. Biochem. Biophys., 340, 208-214 (1997)

277

 
 

   
 1.14.99.5 (transferred to EC 1.14.19.1)    stearoyl-CoA desaturase

278

::

  ;    < 

::4

   
 1.14.99.6 (transferred to EC 1.14.19.2)    acyl-[acyl-carrier-protein] desaturase

279

?     

::8

   
 1.14.99.7    squalene,hydrogen-donor:oxygen oxidoreductase (2,3-epoxidizing)    squalene monooxygenase    hydroxylase, squalene oxygenase, squalene monosqualene 2,3-oxidocyclase squalene epoxidase squalene hydroxylase squalene oxydocyclase (EC 1.14.99.7 together with EC 5.4.99.7, was formerly known as squalene oxydocyclase) squalene-2,3-epoxidase squalene-2,3-epoxide cyclase
    9029-62-3

     Rattus norvegicus [1-9, 11, 13, 15, 18, 19, 20] Saccharomyces cerevisiae (enzyme is depressed in anaerobically grown cells [10]) [10, 14] Sus scrofa [19] Candida albicans [11, 12] Candida parapsilosis [11] Trichophyton rubrum [16] Homo sapiens (HepG2 cells [21]) [17, 21, 22, 23] Nicotiana tabacum [24]

280

1.14.99.7

Squalene monooxygenase

!    "
 # 

squalene + AH2 + O2 = (S)-squalene-2,3-epoxide + A + H2 O ( proposed mechanism [19])    epoxidation oxidation redox reaction reduction       squalene + electron donor + O2 ( may be rate-limiting step in cholesterol biosynthesis in non-cholesterogenic tissues [6]; first oxygenase and last nonsterol reaction of sterol biosynthesis [7]) (Reversibility: ? [6]) [6, 7] $ (S)-squalene-2,3-epoxide + oxidized electron donor + H2 O [6, 7]      1,1-bisnorsqualene + NADPH + O2 (Reversibility: ? [1]) [1] $ 1,1-bisnor-squalene-2,3-epoxide + NADP+ H2 O [2]  1-methylsqualene + NADPH + O2 (Reversibility: ? [2]) [2] $ 1-methyl-squalene-2,3-epoxide + NADP+ + H2 O [2]  1-norsqualene + NADPH + O2 (Reversibility: ? [2]) [2] $ 1-norsqualene-2,3-epoxide + NADP+ H2 O [2]  10,11,14,15-tetrahydrosqualene + NADPH + O2 (Reversibility: ? [2]) [2] $ 10,11,14,15-tetrahydro-(S)-squalene-2,3-epoxide + NADP+ + H2 O [2]  10,11-dihydrosqualene + NADPH + O2 (Reversibility: ? [1]) [1] $ 10,11-dihydro-(S)-squalene-2,3-epoxide + NADP+ + H2 O [1]  2,3-dihydrosqualene + NADPH + O2 (Reversibility: ? [2]) [2] $ ?  2,3-oxidosqualene + NADPH + O2 ( N-terminal truncated recombinant enzyme [15]) (Reversibility: ? [15]) [15] $ 2,3,22,23-dioxidosqualene + NADP+ + H2 O [15]  6,7,18,19-tetrahydrosqualene + NADPH + O2 (Reversibility: ? [2]) [2] $ 6,7,18,19-tetrahydro-(S)-squalene-2,3-epoxide [2]  squalene + NAD(P)H + O2 ( NADPH is preferred [10, 11]; specific for NADPH [14]; NADH is preferred [11]; NADPH is slightly preferred [16]) (Reversibility: ? [4, 6-12, 16]) [4, 6-12, 14, 15, 16] $ (S)-squalene-2,3-epoxide + NADP+ + H2 O [4, 6-12, 14, 15, 16]

281

Squalene monooxygenase

1.14.99.7

2  (E)-N-(6,6-dimethylhept-2-en-4-ynyl)-N-methyl-1-naphthalenemethaneamine hydrochloride ( i.e. compound SF 86-327, non-competitive inhibition [11]) [11] (E)-N-ethyl-N-(6,6-dimethyl-2-hepten-4-ynyl)-3-[2-methyl-2-(3-thienylmethoxy)propyloxy]benzylamine hydrochloride ( trivial name FR194738, 0.0000098 mM, 50% inhibition of enzyme activity in HepG2 cell homogenate [21]) [21] (E)-N-methyl-N-(3-phenylprop-2-enyl)-1-naphthalenemethaneamine ( i.e. naftifine, non-competitive inhibition [11]) [11] 1,2,6-tri-O-galloyl-b-d-glucose ( 0.00063 mM, 50% inhibition [20]) [20] 1,6-di-O-galloyl-O-cinnamoyl-b-d-glucose ( 0.00058 mM, 50% inhibition [20]) [20] Cu2+ ( 5 mM, 99% inhibition [12]) [12] H2 O2 ( inhibition above 2 mM [10]) [10] Mega-8 ( 0.3%, 19% inhibition [16]) [16] N-ethylmaleimide ( 1 mM, 35% inhibition [12]) [12] NB-598 ( 0.045 mM, 50% inhibition, partial non-competitive [14]; 0.0000032 mM, 50% inhibition of microsomal enzyme, 0.0000019 mM, 50% inhibition of N-terminal truncated recombinant enzyme [15]) [14, 15] SDZ 87-469 ( 0.000020 mM, 50% inhibition [16]) [16] Triton X-100 ( 0.02%, 50% inhibition [14]; 0.3%, 71% inhibition [16]) [12, 14, 16] amorolfine ( 0.03 mM, 505 inhibition [16]) [16] antimycin A ( 0.1 mM, 44% inhibition [12]) [12] bovine serum albumin [12] chloromercuriphenylsulfonate ( 1 mM, 35% inhibition [12]) [12] deoxycholate [12] dimethyltelluride ( approx. 0.0001 mM, 50% inhibition of recombinant enzyme, 0.1 mM, complete inhibition, preincubation with 1 mM glutathione maintains 50% of initial activity [22]) [22] dimethyltellurium dichloride ( approx. 0.0001 mM, 50% inhibition of recombinant enzyme, 0.1 mM, complete inhibition, preincubation with 1 mM glutathione maintains 50% of initial activity [22]) [22] epicatechin-3-O-gallate ( 0.0013 mM, 50% inhibition [18]) [18] epigallocatechin-3-O-gallate ( 0.00069 mM, 50% inhibition [18]) [18, 19] farnesyl gallate ( 0.0015 mM, 50% inhibition [18]) [18] gallocatechin-3-O-gallate ( 0.00067 mM, 50% inhibition [18]) [18] geranyl gallate ( 0.0125 mM, 50% inhibition [18]) [18] geranylgeranyl gallate ( 0.0045 mM, 50% inhibition [18]) [18] hydroxymercuribenzoate ( 1 mM, 35% inhibition [12]) [12] methylselenol ( 0.095 mM, 50% inhibition of recombinant enzyme, 1 mM, complete inhibition [23]) [23] n-dodecyl gallate ( 0.000061 mM, 50% inhibition [18]) [18]

282

1.14.99.7

Squalene monooxygenase

phenylarsine oxide ( recombinant enzyme, glutathione and 2,3-dimercaptopropanol protect almost completely [22]) [22] phenylbutyl gallate ( 0.0613 mM, 50% inhibition [18]) [18] phenyldecyl gallate ( 0.0153 mM, 50% inhibition [18]) [18] phenylhexyl gallate ( 0.0119 mM, 50% inhibition [18]) [18] phenyloctyl gallate ( 0.0125 mM, 50% inhibition [18]) [18] procyanidin B-2 3,3'-di-O-gallate ( 0.00054 mM, 50% inhibition [20]) [20] procyanidin B-5 3,3'-di-O-gallate ( 0.00055 mM, 50% inhibition [20]) [20] rotenone ( 0.1 mM, 67% inhibition [12]) [12] selenite ( 0.037 mM, 50% inhibition of recombinant enzyme, 2,3-dimercaptopropanol and dithiothreitol increase inhibition [23]) [23] selenite ( recombinant enzyme [17]) [17] selenium dioxide ( recombinant enzyme [17]) [17] tellurite ( 17 mM, 50% non-competitive inhibition of the recombinant enzyme [17]; 0.01 mM, 50% inhibition of recombinant enzyme, 10 mM, 95% inhibition, glutathione and 2,3-dimercaptopropanol protect almost completely [22]) [17, 22] tellurium dioxide ( 37 mM, 50% inhibition of the recombinant enzyme [17]) [17] terbinafine ( 0.18 mM, 50% inhibition, non-competitive [14]; 0.0000158 mM, 50% inhibition, non-competitive vs. squalene [16]) [14, 16, 24] theasinesin A ( 0.00013 mM, 50% inhibition [18]) [18] tolciclate ( 0.000028 mM, 50% inhibition [16]) [16] tolnaftate ( 0.0000515 mM, 50% inhibition [16]) [16] trisnorsqualene alcohol ( 0.004 mM, 50% inhibition [19]) [19] trisnorsqualene cyclopropylamine ( 0.002 mM, 50% inhibition [19]) [19] trisnorsqualene difluoromethylidene ( 0.0054 mM, 50% inhibition [19]) [19] trisnorsqualene gallate ( 0.0051 mM, 50% inhibition [18]) [18] Additional information ( strong inhibition by allylamines [16]; cholesterol lowering effect of green tea gallocatechins may be attributed to their potent squalene oxidase inhibition [19]; cholesterol lowering effect of rhubarb, Rhei rhizoma, Rheum palmatum L. and polygnaceae, galloyl glucoses and galloyl proanthocyanidins may be attributed to their potent squalene oxidase inhibition [20]) [16, 19, 20]
"%  1-carba-1-deazaFAD ( can replace FAD as cofactor [7]) [7] FAD ( flavoprotein [5, 7]; required for activity [4, 5, 11, 15]; required for activity [11, 12]; required for activity [11]; FAD is loosely bound [17]) [4, 5, 7, 9, 11, 12, 15, 17] Additional information ( 5-carba-5-deazaFAD cannot replace FAD as cofactor [7]; FMN cannot replace FAD as cofactor [12]; FAD

283

Squalene monooxygenase

1.14.99.7

is not essential for activity, 76.2% activity in the absence of FAD [14]; neither FMN nor riboflavin can replace FAD [15]; FAD stimulates activity but is not essential [16]; electron transfer partner NADPH-cytochrome P450 reductase [17]) [7, 12, 14, 15, 16, 17] &

  2-heptyl-4-hydroxyquinoline N-oxide ( 0.1 mM, 236% stimulation [12]) [12] Mega-9 ( 0.3%, 44% activation [16]) [16] NADPH cytochrome c reductase ( required for activity of N-terminal truncated recombinant enzyme in a concentration-dependent manner [15]) [15] Triton X-100 ( 0.1%, 36fold activation of microsomal enzyme, 0.05%, 34fold activation of N-terminal truncated recombinant enzyme [15]) [15] octyl b-d-glucopyranoside ( 0.3%, 42% activation [16]) [16] Additional information ( 105000 g supernatant, 8.5fold activation of microsomal enzyme, 3fold activation of N-terminal truncated recombinant enzyme [15]) [15] Additional information ( squalene epoxidation system requires a supernatant protein and a phospholipid: requirement for the heat-stable factor can be fully met by phosphatidylserine, phosphatidylglycerol or phosphatidylinositol and partially by other phospholipids, the heat-labile factor required is a protein with 44000 Da [1]; 2 cytoplasmic components can be replaced by Triton X-100 [4]; purification of a soluble 47000 Da activator of liver squalene epoxidase [3]; soluble protein factor from hog liver stimulates activity [13]; reconstitution of squalene epoxidase activity by addition of: NADPH-cytochrome P-450 reductase, EC 1.6.2.4, FAD and Triton X-100 [5,8]; enzyme is not a cytochrome P-450 enzyme [8]) [1, 35, 8, 13] ) &  * +, 0.33 (squalene) [8, 9] 1.1 (squalene) [17] 4.1 (squalene, N-terminal trucated recombinant enzyme [19]) [19] " & *-% , 0.000012 ( activity in microsomes [16]) [16] 0.0000321 ( activity in microsomal fraction [14]) [14] 0.0001 ( activity in cell-free extract [10]) [10] 0.00278 [5] 0.00619 [8, 9] 0.17 ( N-terminal truncated recombinant enzyme [15]) [15] . / *', 0.000014 (NADPH-cytochrome P450 reductase, recombinant enzyme, Km for electron transfer partner NADPH-cytochrone P450 reductase [17]) [17] 0.00043 (FAD) [7] 284

1.14.99.7

Squalene monooxygenase

0.00085 (1-carba-1-deazaFAD) [7] 0.0036 (squalene, N-terminal truncated recombinant enzyme [15]) [15, 19] 0.0043 (O2 ) [10] 0.005 (FAD) [8, 9] 0.011 (squalene) [11] 0.013 (squalene) [8, 9] 0.0133 (squalene) [16] 0.0135 (squalene) [14] 0.016 (squalene) [11] 0.05 (squalene) [11, 12] 0.3 (FAD, recombinant enzyme [17]) [17] 7.7 (squalene, recombinant enzyme [17]) [17] . / *', 0.00000041 (NB-598, N-terminal truncated recombinant enzyme [15]) [15] 0.00003 ((E)-N-(6,6-dimethylhept-2-en-4-ynyl)-N-methyl-1-naphthalenemethaneamine hydrochloride) [11] 0.00004 ((E)-N-(6,6-dimethylhept-2-en-4-ynyl)-N-methyl-1-naphthalenemethaneamine hydrochloride) [11] 0.000075 (terbinafine) [14] 0.00034 ((E)-N-methyl-N-(3-phenylprop-2-enyl)-1-naphthalenemethaneamine) [11] 0.00074 (epigallocatechin-3-O-gallate, non-competitive, non-time dependent inhibition [19]) [19] 0.0011 ((E)-N-methyl-N-(3-phenylprop-2-enyl)-1-naphthalenemethaneamine) [11] 0.0087 (NB-598) [14] 0.077 ((E)-N-(6,6-dimethylhept-2-en-4-ynyl)-N-methyl-1-naphthalenemethaneamine hydrochloride) [11] 1.44 ((E)-N-methyl-N-(3-phenylprop-2-enyl)-1-naphthalenemethaneamine) [11] 0  7.5 ( assay at [5]) [5, 14] 7.5-8.5 ( microsomal and N-terminal truncated recombinant enzyme [15]) [15] )  *
, 30-37 ( activity in crude extract [10]) [10]

  #   ' 1  45000 ( sucrose density gradient centrifugation [8]) [8]

285

Squalene monooxygenase

1.14.99.7

  ? ( x * 47000, SDS-PAGE [5]) [5] monomer ( 1 * 51000, most of the enzyme behaves as a monomer, SDS-PAGE [8]) [8, 9] Additional information ( enzyme system consists of squalene epoxidase which is distinct from hemoproteins such as cytochrome P-450 isozymes, and a flavoprotein identical with NADPH-cytochrome P-450 reductase [9]) [9]

 2 %$ %' %

% liver [1, 2, 4-9, 11, 13, 17] Additional information ( very low activity in non-cholesterogenic tissues: brain, muscle, lung, placenta, kidney [6]; cell suspension [24]) [6, 24] 3#

membrane [9] microsome [1, 4-6, 8, 9, 12, 13, 14, 16] $"

(solubilization [4]; partial [7]; DEAE-cellulose, alumina gel, hydroxylapatite, CM-Sephadex C-50, Blue Sepharose 4B [5]; recombinant enzyme, NiNTA-agarose, Blue Sepharose CL-6B [15]) [4, 5, 7-9, 15, 18, 19, 20] (recombinant enzyme [17, 22]) [17, 22, 23]  

(reconstitution of purified enzyme with the addition of NADPH-cytochrome P450 reductase, FAD and Triton X-100 [5,8]) [5, 8]
 

(expression of cDNA in Escherichia coli [15]) [15, 18, 19, 20] (expression of cDNA in Escherichia coli [17]) [17, 22, 23]

4  5    "

, freezing and thawing once, 20% loss of activity [8]    , -70 C, 20 mM Tris-HCl buffer, pH 7.4, 0.5% Triton X-100, several weeks [8] , -70 C, 50 mM Tris-HCl buffer, pH 7.4, 0.5% Triton X-100, several weeks [9] , -25 C, overnight, 2fold decrease of activity in crude extracts [10] , -20 C, 5 d, 75% loss of activity [12] , stable for several months in liquid nitrogen [12]

286

1.14.99.7

Squalene monooxygenase

"  [1] Tai, H.H.; Bloch, K.: Squalene epoxidase of rat liver. J. Biol. Chem., 247, 3767-3773 (1972) [2] Van Tamelen, E.E.; Heys, J.R.: Enzymic epoxidation of squalene variants. J. Am. Chem. Soc., 97, 1252-1253 (1975) [3] Ferguson, J.B.; Bloch, K.: Purification and properties of a soluble protein activator of rat liver squalene epoxidase. J. Biol. Chem., 252, 5381-5385 (1977) [4] Ono, T.; Bloch, K.: Solubilization and partial characterization of rat liver squalene epoxidase. J. Biol. Chem., 250, 1571-1579 (1975) [5] Ono, T.; Takahashi, K.; Odani, S.; Konno, H.; Imai, Y.: Purification of squalene epoxidase from rat liver microsomes. Biochem. Biophys. Res. Commun., 96, 522-528 (1980) [6] Astruc, M.; Tabacik, C.; Descomps, B.; Crastes de Paulet, A.: Squalene epoxidase and oxidosqualene lanosterol-cyclase activities in cholesterogenic and non-cholesterogenic tissues. Biochim. Biophys. Acta, 487, 204-211 (1977) [7] Jordan, D.B.: Squalene oxidase: the elusive flavoenzyme of sterol biosynthesis. Flavins and Flavoproteins (Proc. Int. Symp., 10th, Meeting Date 1990, Curti, B., Ronchi S., Zanetti, G., eds.) de Gruyter, Berlin, New York, 865-868 (1991) [8] Ono, T.; Nakazono, K.; Kosaka, H.: Purification and partial characterization of squalene epoxidase from rat liver microsomes. Biochim. Biophys. Acta, 709, 84-90 (1982) [9] Ono, T.; Imai, Y.: Squalene epoxidase from rat liver microsomes. Methods Enzymol., 110, 375-380 (1985) [10] M'Baya, B.; Karst, F.: In vitro assay of squalene epoxidase of Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun., 147, 556-564 (1987) [11] Ryder, N.S.; Dupont, M.C.: Inhibition of squalene epoxidase by allylamine antimycotic compounds. A comparative study of the fungal and mammalian enzymes. Biochem. J., 230, 765-770 (1985) [12] Ryder, N.S.; Dupont, M.C.: Properties of a particulate squalene epoxidase from Candida albicans. Biochim. Biophys. Acta, 794, 466-471 (1984) [13] Lin, L.F.H.: Roles of phospholipid and detergent in soluble protein activation of squalene epoxidase. Biochemistry, 19, 5135-5140 (1980) [14] Satoh, T.; Horie, M.; Watanabe, H.; Tsuchiya, Y.; Kamei, T.: Enzymic properties of squalene epoxidase from Saccharomyces cerevisiae. Biol. Pharm. Bull., 16, 349-352 (1993) [15] Nagumo, A.; Kamei, T.; Sakakibara, J.; Ono, T.: Purification and characterization of recombinant squalene epoxidase. J. Lipid Res., 36, 1489-1497 (1995) [16] Favre, B.; Ryder, N.S.: Characterization of squalene epoxidase activity from the dermatophyte Trichophyton rubrum and its inhibition by terbinafine and other antimycotic agents. Antimicrob. Agents Chemother., 40, 443-447 (1996)

287

Squalene monooxygenase

1.14.99.7

[17] Laden, B.P.; Tang, Y.; Porter, T.D.: Cloning, heterologous expression, and enzymological characterization of human squalene monooxygenase. Arch. Biochem. Biophys., 374, 381-388 (2000) [18] Abe, I.; Kashiwagi, Y.; Noguchi, H.: Inhibition of vertebrate squalene epoxidase by isoprenyl gallates and phenylalkyl gallates. Bioorg. Med. Chem. Lett., 10, 2525-2528 (2000) [19] Abe, I.; Seki, T.; Umehara, K.; Miyase, T.; Noguchi, H.; Sakakibara, J.; Ono, T.: Green tea polyphenols: novel and potent inhibitors of squalene epoxidase. Biochem. Biophys. Res. Commun., 268, 767-771 (2000) [20] Abe, I.; Seki, T.; Noguchi, H.; Kashiwada, Y.: Galloyl esters from rhubarb are potent inhibitors of squalene epoxidase, a key enzyme in cholesterol biosynthesis. Planta Med., 66, 753-756 (2000) [21] Sawada, M.; Matsuo, M.; Hagihara, H.; Tenda, N.; Nagayoshi, A.; Okumura, H.; Washizuka, K.; Seki, J.; Goto, T.: Effect of FR194738, a potent inhibitor of squalene epoxidase, on cholesterol metabolism in HepG2 cells. Eur. J. Pharmacol., 431, 11-16 (2001) [22] Laden, B.P.; Porter, T.D.: Inhibition of human squalene monooxygenase by tellurium compounds: evidence of interaction with vicinal sulfhydryls. J. Lipid Res., 42, 235-240 (2001) [23] Gupta, N.; Porter, T.D.: Inhibition of human squalene monooxygenase by selenium compounds. J. Biochem. Mol. Toxicol., 16, 18-23 (2002) [24] Wentzinger, L.F.; Bach, T.J.; Hartmann, M.A.: Inhibition of squalene synthase and squalene epoxidase in tobacco cells triggers an up-regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase. Plant Physiol., 130, 334-346 (2002)

288

      *#

,

::7

   
 1.14.99.8 (deleted, included in EC 1.14.14.1)    arene monooxygenase (epoxidizing)

289

 8a    

:::

   
 1.14.99.9    steroid,hydrogen-donor:oxygen oxidoreductase (17a-hydroxylating)    steroid 17a-monooxygenase    17a-hydroxylase-C17,20 lyase CYP 17 [4] CYPXVII EC 1.14.1.7 (formerly) EC 1.99.1.9 (formerly) P450 17 [2-4, 9] P450 -C17 P450 c17 [6, 7, 13, 18, 21] Steroid 17-a-hydroxylase/17,20 lyase cytochrome P-450 (P450 17a,lyase) cytochrome P450 17a steroid 17a-hydroxylase
    9029-67-8

    





290

Homo sapiens [1-11, 14, 17-19, 22, 23, 31] Rattus norvegicus [2, 4, 5, 7, 8, 12, 15, 16, 23, 32, 33] Bos taurus [9, 21, 24] Cavia porcellus [10, 25, 32] Mesocricetus auratus [13] Sus scrofa [5, 20, 26-29] Herpesvirus simiae (cynomolgus monkey [5]) [5] Oryctolagus cuniculus [30]

1.14.99.9

Steroid 17a-monooxygenase

!    "
 # 

a steroid + AH2 + O2 = a 17a-hydroxysteroid + A + H20    hydroxylation oxidation redox reaction reduction       pregnenolone + NADPH + O2 (Reversibility: ? [5-9, 12, 13, 15, 17-19, 22-24, 26, 30, 31]) [5-9, 12, 13, 15, 17-19, 2224, 26, 30, 31] $ 17a-hydroxypregnenolone + NADP+ + H2 O ( product eliminates at C20,21 acetate to yield dehydroepiandrosterone [6, 7, 12, 13, 15, 18, 19, 22, 26]; cytochrome b5 also as cofactor [8]) [5-8, 12, 13, 15, 18, 19, 22, 26]  progesterone + NADPH + O2 (Reversibility: ? [2-5, 7, 911, 13-18, 20, 21, 23, 24, 26-28, 30-32]) [2-5, 7, 9-11, 13-18, 20, 21, 23, 24, 26-28, 30-32] $ 17a-hydroxyprogesterone + NADP+ + H2 O ( product eliminates at C20,21 acetate to yield androstenedione [2-5, 7, 9-11, 13-18, 20, 21, 24, 26-28, 32]) [2-5, 7, 9-11, 13-18, 20, 21, 24, 26-28, 30, 32]  Additional information ( catalyzes the last step of androgen biosynthesis in both testes and adrenals [1]; it catalyzes steroid 17a-hydroxylase and 17,20-lyase activities in the biosynthesis of androgens, estrogens and cortisol [6-8]) [1, 6-8] $ ?      pregnenolone + NADPH + O2 (Reversibility: ? [5-9, 12, 13, 15, 17-19, 22-24, 26, 30, 31]) [5-9, 12, 13, 15, 17-19, 2224, 26, 30, 31] $ 17a-hydroxypregnenolone + NADP+ + H2 O ( product eliminates at C20,21 acetate to yield dehydroepiandrosterone [6, 7, 12, 13, 15, 18, 19, 22, 26]; cytochrome b5 also as cofactor [8]) [5-8, 12, 13, 15, 18, 19, 22, 26]  progesterone + NADPH + O2 (Reversibility: ? [2-5, 7, 911, 13-18, 20, 21, 23, 24, 26-28, 30-32]) [2-5, 7, 9-11, 13-18, 20, 21, 23, 24, 26-28, 30-32] $ 17a-hydroxyprogesterone + NADP+ + H2 O ( product eliminates at C20,21 acetate to yield androstenedione [2-5, 7, 9-11, 13-18, 20, 21, 24, 26-28, 32]) [2-5, 7, 9-11, 13-18, 20, 21, 24, 26-28, 30, 32]  Additional information ( both the lyase and hydroxylase activities proceed from a common steroid-binding geometry by an iron oxene mechanism [6]) [6] $ ? 291

Steroid 17a-monooxygenase

1.14.99.9

2  (S)-(-)-1-(4-pyridyl)ethyl 1-adamantanecarboxylate ( at 1.8 nM 50% C17,20-lyase inhibition, at 3.3 nM 50% 17a-hydroxylase inhibition [14]) [14] 1-(imidazol-1-ylmethyl)-4-bromo-9H-9-xanthenone ( at 0.0025 mM 98% inhibition [3]) [3] 1-(imidazol-1-ylmethyl)-4-nitro-9H-9-xanthenone ( at 0.0025 mM 94% inhibition [3]) [3] 1-(imidazol-1-ylmethyl)-9-oxo-9H-4-xanthenecarbonitrile ( at 0.0025 mM 92% inhibition [3]) [3] 1-[(5,7-dibromobenzofuran-2-yl)methyl]imidazole ( at 185 nM 50% inhibition [9]; at 1150 nM 50% inhibition [9]) [9] 1-[(5,7-dichlorobenzofuran-2-yl)methyl]imidazole ( at 180 nM 50% inhibition [9]; at 1300 nM 50% inhibition [9]) [9] 1-[(5-bromobenzofuran-2-yl)methyl]imidazole ( at 380 nM 50% inhibition [9]; at 1540 nM 50% inhibition [9]) [9] 1-[(5-chlorobenzofuran-2-yl)methyl]imidazole ( at 230 nM 50% inhibition [9]; at 1800 nM 50% inhibition [9]) [9] 17-(1H-1,2,3-triazol-1-yl)androsta-4,16-dien-3-one ( at 19 nM 50% inhibition, also potent inhibitor of 5a-reductase [8]; at 9 nM 50% inhibition [8]) [8] 17-(1H-1,2,4-triazol-1-yl)androsta-4,16-dien-3-one ( at 55 nM 50% inhibition, also potent inhibitor of 5a-reductase [8]; at 11 nM 50% inhibition [8]) [8] 17-(1H-imidazol-1-yl)androsta-4,16-dien-3-one ( at 7 nM 50% inhibition, also potent inhibitor of 5a-reductase [8]; at 8 nM 50% inhibition [8]) [5, 8] 17-(2-amino-4-thiazolyl)-androsta-5,16-dien-3b-ol ( type II competitive inhibitor [5]) [5] 17-(3'-pyrazolyl)androsta-4,16-dien-3b-one ( type II competitive inhibitor [5]) [5] 17-(3'-pyrazolyl)androsta-5,16-dien-3b-ol ( type II competitive inhibitor [5]) [5] 17-(3-pyridyl)-5a-androst-16-en-3-one ( at 3 nM 50% inhibition of C17,20-lyase and at 4.7 nM 50% inhibition of 17a-hydroxylase activity [17]) [17] 17-(3-pyridyl)-5a-androst-16-en-3a-ol ( at 2.5 nM 50% inhibition of C17,20-lyase and at 4.3 nM 50% inhibition of 17a-hydroxylase activity [17]) [17] 17-(3-pyridyl)-androst-5-en-3b-ol ( at 23 nM 50% inhibition of C17,20-lyase and at 47 nM 50% inhibition of 17a-hydroxylase activity [17]) [17] 17-(3-pyridyl)-androsta-4,16-dien-3,11-dione ( at 2.9 nM 50% inhibition of C17,20-lyase and at 13 nM 50% inhibition of 17a-hydroxylase activity [17]) [17] 17-(3-pyridyl)androsta-3,5,16-triene ( at 5.6 nM 50% inhibition of C17,20-lyase and at 12.5 nM 50% inhibition of 17a-hydroxylase activity [17]) [17] 292

1.14.99.9

Steroid 17a-monooxygenase

17-(3-pyridyl)androsta-4,16-dien-3-one ( at 2.1 nM 50% inhibition of C17,20-lyase and at 2.8 nM 50% inhibition of 17a-hydroxylase activity [17]) [17] 17-(3-pyridyl)androsta-5,6-dien-3b-ol ( at 2.9 nM 50% inhibition of C17,20-lyase and at 4 nM 50% inhibition of 17a-hydroxylase activity [17]) [5, 17] 17-(3-pyridyl)estra-1,3,5[10],16-tetraen-3-ol ( at 1.8 nM 50% inhibition of C17,20-lyase and at 2.6 nM 50% inhibition of 17a-hydroxylase activity [17]) [17] 17-(5'-isooxazoloyl)androsta-4,16-dien-3b-one ( type II competitive inhibitor [5]) [5] 17-(methylthio)androst-5-en-3b-ol S-oxide ( type I competitive inhibitor [5]) [5] 17-hydroxypregnenolone ( competitive inhibitor of 17a-hydrolase activity [6]) [6] 17a-hydroxy-4-androsten-3-one ( competitive inhibitor of 17a-hydroxylation of pregnenolone and of the subsequent C17,20-side chain cleavage reaction [19]) [19] 17b-(cyclopropylamino)-androst-5-en-3b-ol ( mechanism-based inhibitor, irreversible inhibition [22]) [22] 17b-acetamidoandrost-4-en-3-one [5] 17b-ureidoandrosta-1,4-dien-3-one [5] 2-(1H-imidazol-4-ylmethyl)-9H-carbazole ( suicide inhibitor [5]) [5] 2-(4-pyridyl)propan-2-yl 1-adamantanecarboxylate ( at 2.7 nM 50% C17,20-lyase inhibition, at 8.8 nM 50% 17a-hydroxylase inhibition [14]) [14] 20(S)-20,21-aziridinylpregn-5-en-3b-ol ( type II competitive inhibitor [5]) [5] 20-hydroxyiminopregna-4,14,16-trien-3-one ( at 0.0002 mM 50% inhibition [4]) [4] 20-hydroxyiminopregna-4,16-dien-3-one ( at 0.0001 mM 50% inhibition [4]) [4] 20-hydroxyiminopregna-5,14,16-trien-3b-ol ( at 0.0002 mM 50% inhibition [4]) [4] 20-hydroxyiminopregna-5,16-dien-3b-ol ( at 0.00017 mM 50% inhibition [4]) [4] 20-hydroxy-21-trifluoropregn-4-en-3-one ( at 0.0006 mM 50% inhibition [12]; type I competitive inhibitor [5]) [5, 12] 21-hydroxyimino-21-methylpregn-4-en-3-one ( at 0.0036 mM 50% inhibition [4]) [4] 21-hydroxyiminopregn-4-en-3-one ( at 0.0003 mM 50% inhibition, also 5a-reductase inhibitor [4]) [4] 21-hydroxyiminopregn-5-en-3b-ol ( at 0.00027 mM 50% inhibition [4]; at 0.00276 mM 50% inhibition [4]) [4] 21-hydroxyiminopregna-4,17(20)-dien-3-one ( at 0.00018 mM 50% inhibition, also 5a-reductase inhibitor [4]; at 0.00014 mM 50% inhibition [4]) [4]

293

Steroid 17a-monooxygenase

1.14.99.9

21-hydroxyiminopregna-5,17(29)-dien-3b-ol ( at 0.000077 mM 50% inhibition [4]; at 0.00052 mM 50% inhibition [4]) [4] 21-methylpregn-5-en-3b-ol-21-one ( at 0.0098 mM 50% inhibition [4]) [4] 21-trifluoropregn-4-en-3,20-dione ( at 0.0021 mM 50% inhibition [12]) [12] 21-trifluoropregn-5-en-3b,20-diol ( at 0.0044 mM 50% inhibition [12]) [12] 22-amino-23,24-bisnor-5-cholen-3b-ol ( type II competitive inhibitor [5,24]) [5, 24] 3-(6-chloro-3-methyl-2-indenyl)pyridine ( competitive inhibitor [28]) [28] 3-pyridyl-1a,2,3,7b-tetrahydro-1H-cyclopropa[a]naphthalene ( suicide inhibitor [5]) [5] 3b-acetoxy-17-(3-pyridyl)androsta-5,16-diene ( at 17 nM 50% inhibition of C17,20-lyase and at 18 nM 50% inhibition of 17a-hydroxylase activity [17]) [17] 3b-hydroxy-17-(1H-1,2,3-triazol-1-yl)androsta-5,16-diene ( at 150 nM 94% inhibition [8]; at 10 nM 50% inhibition [8]) [8] 3b-hydroxy-17-(1H-1,2,3-triazol-1-yl)androsta-5,16-diene ( type II competitive inhibitor [5]) [5] 3b-hydroxy-17-(1H-1,2,4-triazol-1-yl)androsta-5,16-diene ( at 150 nM 60% inhibition [8]; at 26 nM 50% inhibition [8]) [8] 3b-hydroxy-17-(1H-imidazol-1-yl)androsta-5,16-diene ( at 150 nM 97% inhibition [8]; at 9 nM 50% inhibition [8]) [8] 3b-hydroxy-17-(1H-imidazol-1-yl)androsta-5,16-diene ( type II competitive inhibitor [5]) [5] 3b-hydroxy-23,24-bisnor-5-cholenic-hydroxamic acid ( at 0.0025 mM 20% inhibition [2]; at 0.125 mM 19% inhibition [2]) [2] 3b-hydroxy-5-androsten-17b-hydroxamic acid ( at 0.0025 mM 17% inhibition [2]; at 0.125 mM 18% inhibition [2]) [2] 4-amino-17b-(cyclopropylamino)-androst-4-en-3-one ( suicide inhibitor [5]) [5] 4-amino-17b-(cyclopropyloxy)-androst-4,6-dien-3-one ( suicide inhibitor [5]) [5] 4-amino-17b-(cyclopropyloxy)-androst-4-en-3-one ( suicide inhibitor [5]) [5] 4-chloro-3,4-dihydro-2-(3-pyridyl)-1-(2H)-naphthalenone ( competitive inhibitor [28]) [28] 4-pyridylmethyl 1-adamantanecarboxylate ( at 18 nM 50% C17,20lyase inhibition, at 43 nM 50% 17a-hydroxylase inhibition [14]) [14] 5-[(3-chlorophenyl-1H-imidazole-1-yl)methyl]-1H-benzimidazole ( suicide inhibitor [5]) [5] CO [28] E-1-methyl-2-(1-hydroxyiminoethyl)-6-methoxy-3,4-dihydronaphthalene ( at 0.0025 mM 7% inhibition [11]) [11] Emulgen 913 ( at 0.2% w/v 94% inhibition [20]) [20] 294

1.14.99.9

Steroid 17a-monooxygenase

Z-1-methyl-2-(1-hydroxyiminoethyl)-6-methoxy-3,4-dihydronaphthalene ( at 0.0025 mM 5% inhibition [11]) [11] cytochrome b5 ( supresses 17a-hydrolase and C17,20-lyase activity below pH 6.3-6.5 [25]) [25] estradiol-17b ( inhibits both and C17,20 lyase activity in testis and duodenum, competitive inhibitor [15]) [15] iodoacetate ( at 1 mM 100% inhibition of 17a-hydroxylation and 50% inhibition of lyase activity [32]) [32] ketoconazole ( at 0.00074 mM 50% inhibition [3]; at 150 nM 67% inhibition [8]; at 0.0019 mM 50% inhibition [12]) [3, 8, 12] nitrogen ( complete inhibition [32]) [32] p-chloromercuribenzoate ( at 1 mM complete inhibition [32]) [32] sodium cholate ( at 0.3% w/v 87% inhibition [20]) [20, 26] transforming growth factor-b1 ( inhibits 17a-hydroxylation in vitro by a noncompetitive mechanism [16]) [16]
"%  NADPH [2-7, 9, 15, 16, 18, 22-26, 29-32] &

  cytochrome b5 ( acts as allosteric facilitator but not as electron donor, increases 17,20-lyase activity [6]; above pH 6.3-6.5 stimulates 17a-hydrolase and C17,20-lyase activity [25]) [6, 8, 25] phosphatidylcholine ( incorporation of P450 into liposomal membranes composed of phosphatidylcholine increases activity [20]) [20] '(   Fe3+ ( heme [6, 14, 24]; heme content more than 0.8 nM/ nM protein [26]; 11.1 nM heme per mg protein [27]; 8 nM per mg protein [29]) [6, 8, 14, 15, 24, 26, 27, 29] ) &  * +, 0.17 (17a-hydroxypregnenolone) [18] 2.9 (progesterone) [18] 15.5 (progesterone) [20] 93 (progesterone, 17a-hydroxylase activity [21]) [21] " & *-% , 0.008 ( lyase activity [29]) [29] 0.023 ( hydroxylase activity [29]) [29] . / *', 1.42e-005 (progesterone, 17a-hydroxylation in testis [15]) [15] 2.32e-005 (progesterone, 17a-hydroxylation in female duodenum [15]) [15] 2.38e-005 (progesterone, 17a-hydroxylation in male duodenum [15]) [15] 8e-005 (pregnenolone, wild type enzyme [10]) [10] 0.00022 (pregnenolone, R200N mutant [10]) [10] 0.00024 (progesterone, wild type enzyme [10]) [10] 295

Steroid 17a-monooxygenase

1.14.99.9

0.00025 (pregnenolone) [10] 0.00031 (progesterone, R200N mutant [10]) [10] 0.0004 (17-hydroxypregnenolone, wild type enzyme [6]) [6] 0.00045 (progesterone) [10] 0.000525 (17a-hydroxyprogesterone, C17,20 lyase activity in testis [15]) [15] 0.00056 (17-hydroxypregnenolone) [8] 0.000637 (17a-hydroxyprogesterone, C17,20 lyase activity in female duodenum [15]) [15] 0.000675 (17a-hydroxyprogesterone, C17,20 lyase activity in male duodenum [15]) [15] 0.0008 (17-hydroxypregnenolone, K89N-mutant expressed in Saccharomyces cerevisiae [6]) [6] 0.0008 (pregnenolone, adrenal gland [26]) [26] 0.0009 (17a-hydroxypregnenolone, adrenal gland [26]) [26] 0.0015 (progesterone) [29] 0.0017 (17a-hydroxypregnenolone) [18] 0.0018 (progesterone, adrenal gland [26]) [26] 0.0023 (progesterone) [31] 0.0024 (17a-hydroxyprogesterone) [29] 0.0025 (17a-hydroxyprogesterone, adrenal gland [26]) [26] 0.0025 (pregnenolone) [31] 0.0063 (progesterone) [18] 0.0077 (pregnenolone) [18] . / *', 0.0000000464 (transforming growth factor-b1) [16] 0.00000012 (3b-hydroxy-17-(1H-imidazol-1-yl)androsta-5,16-diene, noncompetitive inhibitor [8]) [8] 0.0000014 (3b-hydroxy-17-(1H-1,2,3-triazol-1-yl)androsta-5,16-diene, noncompetitive inhibitor [8]) [8] 0.0000019 (17-(1H-imidazol-1-yl)androsta-4,16-dien-3-one, noncompetitive inhibitor [8]) [8] 0.000029 (22-amino-23,24-bisnor-5-cholen-3b-ol, 17a-hydroxylase activity [24]) [24] 0.00004 (3-(6-chloro-3-methyl-2-indenyl)pyridine) [28] 0.000041 (17-(1H-1,2,4-triazol-1-yl)androsta-4,16-dien-3-one, noncompetitive inhibitor [8]) [8] 0.00008 (17-(1H-1,2,3-triazol-1-yl)androsta-4,16-dien-3-one, noncompetitive inhibitor [8]) [8] 0.00023 (3b-hydroxy-17-(1H-1,2,4-triazol-1-yl)androsta-5,16-diene, noncompetitive inhibitor [8]) [8] 0.0003 (17-hydroxypregnenolone, R347H-mutant, inhibiton of 17ahydrolase activity [6]) [6] 0.0003 (4-chloro-3,4-dihydro-2-(3-pyridyl)-1-(2H)-naphthalenone) [28] 0.0005 (17-hydroxypregnenolone, K89N-mutant, inhibiton of 17ahydrolase activity [6]) [6]

296

Steroid 17a-monooxygenase

1.14.99.9

0.0007 (17-hydroxypregnenolone, wild type, inhibiton of 17a-hydrolase activity [6]) [6] 0.0008 (17-hydroxypregnenolone, R358Q-mutant, inhibiton of 17ahydrolase activity [6]) [6] 0.0008 (pregnenolone, competitive substrate with progesterone [31]) [31] 0.00124 (17a-hydroxyandrosten-3-one, C17,20-lyase activity [19]) [19] 0.0032 (progesterone, competitive substrate with pregnenolone [31]) [31] 0.0096 (17a-hydroxyandrosten-3-one, 17a-hydroxylase activity [19]) [19] 0  6.1 ( both 17a-hydroxylation and C17,20-lyase actiivty [25]) [25] 6.6 ( C17,20-lyase activity after addition of cytochrome b5 [25]) [25] 6.9-8.5 ( for 17a-hydroxylation of pregnenolone [31]) [31] 7 ( 17a-hydroxylation after addition of cytochrome b5 [25]) [25] 7.2 ( assay at [7, 18]) [7, 18] 7.3 [28, 29] 7.4 ( assay at [5, 9, 23, 24]) [5, 9, 23, 24] 8.5 ( for 17a-hydroxylation of progesterone [31]) [31] )  *
, 25 ( assay at [24]) [24] 32 [2] 34 [5, 23] 37 [2, 7, 15, 18, 20, 28, 29] ) 

 *
, 30-44 ( hydroxylase and lyase activity decrease proportionally [28]) [28]

  #   ' 1  46000 ( SDS-PAGE 53000 ( SDS-PAGE 54000 ( SDS-PAGE 57000 [33] 59000 ( SDS-PAGE

[20]) [20] [27]) [27] [26]) [26] and denaturating gel filtration [29]) [29]

  monomer ( a, 1 * 57000 [33]) [33] monomer ( a, 1 * 59000 [29]) [29]

297

Steroid 17a-monooxygenase

1.14.99.9

$   "

glycoprotein ( three glucosamine residues per molecule [28]) [28, 29] phospholipoprotein ( 40 nmol phospholipid per mg of protein [29]) [29]

 2 %$ %' %

% adrenal gland ( little lyase activity [26]) [9, 13, 24-27, 30] duodenum [15] ovary ( theca-interstitial cell [16]) [16] testis ( adult and foetal [5]; greater lyase than hydroxylase activity [26]; neonatal [28]) [2-5, 8, 9, 11, 12, 14, 15, 17, 19, 20, 22, 23, 26, 28, 29, 31, 32] 3#

microsome ( membrane-bound [10, 29, 32] ; endoplasmic reticulum [24]) [2-4, 6, 8-10, 12, 14, 15, 19, 21, 22, 24-27, 29, 31, 32] $"

[7, 18] (purification system especially useful for steroid enzyme assays utilizing radiolabeled substrates [23]) [23] [20, 27, 29]
 

(expression in Escherichia coli XL1 [4]) [4] (expression in Escherichia coli, human gene with an (His)4 -tail [18]) [18] (expression in Saccharomyces cerevisiae strain W303B of the K89N-mutant [6]) [6] (expression in Saccharomyces cerevisiae strain AH22, fused with yeast reductase, greater 17a-hydroxylase activity [21]) [21] (expression in COS-1 cells [13]) [13] (expression in Eschericia coli JM109, human gene with an (His)4 -tail [7]) [7] (expression in monkey kidney COS-1 cells [10]) [10] 

 

K89N ( 78% loss of 17,20-lyase activity and 20% loss of 17a-hydroxylase activity [6]) [6] R200N ( increased reactivity towards pregnenolone, converts pregnenolone to 17a-hydroxypregnenolone and dehydroepiandrosterone, at the expense of 17,20-lyase activity towards 17a-hydroxyprogesterone [10]) [10] R347H ( loss of 17,20-lyase activity to a greater extent than 17ahydroxylase activity [6]) [6]

298

1.14.99.9

Steroid 17a-monooxygenase

R358Q ( loss of 17,20-lyase activity to a greater extent than 17ahydroxylase activity [6]) [6] 

medicine ( inhibition of steroid 17a-monooxygenase is an important therapeutic strategy in order to inhibit tumor growth in prostate cancer [1, 4, 5]) [1, 4, 5]

4     , -20 C, 0.25 M sucrose, pH 7.4, 2 months [31] , -70 C, 0.2% Emulgen 913 v/v and 20% glycerol v/v, 6 months, no detectable loss of activity [26, 29]

"  [1] Hartmann, R.W.; Ehmer, P.B.; Haidar, S.; Hector, M.; Jose, J.; Klein, C.D.; Seidel, S.B.; Sergejew, T.F.; Wachall, B.G.; Wachter, G.A.; Zhuang, Y.: Inhibition of CYP 17, a new strategy for the treatment of prostata cancer. Arch. Pharmacol., 335, 119-128 (2002) [2] Haidar, S.; Klein, C.D.; Hartmann, R.W.: Synthesis and evaluation of steroidal hydroxamic acids as inhibitors of P450 17 (1 a-hydroxylase/C17-20lyase). Arch. Pharmacol., 334, 138-140 (2001) [3] Recanatini, M.; Bisi, A.; Cavalli, A.; Belluti, F.; Gobbi, S.; Rampa, A.; Valenti, P.; Palzer, M.; Palusczak, A.; Hartmann, R.W.: A new class of nonsteroidal aromatase inhibitors: design and synthesis of chromone and xanthone derivatives and inhibition of the P450 enzymes aromatase and 17a-hydroxylase/C17,20-lyase. J. Med. Chem., 44, 672-680 (2001) [4] Hartmann, R.W.; Hector, M.; Haidar, S.; Ehmer, P.B.; Reichert, W.; Jose, J.: Synthesis and evaluation of novel steroidal oxime inhibitors of P450 17 (17a-hydroxylase/C17-20-lyase) and 5a-reductase types 1 and 2. J. Med. Chem., 43, 4266-4277 (2000) [5] Njar, V.C.; Brodie, A.M.: Inhibitors of 17a-hydroxylase/17,20-lyase (CYP17): potential agents for the treatment of prostate cancer. Curr. Pharm. Des., 5, 163-180. (1999) [6] Auchus, R.J.; Miller, W.L.: Molecular modeling of human P450c17 (17a-hydroxylase/17,20-lyase): insights into reaction mechanisms and effects of mutations. Mol. Endocrinol., 13, 1169-1182 (1999) [7] Brock, B.J.; Waterman, M.R.: The use of random chimeragenesis to study structure/function properties of rat and human P450c17. Arch. Biochem. Biophys., 373, 401-408 (2000) [8] Njar, V.C.; Kato, K.; Nnane, I.P.; Grigoryev, D.N.; Long, B.J.; Brodie, A.M.: Novel 17-azolyl steroids, potent inhibitors of human cytochrome 17 a-hy-

299

Steroid 17a-monooxygenase

[9]

[10]

[11]

[12] [13]

[14]

[15] [16] [17]

[18]

[19] [20]

300

1.14.99.9

droxylase-C17,20-lyase (P450 (17) a): potential agents for the treatment of prostate cancer. J. Med. Chem., 41, 902-912 (1998) Bahshwan, S.A.; Owen, C.P.; Nicholls, P.J.; Smith, H.J.; Ahmadi, M.: Some 1[(benzofuran-2-yl)methyl]imidazoles as inhibitors of 17a-hydroxylase: 17,20-lyase (P450 17) and their specificity patterns. J. Pharm. Pharmacol., 50, 1109-1116 (1998) Beaudoin, C.; Lavallee, B.; Tremblay, Y.; Hu, D.W.; Breton, R.; De Launoit, Y.; Belanger, A.: Modulation of 17 a-hydroxylase/17,20-lyase activity of guinea pig cytochrome P450c17 by site-directed mutagenesis. DNA Cell Biol., 17, 707-715 (1998) Zhuang, Y.; Zapp, J.; Hartmann, R.W.: Synthesis of Z- and E-1-methyl-2-(1hydroximinoethyl)-6-methoxy-3,4-dihydronaphthalene and evaluation as inhibitors of 17 a-hydroxylase-C17,20-lyase (P450 17). Arch. Pharmacol., 330, 359-361 (1997) Njar, V.C.; Klus, G.T.; Johnson, H.H.; Brodie, A.M.: Synthesis of novel 21trifluoropregnane steroids: inhibitors of 17a-hydroxylase/17,20-lyase (17 alyase). Steroids, 62, 468-473 (1997) Cloutier, M.; Fleury, A.; Courtemanche, J.; Ducharme, L.; Mason, J.I.; Lehoux, J.G.: Characterization of the adrenal cytochrome P450C17 in the hamster, a small animal model for the study of adrenal dehydroepiandrosterone biosynthesis. DNA Cell Biol., 16, 357-368 (1997) Chan, F.C.; Potter, G.A.; Barrie, S.E.; Haynes, B.P.; Rowlands, M.G.; Houghton, J.; Jarman, M.: 3- and 4-pyridylalkyl adamantanecarboxylates: inhibitors of human cytochrome P450(17a) (17 a-hydroxylase/C17,20-lyase). Potential nonsteroidal agents for the treatment of prostatic cancer. J. Med. Chem., 39, 3319-3323 (1996) Dalla Valle, L.; Ramina, A.; Vianello, S.; Belvedere, P.; Colombo, L.: Kinetic analysis of duodenal and testicular cytochrome P450c17 in the rat. J. Steroid Biochem. Mol. Biol., 58, 577-584 (1996) Fournet, N.; Weitsman, S.R.; Zachow, R.J.; Magoffin, D.A.: Transforming growth factor-b inhibits ovarian 17a-hydroxylase activity by a direct noncompetitive mechanism. Endocrinology, 137, 166-174 (1996) Potter, G.A.; Barrie, S.E.; Jarman, M.; Rowlands, M.G.: Novel steroidal inhibitors of human cytochrome P45017 a (17a-hydroxylase-C17,20-lyase): potential agents for the treatment of prostatic cancer. J. Med. Chem., 38, 24632471 (1995) Imai, T.; Globerman, H.; Gertner, J.M.; Kagawa, N.; Waterman, M.R.: Expression and purification of functional human 17 a-hydroxylase/17,20lyase (P450c17) in Escherichia coli. Use of this system for study of a novel form of combined 17 a-hydroxylase/17,20-lyase deficiency. J. Biol. Chem., 268, 19681-19689 (1993) Bicikova, M.; Hampl, R.; Hill, M.; Starka, L.: Inhibition of steroid 17a-hydroxylase and C17,20-lyase in the human testis by epitestosterone. J. Steroid Biochem. Mol. Biol., 46, 515-518 (1993) Kuwada, M.; Sone, Y.; Kitajima, R.: Purification from adult pig testicular P450 and 17 a-hydroxylase activity of P-450 containing liposomal membranes. Biochem. Biophys. Res. Commun., 196, 816-824 (1993)

1.14.99.9

Steroid 17a-monooxygenase

[21] Sakaki, T.; Shibata, M.; Yabusaki, Y.; Murakami, H.; Ohkawa, H.: Genetically engineered P450 monooxygenases: construction of bovine P450c17/yeast reductase fused enzymes. DNA Cell Biol., 9, 27-36 (1990) [22] Angelastro, M.R.; Laughlin, M.E.; Schatzman, G.L.; Bey, P.; Blohm, T.R.: 17b-(Cyclopropylamino)-androst-5-en-3 b-ol, a selective mechanism-based inhibitor of cytochrome P450(17 a) (steroid 17a-hydroxylase/C17-20 lyase). Biochem. Biophys. Res. Commun., 162, 1571-1577 (1989) [23] Schatzman, G.L.; Laughlin, M.E.; Blohm, T.R.: A normal phase high-performance liquid chromatography system for steroid 17a-hydroxylase/C17-20 lyase (cytochrome P-45021scc ) assays. Anal. Biochem., 175, 219-226 (1988) [24] Sheets, J.J.; Zuber, M.X.; McCarthy, J.L.; Vickery, L. E.; Waterman, M.R.: Discriminatory inhibition of adrenocortical 17a-hydroxylase activity by inhibitors of cholesterol side chain cleavage cytochrome P-450. Arch. Biochem. Biophys., 242, 297-305 (1985) [25] Shinzawa, K.; Kominami, S.; Takemori, S.: Studies on cytochrome P-450 (P450 17a,lyase) from guinea pig adrenal microsomes. Dual function of a single enzyme and effect of cytochrome b5. Biochim. Biophys. Acta, 833, 151-160 (1985) [26] Nakajin, S.; Shinoda, M.; Haniu, M.; Shively, J.E.; Hall, P.F.: C21 steroid side chain cleavage enzyme from porcine adrenal microsomes. Purification and characterization of the 17 a-hydroxylase/C17,20-lyase cytochrome P-450. J. Biol. Chem., 259, 3971-3976 (1984) [27] Nakajin, S.; Shinoda, M.; Hall, P.F.: Purification and properties of 17a-hydroxylase from microsomes of pig adrenal: a second C21 side-chain cleavage system. Biochem. Biophys. Res. Commun., 111, 512-517 (1983) [28] Nakajin, S.; Shively, J.E.; Yuan, P.M.; Hall, P.F.: Microsomal cytochrome P450 from neonatal pig testis: two enzymatic activities (17a-hydroxylase and c17,20-lyase) associated with one protein. Biochemistry, 20, 4037-4042 (1981) [29] Nakajin, S.; Hall, P.F.: Microsomal cytochrome P-450 from neonatal pig testis. Purification and properties of a C21 steroid side-chain cleavage system (17a-hydroxylase-C17,20 lyase). J. Biol. Chem., 256, 3871-3876 (1981) [30] Fevold, H.R.; Wilson, P.L.; Slanina, S.M.: ACTH-stimulated rabbit adrenal 17a-hydroxylase. Kinetic properties and a comparison with those of 3b-hydroxysteroid dehydrogenase. J. Steroid Biochem., 9, 1033-1041 (1978) [31] Fan, D.F.; Oshima, H.; Troen, B.R.; Troen, P.: Studies of the human testis. IV. Testicular 20 a-hydroxysteroid dehydrogenase and steroid 17a-hydroxylase. Biochim. Biophys. Acta, 360, 88-99 (1974) [32] Lynn, W.S.; Brown, R.H.: The conversion of progesterone to androgens by testes. J. Biol. Chem., 232, 1015-1031 (1958) [33] Namiki, M.; Kitamura, M.; Buczko, E.; Dufau, M.L.: Rat testis P-45017a cDNA: the deduced amino acid sequence, expression and secondary structural configuration. Biochem. Biophys. Res. Commun., 157, 705-712 (1988)

301

     

::=

   
 1.14.99.10    steroid,hydrogen-donor:oxygen oxidoreductase (21-hydroxylating)    steroid 21-monooxygenase    17a-hydroxyprogesterone 21-hydrolase 21-hydroxylase 21-hydroxylase cytochrome P-450 cytochrome P-450 specific for steroid C-21 hydroxylation EC 1.14.1.8 (formerly) EC 1.99.1.11 (formerly) P-450(C21) P450 -C21 P450 -C21B Steroid 21-hydroxylase cytochrome P-450-linked mixed function oxidase system cytochrome P-450C-21 steroid 21-hydroxylase system steroid 21-hydroxylation system
    9029-68-9

    



Bos taurus (beef [1, 2, 3, 5, 7, 9, 10]) [1, 2, 3, 5, 7, 9, 10] Sus scrofa (pig [4, 8]) [4, 8] Oncorhynchus mykiss (rainbow trout [6]) [6] Homo sapiens (man [11]) [11]

!    "
 # 

a steroid + AH2 + O2 = a 21-hydroxysteroid + A + H2 O 302

1.14.99.10

Steroid 21-monooxygenase

   hydroxylation oxidation redox reaction reduction       17-hydroxyprogesterone + NADPH + O2 (Reversibility: ? [6]) [6] $ corticosteroids + NADP+ + H2 O [6]  steroid + electron donor + O2 (essential step in synthesis of steroid hormones by adrenal gland) [1] $ 21-hydroxysteroid + oxidized electron donor + H2 O [1]      (+)-benzphetamine + NADH + O2 (Reversibility: ? [2]) [2] $ N-demethyl-benzphetamine + NAD+ + H2 O [2]  11,17-dihydroxyprogesterone + NADH + O2 (Reversibility: ? [1]) [1] $ cortisol + NAD+ + H2 O [1]  11b-hydroxyprogesterone + NADH + O2 (Reversibility: ? [1]) [1] $ corticosterone + NAD+ + H2 O [1]  17a-hydroxyprogesterone + NADH + O2 (Reversibility: ? [1, 2, 5, 6, 8]) [1, 2, 5, 6, 8] $ 17,21-dihydroxyprogesterone + NAD+ + H2 O [1, 2, 6, 8]  17a-hydroxyprogesterone + NADPH + O2 (Reversibility: ? [9, 10]) [9, 10] $ 11-deoxycortisol + NADP+ + H2 O [9, 10]  D5 -pregnen-3b,17a-diol-20-one + NADH + O2 [1] $ ? + NAD+ + H2 O  D5 -pregnen-3b-ol-20-one + NADH + O2 ( no substrate of cytochrome P-450-linked mixed function oxidase system [2]) (Reversibility: ? [1]) [1] $ deoxycorticosterone + NAD+ + H2 O ( very small yields, presumably via dehydrogenation followed by C-21 hydroxylation [1]) [1]  progesterone + NADH + O2 (Reversibility: ? [1, 2, 5, 8]) [1, 2, 5, 8] $ deoxycorticosterone + NAD+ + H2 O [1, 2, 8]  progesterone + NADPH + O2 (Reversibility: ? [9, 10]) [9, 10] $ deoxycorticosterone + NAD+ + H2 O [9, 10] 2  CuSO4 ( 50% inhibition at 1 mM [1]) [1, 3] HgCl2 ( complete inhibition at 0.1 mM [1]) [1] RU38486 [7] antibody to NADPH-cytochrome P-450 reductase [2] antibody to cytochrome P-450BPA [2] antimycin A ( 15% inhibition at 1mg per l [1]) [1] ascorbate ( 10% inhibition at 10 mM [1]) [1, 3]

303

Steroid 21-monooxygenase

1.14.99.10

azide ( 9% inhibition at 1 mM [1]) [1] carbon monoxide ( dark 65% inhibition at 90%, light 57% inhibition at 90% [1]) [1, 2] cyanide ( 14% inhibition at 1 mM [1]) [1] cytochrome c ( complete inhibition at 0.1 mM, 0.003 mM [1]) [1] diethylaminoethyldiphenylpropylacetic acid SKF 525 A ( 9% inhibition at 1 mM [1]) [1] p-chloromercuribenzoate ( complete inhibition at 1 mM, but not inhibitory at 0.1 mM [1]) [1] phenylisocyanide ( inhibition at 0.5 mM [2]) [2] progesterone [6] sodium o-(3-hydroxymercuri-2-methoxypropyl)carbamyl-phenoxyacetc acid (mersalyl) ( complete inhibition at 1 mM [1]) [1] Additional information ( not inhibited by iodoacetate, o-iodosobenzoate, glutathione, EDTA, dipyridyl, quinacrine, ribonuclease, cytochrome c + CN- [1]; not inhibited by catalase [1, 2]; not inhibited by superoxide dismutase, cytochrome b5, NADH-cytochrome b5 reductase [2]; not inhibited by metal ions such as Cr3+, Fe3+ , Zn2+ , Pb2+ , Mn2+ , Co2+ at 1 mM [1]) [1, 2]
"%  NADH ( NADH is 50% as effective as NADPH [1]) [1] NADPH [1] cytochrome p450 S21 ( steroid 21-hydroxylase system consists of cytochrome P-450S21, NADPH-cytochrome P-450 reductase (EC 1.6.2.4) and steroid 21-monooxygenase (EC 1.14.99.10) [2]) [2] heme ( 0.8 mol/mol peptide [8]) [8] &

  2-mercaptoethanol [3] EDTA [3] Emulgen 911 [2] Emulgen 913 ( maximum with 0.008% v/v [2]) [2] GSH [3] GSSG [3] l-cysteine [3] l-cystine [3] Triton X-100 [2] bovine serum albumin [3] dithiothreitol [3] lysophosphatidylcholine [2] sodium cholate [2] sodium deoxycholate [2] ) &  * +, 10.3 (progesterone) [9] 19.3 (17a-hydroxyprogesterone) [9]

304

1.14.99.10

Steroid 21-monooxygenase

" & *-% , 0.003 ( 21-hydroxylation of progesterone at 26 C [5]) [5] 0.0038 ( 21-hydroxylation of 17a-hydroxyprogesterone at 26 C [5]) [5] 0.0052 ( N-demethylation of (+)-benzphetamine [2]) [2] 0.0077 ( 21-hydroxylation of progesterone [2]) [2] 0.0144 ( 21-hydroxylation of progesterone [10]) [10] 0.0195 ( cytochrome P-450-linked mixed function oxidase system from adrenal gland, 21-hydroxylation of 17a-hydroxyprogesterone [2]) [2] 0.0452 ( 21-hydroxylation of 17a-hydroxyprogesterone [10]) [10] . / *', 0.0038 (17-hydroxyprogesterone, head kidney microsomes [6]) [6] 0.0055 (17-hydroxyprogesterone) [8] 0.0055 (progesterone) [8] 0.031 (17-hydroxyprogesterone, liver microsomes [6]) [6] 0  6.5-7 [1] 7.4 [2] 0 

 5.5-8 ( about 50% of activity maximum at pH 5.5 and 8.0 [1]) [1] 7.5-8.5 [6]

  #   ' 1  47000 ( SDS-PAGE [9]) [9] 47500 ( SDS-PAGE [2]) [2] 48870 ( calculation form amino acid composition [10]) [10] 52000 ( SDS-PAGE [5,10]) [5, 10] 54000 ( SDS-PAGE [4]) [4] 54000 ( SDS-PAGE, chromatography on Sephadex [8]) [8]   Additional information ( steroid 21-hydroxylase system consists of cytochrome P-450S21, NADPH-cytochrome P-450 reductase, EC 1.6.2.4, and steroid 21-monooxygenase, EC 1.14.99.10 [2]; amino acid analysis [10]; monomer-dimer equilibrium [5]) [2, 5, 10] Additional information ( amino acid analysis, N-terminal sequence, comparison with other cytochromes P-450 [8]) [8] $   "

glycoprotein ( 3.6% carbohydrate [2]) [2]

305

Steroid 21-monooxygenase

1.14.99.10

 2 %$ %' %

% adrenal cortex [8-10] adrenal gland [1-5] interrenal cell [6] liver [6] 3#

microsome [1-6, 8-10] $"

[9, 10] (enzyme system consisting of cytochrome P-450S21 and NADPH-cytochrome P-450 reductase [2]) [2, 5] [8]
 

[11]

4     , -70 C, 0.2 M potassium phospate, pH 7.4, 20% glycerol, 0.1 mM EDTA, 3 months without loss of activity [2] , 0 C, 50 mM Tris, pH 7.2, 20% glycerol, 0.15% Emulgen, 0.1 mM dithiothreitol, 0.1 mM EDTA, several weeks spectroscopically stable [9] , 25 C, 50 mM Tris, pH 7.2, 20% glycerol, 0.15% Emulgen, 0.1 mM dithiothreitol, 0.1 mM EDTA, several hours spectroscopically stable, without Emulgen 50% precipitation after 30 min [9]

"  [1] Ryan, K.J.; Engel, L.L.: Hydroxylation of steroids at carbon 21. J. Biol. Chem., 225, 103-114 (1957) [2] Hiwatashi, A.; Ichikawa, Y.: Purification and reconstitution of the steroid 21-hydroxylase system (cytochrome P-450-linked mixed function oxidase system) of bovine adrenocortical microsomes. Biochim. Biophys. Acta, 664, 33-48 (1981) [3] Greenfield, N.; Ponticorvo, L.; Chasalow, F.; Lieberman, S.: Activation and Inhibition of the adrenal steroid 21-hydroxylation system by cytosolic constituents: influence of glutathione, glutathione reductase, and ascorbate. Arch. Biochem. Biophys., 200, 232-244 (1980) [4] Haniu, M.; Yanagibashi, K.; Hall, P.F.; Shively, J.E.: Complete amino acid sequence of 21-hydroxylase cytochrome P-450 from porcine adrenal mircosomes. Arch. Biochem. Biophys., 254, 380-384 (1987)

306

1.14.99.10

Steroid 21-monooxygenase

[5] Narasimhulu, S.; Eddy, C.R.: Adrenal microsomal hydroxylating system: purification and substrate binding properties of cytochrome P-450C-21. Biochemistry, 24, 4287-4294 (1985) [6] Blom, S.; Förlin, L.; Andersson, T.B.: Characterisation of head kidney and liver microsomal 17-hydroxyprogesterone 21-hydrolase activity in rainbow trout (Oncorhynchus mykiss) and the lack of regulatory effects of ACTH. Fish Physiol. Biochem., 24, 1-8 (2001) [7] Belanger, A.; Tremblay, Y.; Vallee, M.; Provencher, P.H.; Perron, S.: Regulation of 21-hydroxylase activity by steroids. Endocr. Res., 21, 329-41 (1995) [8] Yuan, P.M.; Nakajin, S.; Haniu, M.; Shinoda, M.; Hall, P.F.; Shively, J.E.: Steroid 21-hydroxylase (cytochrome P-450) from porcine adrenocortica microsomes: microsequence analysis of cysteine-containing peptides. Biochemistry, 22, 143-149 (1983) [9] Kominami, S.; Ochi, H.; Kobayashi, Y.; Takemori, S.: Studies on the steroid hydroxylation system in adrenal cortex microsomes. J. Biol. Chem., 255, 3386-3394 (1980) [10] Bumpus, J. A.; Dus, K.: Bovine adrenocortical microsomal hemeproteins P45017a and P-450C-21: Isolation, partial characterization, and comparison to P-450SCC. J. Biol. Chem., 257, 12696-12704 (1982) [11] Higashi, Y; Yoshioka, H.; Yamane, M.; Gotoh, O.; Fuji-Kuriyama, Y.: complete nucleotide sequence of two steroid 21-hydroxylase genes tandemly arranged in human chromosomes: a pseudogene and a genuine gene. Proc. Natl. Acad. Sci. USA, 83, 2841-2845 (1986)

307

 4b    

::

   
 1.14.99.11    estradiol-17b,hydrogen-donor:oxygen oxidoreductase (6b-hydroxylating)    estradiol 6b-monooxygenase    EC 1.14.1.10 (formerly) EC 1.99.1.8 (formerly) estradiol 6b-hydroxylase oxygenase, estradiol 6b-mono
    9029-70-3

     Mus musculus [1] Gallus gallus [2]

!    "
 # 

estradiol-17b + AH2 + O2 = 6b-hydroxyestradiol-17b + A + H2 O    oxidation redox reaction reduction       estradiol-17b + NADPH + O2 ( estradiol-17b i.e. estra1,3,5(10)-triene-3,17b-diol [1]) (Reversibility: ? [1]) [1, 2] $ 6b-hydroxyestradiol-17b + NADP+ + H2 O ( 6b-hydroxyestradiol i.e. 6b-hydroxyestra-1,3,5(10)-triene-3,17b-diol [1]) [1]

308

1.14.99.11

Estradiol 6b-monooxygenase

     estradiol-17b + NADPH + O2 ( estradiol-17b i.e. estra1,3,5(10)-triene-3,17b-diol [1]) (Reversibility: ? [1]) [1, 2] $ 6b-hydroxyestradiol-17b + NADP+ + H2 O ( 6b-hydroxyestradiol i.e. 6b-hydroxyestra-1,3,5(10)-triene-3,17b-diol [1]) [1]
"%  NADPH [1, 2]

 2 %$ %' %

% liver [1, 2] 3#

microsome [1, 2] $"

[2]

"  [1] Muller, G.C.; Rumney, G.: Formation of 6b-hydroxy and 6-keto derivatives of estradiol-16-C14 by mouse liver microsomes. J. Am. Chem. Soc., 79, 10041005 (1957) [2] Hokama, Y.; Koga, N.; Yoshimura, H.: Purification and characterization of two forms of chicken liver cytochrome P-450 induced by 3,4,5,3',4'-pentachlorobiphenyl. J. Biochem., 104, 355-361 (1988)

309

     !(8      

::

   
 1.14.99.12    androst-4-ene-3,17-dione-hydrogen-donor:oxygen oxidoreductase (13-hydroxylating, lactonizing)    androst-4-ene-3,17-dione monooxygenase    4-androstene-3,17-dione monooxygenase androst-4-ene-3,17-dione 17-oxidoreductase androst-4-ene-3,17-dione hydroxylase androstene-3,17-dione hydroxylase androstenedione monooxygenase oxygenase, androstenedione mono
    37256-74-9

     Penicillium lilacinum [1] Cylindrocarpon radicicola (the steroid monooxygenase also has androstenedione monooxygenase activity [2]) [2] Rattus norvegicus [3]

!    "
 # 

androst-4-ene-3,17-dione + AH2 + O2 = 3-oxo-13,17-secoandrost-4-ene17,13a-lactone + A + H2 O    oxidation redox reaction reduction

310

1.14.99.12

Androst-4-ene-3,17-dione monooxygenase

     androst-4-ene-3,17-dione + NADPH + O2 (Reversibility: ? [1, 2]) [1, 2] $ 3-oxo-13,17-secoandrost-4-ene-17,13a-lactone + NADP+ + H2 O ( testololactone [1, 2]) [1, 2] 2  5-pregnene-3b,20a-diol [2] 5a-androstane-17b-ol ( exhibits an I50 of 0.046 mM against androstendione 6b-hydroxylase activity and an I50 of 0.026 against androstendione 16a-hydroxylase activity and an I50 of 0.047 against androstendione 16b-hydroxylase activity [3]) [3] 5a-androstane-3b-ol ( exhibits an I50 of 0.11 mM against androstendione 6b-hydroxylase activity and an I50 of 0.071 against androstendione 16a-hydroxylase activity [3]) [3] 5a-androstane-3b-ol-17-one ( exhibits an I50 of 0.12 mM against androstendione 6b-hydroxylase activity and an I50 of 0.14 against androstendione 16b-hydroxylase activity [3]) [3] 5b-androstane-17b-ol ( exhibits an I50 of 0.11 mM against androstendione 6b-hydroxylase activity and an I50 of 0.065 against androstendione 16a-hydroxylase activity [3]) [3] 5b-androstane-3-one-17-one ( exhibits an I50 of 0.084 mM against androstendione 7a-hydroxylase activity [3]) [3] 5b-androstane-3b-ol ( exhibits an I50 of 0.027 mM against androstendione 6b-hydroxylase activity and an I50 of 0.007 against androstendione 16a-hydroxylase activity [3]) [3] 5b-androstane-3b-ol-17-one ( exhibits an I50 of 0.047 mM against androstendione 6b-hydroxylase activity and an I50 of 0.06 mM against androstendione 7a-hydroxylase activity and an I50 of 0.051 against androstendione 16a-hydroxylase activity and an I50 of 0.087 against androstendione 16b-hydroxylase activity [3]) [3] 5b-androstane-3b-ol-17-one ( exhibits an I50 of 0.095 against androstendione 16a-hydroxylase activity [3]) [3] CuSO4 [1] ZnSO4 [1] dehydroepiandrosterone [2] p-mercuriphenylsulfonate ( in presence of NADPH [1]) [1] pregnenolone [2] progesterone [2] testosterone [2] testosterone acetate [2] Additional information ( 5b-reduced steroids are more potent inhibitors than 5a-reduced epimers [3]) [3]
"%  NADPH ( absolute requirement [1, 2]) [1, 2]

311

Androst-4-ene-3,17-dione monooxygenase

1.14.99.12

) &  * +, 27 (testololactone, formed [2]) [2] . / *', 0.002 (NADPH) [2] 0.04 (androst-4-ene-3,17-dione) [2] 0  6.5 [2] 0 

 5.7-7.8 ( about 50% of activity maximum at pH 5.7 and 7.8 [2]) [2] )  *
, 30 ( assay at [1]) [1]

 2 %$ %' %

% liver [3] mycelium [1] 3#

microsome [3] $"

[1]

"  [1] Prairie, R.L.; Talalay, P.: Enzymatic formation of testololactone. Biochemistry, 2, 203-208 (1963) [2] Itagaki, E.: Studies on steroid monooxygenase from Cylindrocarpon radicicola ATCC 11011. Oxygenative lactonization of androstenedione to testololactone. J. Biochem., 99, 825-832 (1986) [3] Murray, M.; Mehta, I.: Structure-activity relationships in the in vitro modulation of rat hepatic microsomal androst-4-ene-3,17,dione hydroxylase activities by derivatives of 5a- and 5b-androstane. J. Steroid Biochem., 35, 465471 (1999)

312

! 0   #     

::!

   
 1.14.99.13 (transferred to EC 1.14.13.23)    3-hydroxybenzoate 4-monooxygenase

313

$   a    

::

   
 1.14.99.14    progesterone,hydrogen-donor:oxygen oxidoreductase (11a-hydroxylating)    progesterone 11a-monooxygenase    progesterone 11a-hydroxylase
    37256-77-2

     Aspergillus ochraceus (strain NRRL 405 [1, 4]; strain TS [3, 4, 9]; strain G8 [9]) [1, 3, 4, 9] Bacillus megaterium [1] Rhizopus arrhizus [4] Rhizopus nigricans (ATCC 6277b [2, 5-7]) [2, 4-10] Rhizopus stolonifer [9]

!    "
 # 

progesterone + AH2 + O2 = 11a-hydroxyprogesterone + A + H2 O    oxidation redox reaction reduction       progesterone + NADPH + O2 ( activity dependent upon NADPH and O2 [4]; necessary step in the synthesis of cortico-steroid hormones [7]; multicomponent monooxygenase system [10]) (Reversibility: ? [1-10]) [1-10] $ 11a-hydroxyprogesterone + NADP+ + H2 O [1-10] 314

1.14.99.14

Progesterone 11a-monooxygenase

     11a-hydroxyprogesterone + NADPH + O2 (Reversibility: ? [1]) [1] $ 6b,11a-dihydroxyprogesterone [1]  19-nortestosterone + NADPH + O2 (Reversibility: ? [1]) [1] $ 11a-hydroxy-19-nortestosterone + NADP+ + H2 O  progesterone + NADPH + O2 (Reversibility: ? [1-10]) [1-10] $ 11a-hydroxyprogesterone + NADP+ + H2 O [1-10]  Additional information ( 11a-hydroxylase induced by progesterone or 19-nortestosterone is free from associated 6b-hydroxylase or 17b-hydroxysteroid dehydrogenase activity, but induction with 11a-hydroxyprogesterone yields an enzyme with both 6b- and 11a-hydroxylase activity [1]; multi-enzyme complex contains cytochrome P450 and NADPH cytochrome c reductase [7]) [1, 7] $ ? 2  N-methyl maleimide [4] SKF-525A [4] carbon monoxide [4] cytochrome c [4] ketoconazole [10] metyrapone [3, 4, 10] p-chloromercuribenzoate [4]
"%  NADH ( NADH is far less efficient than NADPH [4]) [4] NADPH [4] cytochrome P450 [2-6, 8, 10] &

  11a-hydroxyprogesterone ( induced by [1]) [1] corn steep liquor [9] cyanide [3] ethanol [9] isopropanol [9] malt extract [9] methanol [9] n-butanol [9] nortestosterone ( induced by [1]) [1] peptone [9] phenol [9] progesterone ( best inducer [1]) [1] sodium-meta-periodate [3] tryptone [9] " & *-% , 0.0484 ( cytosol [4]) [4] 0.388 ( microsomes [4]) [4] 315

Progesterone 11a-monooxygenase

1.14.99.14

0  7.7 [4] )  *
, 22 [8]

  #     dimer ( 2 components, rhizoporedoxin and rhizoporedoxin reductase, DEAE-cellulose chromatography [5]) [5]

 2 %$ %' %

% conidium [9] mycelium [1, 4-6, 8, 9] 3#

cytosol [4, 8] endoplasmic reticulum [6, 7] membrane [5-7, 10] microsome [4, 5, 7] $"

(enzyme complex [7]) [6, 7] 

synthesis ( membrane-associated enzymes catalyse several reactions of industrial interest, including steroid hydroxylation [6, 7]; immobilisation of membrane-bound multi-enzyme complexes for industrial use [7]) [6, 7]

4  )  30 ( loses potency at higher temperatures [1]) [1] 5    "

, immobilization often results in loss of enzyme activity [9] , resistant to DNase [8]

316

1.14.99.14

Progesterone 11a-monooxygenase

"  [1] Shibahara, M.; Moody, J.A.; Smith, L.L.: Microbial hydroxylations. V. 11ahydroxylation of progesterone by cell-free preparations of Aspergillus ochraceus. Biochim. Biophys. Acta, 202, 172-179 (1979) [2] Hanisch, W.H.; Dunnill, P.: Regeneration of the progesterone 11a-hydroxylase of Rhizopus nigricans by the use of periodate. Biotechnol. Lett., 2, 123126 (1980) [3] Ghosh, D.; Samanta, T.B.: 11 a-Hydroxylation of progesterone by cell free preparation of Aspergillus ochraceus TS. J. Steroid Biochem., 14, 1063-1067 (1981) [4] Jayanthi, C.R.; Madyastha, P.; Madyastha, K.M.: Microsomal 11a-hydroxylation of progesterone in Aspergillus ochraceus: Part I: Characterization of the hydroxylase system. Biochem. Biophys. Res. Commun., 106, 1262-1268 (1982) [5] Cresnar, B.; Breskvar, K.; Hudnik-Plevnik, T.: Resolution and reconstitution of the NADPH-cytochrome c (P-450) reductase induced by progesterone in Rhizopus nigricans. Biochem. Biophys. Res. Commun., 133, 1057-1063 (1985) [6] Broad, D.F.; Pontin, S.; Dunnill, P.: Studies for the large scale isolation of membrane-associated progesterone 11a-hydroxylase:enzyme stability in Rhizopus nigricans ATCC 6277b cells. Enzyme Microb. Technol., 9, 546548 (1986) [7] Bonnerjea, J.; Pontin, S.; Hoare, M.; Dunnill, P.: Poly(ethylene glycol) precipitation and two aquaeous-phase separation of progesterone 11-a hydroxylase from Rhizopus nigricans. Appl. Microbiol. Biotechnol., 27, 362-365 (1988) [8] Lenasi, H.; Hudnik-Plevnik, T.: Identification and partial characterization of cytosolic progesterone-binding sites in the filamentous fungus Rhizopus nigricans. Arch. Biochem. Biophys., 330, 80-86 (1996) [9] Dutta, T.K.; Samanta, T.B.: Bioconversion of progesterone by the activated immobilized conidia of Aspergillus ochraceus TS. Curr. Microbiol., 39, 309312 (1999) [10] Kunic, B.; Makovec, T.; Breskvar, K.: Comparison of two monooxygenase systems with cytochrome P450 in filamentous fungus Rhizopus nigricans. Pflugers Arch., 439, R107-108 (2000)

317

 '  #     *  

,

::

   
 1.14.99.15    4-methoxybenzoate,hydrogen-donor:oxygen oxidoreductase (O-demethylating)    4-methoxybenzoate monooxygenase (O-demethylating)    4-methoxybenzoate 4-monooxygenase (O-demethylating) 4-methoxybenzoate O-demethylase oxygenase, 4-methoxybenzoate 4-mono- (O-demethylating) p-anisic O-demethylase piperonylate-4-O-demethylase
    37256-78-3

     Pseudomonas putida [1-10]

!    "
 # 

4-methoxybenzoate + AH2 + O2 = 4-hydroxybenzoate + formaldehyde + A + H2 O ( the bacterial enzyme is a two-component enzyme, consisting of an iron-sulfur flavoprotein (FMN), NADH-putidamonooxin-reductase and a ferredoxin-type, oxygen-activating protein, putidamonooxin [1, 2]; a terminal oxygenase [3])    N-demethylation O-demethylation S-demethylation dealkylation

318

1.14.99.15

4-Methoxybenzoate monooxygenase (O-demethylating)

oxidation redox reaction reduction       4-methoxybenzoate + NADH + O2 (Reversibility: ? [1-3, 5-9, 10]) [1-3, 5-9, 10] $ 4-hydroxybenzoate + NAD+ + H2 O + formaldehyde      3,4-dimethoxybenzoate + NADH + O2 (Reversibility: ? [1, 9]) [1, 9] $ 4-hydroxy-3-methoxybenzoate + NAD+ + H2 O + formaldehyde  3,4-methylenedioxybenzoate + NADH + O2 ( piperonylate [1, 9]) (Reversibility: ? [1, 9]) [1, 9] $ 3,4-dihydroxybenzoate + NAD+ + H2 O + formaldehyde ( protocatechuate [1, 9]) [1, 9]  3-hydroxybenzoate + NADH + O2 ( partial uncoupler [1, 2, 9]) (Reversibility: ? [1, 2, 9]) [1, 2, 9] $ 3,4-dihydroxybenzoate + NAD+ + H2 O ( in the uncoupled part of the reaction, 3-hydroxybenzoate is not hydroxylated and H2 O2 is a product, too [1, 2, 9]) [1, 2, 9]  3-methoxybenzoate + NADH + O2 ( partial uncoupler [1, 9]) (Reversibility: ? [1, 9]) [1, 9] $ 3-hydroxybenzoate + NAD+ + H2 O + formaldehyde ( in the uncoupled part of the reaction 3-methoxybenzoate is not hydroxylated, and H2 O2 is a product, too [1]) [1, 9]  3-nitro-4-methoxybenzoate + NADH + O2 (Reversibility: ? [1, 2, 5-7, 10]) [1, 2, 5-7, 10] $ 3-nitro-4-hydroxybenzoate + NAD+ + H2 O + formaldehyde  3-phenyl-4-methoxybenzoate + NADH + O2 ( partial uncoupler [1]) (Reversibility: ? [1, 10]) [1, 10] $ 3-phenyl-4-hydroxybenzoate + NAD+ + H2 O + formaldehyde ( in the uncoupled part of the reaction 3-phenyl-4-methoxybenzoate is not hydroxylated and H2 O2 is also a product of the reaction [1]) [1]  4-aminobenzoate + NADH + O2 (Reversibility: ? [1, 3]) [1, 3] $ 4-amino-3-hydroxybenzoate + NAD+ + H2 O  4-ethoxybenzoate + NADH + O2 (Reversibility: ? [1, 8, 9]) [1, 8, 9] $ 4-hydroxybenzoate + acetaldehyde + NAD+ + H2 O  4-hydroxy-3-methoxybenzoate + NADH + O2 ( vanillate, partial uncoupler [1,9]) (Reversibility: ? [1, 9]) [1, 9] $ ?  4-hydroxybenzoate + NADH + O2 (Reversibility: ? [1-3, 5, 6, 8]) [1-3, 5, 6, 8] $ 3,4-dihydroxybenzoate + NAD+ + H2 O ( ring hydroxylation [5]) [1-3, 5, 6, 8]

319

4-Methoxybenzoate monooxygenase (O-demethylating)

1.14.99.15

 4-methoxybenzoate + NADH + O2 (Reversibility: ? [1-3, 5-10]) [1-3, 5-10] $ 4-hydroxybenzoate + NAD+ + H2 O + formaldehyde  4-methylbenzoate + NADH + O2 ( p-toluate, partial uncoupler [1, 3, 6]) (Reversibility: ? [1, 3, 6, 10]) [1, 3, 6, 10] $ 4-carboxybenzylalcohol + NAD+ + H2 O ( in the uncoupled part of the reaction, p-toluate is not hydroxylated and H2 O2 is a product, too [1, 3, 6]) [1, 3, 6]  4-methylmercaptobenzoate + NADH + O2 (Reversibility: ? [1]) [1] $ ?  4-trifluoromethylbenzoate + NADH + O2 (Reversibility: ? [5, 6, 10]) [5, 6, 10] $ ?  4-vinylbenzoate + NADH + O2 ( external dioxygenase reaction by substrate induced modulation [1,2]) (Reversibility: ? [1, 2]) [1, 2] $ 4-glycylbenzoate + NAD+ + H2 O  N,N'-dimethyl-4-aminobenzoate + NADH + O2 (Reversibility: ? [1, 9]) [1, 9] $ 4-aminobenzoate + NAD+ + H2 O + formaldehyde  N-methyl-4-aminobenzoate + NADH + O2 (Reversibility: ? [1, 69]) [1, 6-9] $ 4-aminobenzoate + NAD+ + H2 O + formaldehyde  alkylbenzoates (Reversibility: ? [9]) [9] $ ?  benzoate + NADH + O2 (Reversibility: ? [10]) [10] $ ?  Additional information ( overview: substrates being absolutely planar aromatic rings with a directly bound dissociable carboxy group are oxygenated under stoichiometric consumption of O2 and NADH [1]; overview: substrates that are not oxygenized while NADH-oxidation and O2 -consumption are catalyzed, such as: benzoate, 3-chlorobenzoate, 4-chlorobenzoate, 2-hydroxybenzoate, 4-bromobenzoate, 2-aminobenzoate, 3-aminobenzoate, 4-trifluoromethylbenzoate, 4-tert-butylbenzoate [1-4, 6]) [1-4, 6, 9] $ Additional information ( uncoupling substrates are not oxygenized, NAD+ and H2 O2 being the only products of the reaction [1-4, 6, 9]) [1-4, 6, 9] 2  1,10-phenanthroline [7] 2,2'-dipyridyl [7] 3-methoxybenzoate [9] 4-(2-pyridylazo)resorcinol [5] 4-tert-butylbenzoate ( competitive inhibitor of the O-demethylation of 3-nitro-4-methoxybenzoate, hinders O2 -binding or O2 -activation [5]) [5] 4-trifluoromethylbenzoate [9]

320

1.14.99.15

4-Methoxybenzoate monooxygenase (O-demethylating)

5,5'-dithiobis(2-nitrobenzoate) [7] 8-hydroxyquinoline [5, 7] Cd2+ [5] Co2+ [5] Cu2+ [5] EDTA [7] Hg2+ [5] KCN [7] Ni2+ [5] Zn2+ [5] amytal [5, 7] atebrin [5, 7] bathocuproinedisulfonate [5, 7] bathophenanthrolinedisulfate ( preferentially inhibiting putidamonooxin [5]) [5, 7] benzoate ( competitive inhibition [9]) [9] cumylhydroperoxide [5] diethyldithiocarbamate [7] iodosobenzene [5] oxidized putidamonooxin ( 60% inhibition with 3-nitro-4-methoxybenzoate as substrate, fully reactivated by Fe2+ and sulfhydryl-reagents [3]) [3] p-chloromercuribenzoate ( reversible by GSH [7]) [5, 7] rotenone [5, 7] thenoyl trifluoroacetone [7]
"%  FMN ( prosthetic group of the oxidoreductase [3]) [1, 3, 5, 7-9] NADH ( requirement, can be replaced by NADPH with 40% efficiency [1, 5]) [1-5, 7-9] NADPH ( can replace NADH with 40% efficiency [1, 5]) [1, 5] &

  putidamonooxin ( essential part of the enzyme system, O2 -activating 2Fe-2S-protein, identified by EPR and Mössbauer spectroscopy [1, 3]) [19] '(   Fe2+ ( mononuclear non-heme iron protein, 2Fe-2S cluster [1, 3]; both enzyme components are iron-sulfur proteins [1-9]) [1-9] " & *-% , 0.002 ( 4-hyroxybenzoate, reductase [9]) [9] 0.004 ( 4-hydroxybenzoate, monooxygenase [9]) [9] 0.008 ( 3-methoxybenzoate, 4-methoxybenzoate, cell-free extract [9]) [9] 0.009 ( N,N-dimethyl-4-aminobenzoate [9]) [9] 0.018 ( superoxide anion instead of substrate, putidamonooxin [5]) [5] 321

4-Methoxybenzoate monooxygenase (O-demethylating)

1.14.99.15

0.021 ( 4-hydroxy-3-methoxybenzoate [9]) [9] 0.023 ( 3-hydroxybenzoate, reconstituted enzyme [9]) [9] 0.024 ( 4-ethoxybenzoate, N-methyl-4-aminobenzoate [9]) [9] 0.025 ( 3,4-dimethoxybenzoate [9]) [9] 0.04 ( 4-methoxybenzoate, cell-free extract [9]) [9] 0.055 ( 4-hydroxybenzoate [9]) [9] 0.077 ( 4-hydroxybenzoate, reconstituted enzyme [9]) [9] 0.11 ( 3-methoxybenzoate [9]) [9] 0.178 ( N-methyl-4-aminobenzoate [9]) [9] 0.192 ( 3-nitro-4-methoxybenzoate, putidamonooxin [5]) [5] 0.27 ( 4-methoxybenzoate, putidamonooxin [5]) [5] 0.314 ( 4-ethoxybenzoate, piperonylate [9]) [9] 0.345 ( 3,4-dimethoxybenzoate [9]) [9] 0.36 ( 4-methoxybenzoate [9]) [9] 10.97 [7] 21.96 ( 4-methoxybenzoate [7]) [7] 194.5 ( 3-phenyl-4-[2H3]-methoxybenzoate, product formation, reaction in H2 O [10]) [10] 207.8 ( 4-trifluoromethylbenzoate, oxygen uptake, reaction in D2 O [10]) [10] 239.8 ( 3-phenyl-4-[2H3]-methoxybenzoate, product formation, reaction in D2 O [10]) [10] 265.6 ( benzoate, oxygen uptake, reaction in D2 O [10]) [10] 295.1 ( benzoate, oxygen uptake, reaction in H2 O [10]) [10] 303.9 ( 3-phenyl-4-[1H3]-methoxybenzoate, product formation, reaction in H2 O [10]) [10] 305.2 ( 4-methylbenzoate, NADH oxidation, reaction in H2 O [10]) [10] 310.1 ( 3-phenyl-4-[2H3]-methoxybenzoate, oxygen uptake, reaction in D2 O [10]) [10] 310.1 ( 3-phenyl-4-[2H3]-methoxybenzoate, oxygen uptake, reaction in H2 O [10]) [10] 315.5 ( 4-trifluoromethylbenzoate, NADH oxidation, reaction in D2 O [10]) [10] 317.2 ( 4-methylbenzoate, NADH oxidation, reaction in D2 O [10]) [10] 317.4 ( 3-phenyl-4-[1H3]-methoxybenzoate, product formation, reaction in D2 O [10]) [10] 326.7 ( 3-phenyl-4-[1H3]-methoxybenzoate, oxygen uptake, reaction in H2 O [10]) [10] 329.2 ( 3-phenyl-4-[1H3]-methoxybenzoate, oxygen uptake, reaction in D2 O [10]) [10] 339.1 ( benzoate, NADH oxidation, reaction in D2 O [10]) [10] 340.2 ( 3-phenyl-4-[2H3]-methoxybenzoate, NADH oxidation, reaction in H2 O [10]) [10] 345 ( 4-trifluoromethylbenzoate, oxygen uptake, reaction in H2 O [10]) [10] 322

1.14.99.15

4-Methoxybenzoate monooxygenase (O-demethylating)

345.6 ( benzoate, NADH oxidation, reaction in H2 O [10]) [10] 353.5 ( 3-phenyl-4-[2H3]-methoxybenzoate, NADH oxidation, reaction in D2 O [10]) [10] 360.4 ( 3-phenyl-4-[1H3]-methoxybenzoate, NADH oxidation, reaction in H2 O [10]) [10] 367 ( 4-methylbenzoate, oxygen uptake, reaction in H2 O [10]) [10] 368 ( 4-methylbenzoate, oxygen uptake, reaction in D2 O [10]) [10] 380.2 ( 3-phenyl-4-[1H3]-methoxybenzoate, NADH oxidation, reaction in D2 O [10]) [10] 391.5 ( 4-methoxybenzoate, oxygen uptake, reaction in D2 O [10]) [10] 394.1 ( 4-methoxybenzoate, oxygen uptake, reaction in H2 O [10]) [10] 402.6 ( 3-nitro-4-methoxybenzoate, NADH oxidation, reaction in D2 O [10]) [10] 409.7 ( 4-trifluoromethylbenzoate, NADH oxidation, reaction in H2 O [10]) [10] 422.1 ( 3-nitro-4-methoxybenzoate, NADH oxidation, reaction in H2 O [10]) [10] 432.4 ( 3-nitro-4-methoxybenzoate, oxygen uptake, reaction in H2 O [10]) [10] 432.7 ( 3-nitro-4-methoxybenzoate, oxygen uptake, reaction in D2 O [10]) [10] 442.2 ( 4-methoxybenzoate, NADH oxidation, reaction in D2 O [10]) [10] 445 ( 4-methoxybenzoate, NADH oxidation, reaction in H2 O [10]) [10] 450.9 ( 3-nitro-4-methoxybenzoate, product formation, reaction in H2 O [10]) [10] 487.3 ( 3-nitro-4-methoxybenzoate, product formation, reaction in D2 O [10]) [10] . / *', 7e-005 (4-methoxybenzoate) [5, 6] 0.0003 (3-nitro-4-methoxybenzoate) [5, 6] 0.00063 (NADH, reconstituted enzyme [1, 5]) [1, 5] 0.0007 (3-nitro-4-methoxybenzoate) [7] 0.0014 (4-methoxybenzoate) [7, 9] 0.0019 (O2, + 4-methoxybenzoate [9]) [9] 0.0038 (O2, + 3,4-dimethoxybenzoate [9]) [9] 0.0058 (4-methylaminobenzoate) [5, 6] 0.0062 (4-methylbenzoate, reaction in D2 O [10]) [10] 0.008 (NADH, reductase [7]) [7] 0.009 (4-methylbenzoate, reaction in H2 O [10]) [5, 6, 10] 0.01 (O2, + 4-ethoxybenzoate [9]) [9] 0.0144 (4-trifluoromethylbenzoate, reaction in D2 O [10]) [10] 0.024 (4-trifluoromethylbenzoate, reaction in H2 O [10]) [5, 6, 10]

323

4-Methoxybenzoate monooxygenase (O-demethylating)

1.14.99.15

0.029 (4-hydroxybenzoate) [5, 6] 0.03 (putidamonooxin) [1] 0.0367 (benzoate, reaction in H2 O [10]) [10] 0.051 (O2, + N-methyl-4-aminobenzoate [9]) [9] 0.055 (O2, + 4-methylbenzoate [9]; + benzoate [5, 6]) [5, 6, 9] 0.055 (benzoate, reaction in D2 O [10]) [10] 0.077 (N-methyl-4-aminobenzoate) [7, 9] 0.14 (NADPH, reconstituted enzyme [1, 5]) [1, 5] 0  8 [1, 7, 9] 0 

 7.3-9 ( about half-maximal activity at pH 7.3 and 9.0, reductase [7]) [7] )  *
, 30 ( assay at [1-5, 7-9]) [1-5, 7-9]

  #     dimer ( a2 , 2 * 52000, putidamonooxin, SDS-PAGE [8]) [8] oligomer ( 3-4 * 41500, putidamonooxin, SDS-PAGE [1, 6]; 3-4 * 33000-45000, putidamonooxin, SDS-PAGE [6]) [1, 6] $   "

glycoprotein ( 30% carbohydrate in each of the 50000 Da subunits [7]) [7]

 2 %$ %' %

$"

[1, 6, 7]

4    , aerobic conditions, 0-4 C, 50% activity lost within 24 h [1] , putidamonooxin and NADH-reductase, extremely O2 -sensitive, GSH, DTT, 2-mercaptoethanol prevent putidamonooxin oxidation of putdamonooxin [3] , putidamonooxin and NADH-reductase, extremely O2 -sensitive, purification and storage of the reductase under anaerobic conditions [7]

324

1.14.99.15

4-Methoxybenzoate monooxygenase (O-demethylating)

5    "

, 4-methoxybenzoate, stabilizes [1] , NADH, not NADPH or substrate stabilize the reductase [9] , ethanol, 5-15% v/v, stabilizes activity in buffer and crude extract [1, 7] , substrate or substrate analogues stabilize putidamomooxin by preventing loss of Fe2+ [3, 9]    , -20 C, freeze-dried partially purified reductase stable for months without loss of activity, purified reductase is 3-4 weeks stable after addition of NADH in N2 -atmosphere [1, 7] , 0-4 C, more than 24 days stable in crude extract under anaerobic conditions [1] , 4 C, concentrated putidamonooxin stable for several months in N2 -atmosphere with DTT and dithionite [7]

"  [1] Bernhardt, F.H.; Bill, E.; Trautwein, A.X.; Twilfer, H.: 4-Methoxybenzoate monooxygenase from Pseudomonas putida: isolation, biochemical properties, substrate specificity, and reaction mechanisms of the enzyme components. Methods Enzymol., 161, 281-294 (1988) [2] Wende, P.; Pfleger, K.; Bernhardt, F.H.: Dioxygen activation by putidamonooxin: substrate-modulated reaction of activated dioxygen. Biochem. Biophys. Res. Commun., 140, 527-532 (1982) [3] Twilfer, H.; Bernhardt, F.H.; Gersonde, K.: An electron-spin-resonance study on the redox-active centers of the 4-methoxybenzoate monooxygenase from Pseudomonas putida. Eur. J. Biochem., 119, 595-602 (1981) [4] Bernhardt, F.H.; Kuthan, H.: Dioxygen activation by putidamonooxin. The oxygen species formed and released under uncoupling conditions. Eur. J. Biochem., 120, 547-555 (1981) [5] Bernhardt, F.H.; Nastainczyk, W.; Seydewitz, U.: Kinetic studies on a 4methoxybenzoate O-demethylase from Pseudomonas putida. Eur. J. Biochem., 72, 107-115 (1977) [6] Bernhardt, F.H.; Heymann, E.; Traylor, P.S.: Chemical and spectral properties of putidamonooxin, the iron-containing and acid-labile-sulfur-containing monooxygenase of a 4-methoxybenzoate O-demethylase from Pseudomonas putida. Eur. J. Biochem., 92, 209-223 (1978) [7] Bernhardt, F.H.; Pachowsky, H.; Staudinger, H.: A 4-methoxybenzoate Odemethylase from Pseudomonas putida. A new type of monooxygenase system. Eur. J. Biochem., 57, 241-256 (1975) [8] Bernhardt, F.H.; Ruf, H.H.; Ehrig, H.: A 4-methoxybenzoate monooxygenase system from Pseudomonas putida. Circular dichroism studies on the iron±sulfur protein. FEBS Lett., 43, 53-55 (1974) [9] Bernhardt, F.H.; Erdin, N.; Staudinger, H.; Ullrich, V.: Interactions of substrates with a purified 4-methoxybenzoate monooxygenase system (O-de-

325

4-Methoxybenzoate monooxygenase (O-demethylating)

1.14.99.15

methylating) from Pseudomonas putida. Eur. J. Biochem., 35, 126-134 (1973) [10] Twilfer, H.; Sandfort, G; Bernhardt, F.H.: Substrate and solvent isotope effects on the fate of the active oxygen species in substrate-modulated reactions of putidamonooxin. Eur. J. Biochem., 267, 5926-5934 (2000)

326

'     

::4

   
 1.14.99.16 (transferred to EC 1.14.13.72)    methylsterol monooxygenase

327

5       

   
 1.14.99.17 (transferred to EC 1.14.16.5)    glyceryl-ether monooxygenase

328

::8


'$         

::7

   
 1.14.99.18 (shortly after the editorial deadline this EC number was deleted by the IUBMB)    N-acetylneuraminate,hydrogen-donor:oxygen oxidoreductase (N-acetyl-hydroxylating)    CMP-N-acetylneuraminate monooxygenase    CMP-N-acetylneuraminic acid:NADH oxidoreductase (N-acetyl-hydroxylating) CMP-Neu5Ac hydroxylase N-acetylneuraminate,hydrogen-donor:oxygen oxidoreductase (N-acetyl-hydroxylating) N-acetylneuraminic monooxygenase cytidine-5'-monophosphate-N-acetylneuraminic acid hydroxylase oxygenase, cytidine monophosphoacetylneuraminate mono
    116036-67-0

     no activity in Homo sapiens (the CMAH gene is inactivated shortly before the time when the brain expansion began in humankind's ancestry, 2.1-2.2 million years ago [11]; the human CMP-N-acetylneuraminic acid hydroxylase is inactive because of a partial deletion in the hydroxylase gene [12]) [11, 12] Sus scrofa [1, 6, 7, 17, 18, 21, 24] Mus musculus [2, 3, 4, 7, 9, 10, 12, 14, 15, 16, 22, 23] Asterias rubens [5, 8, 13, 19, 20] Bos taurus [7] Rattus norvegicus [23]

329

!    "
 # 

CMP-N-acetylneuraminate + AH2 + O2 = CMP-N-glycoloylneuraminate + A + H2 O    oxidation redox reaction reduction       CMP-N-acetylneuraminate + NADH + O2 (, the enzyme is the key for regulation of the overall velocity of CMP-NeuAc hydroxylation and consequently for the expression of N-glycoloylneuraminic acid glycoconjugates [9]; , binding of CMP-N-acetylneuraminate to CMP-Nacetylneuraminate hydroxylase changes conformation of the enzyme so as to construct a recognition site for cytochrome b5, followed by the formation of a ternary complex through this domain. Then the transport of electrons from NAD(P)H to the enzyme through cytochrome b5 takes place, CMP-N-acetylneuraminate is converted to CMP-N-glycoloylneuraminic acid and finally the ternary complex dissociates into its components to release CMP-N-glycoloylneuraminic acid [14]; , a regulation of CMP-N-acetylneuraminate hydroxylation and thus the ratio of glycoconjugate-bound N-acetylneuraminate and N-acetylglycoloylneuraminate might occur by varying the amount of hydroxylase protein within the cell, possibly by controlling the expression of the hydroxylase gene [15]; , key enzyme for the expression of N-glycoloylneuraminic acid [16]; , the biosynthesis of the sialic acid N-glycolylneuraminic acid occurs by the action of cytidine monophosphate-N-acetylneuraminate hydroxylase. Incorporation of N-glycoloylneuraminic acid into glycoconjugates is generally controlled by the amount of hydroxylase protein expressed in a tissue [17]; , the enzyme plays a decisive role in governing the relative amounts of N-acetylneuraminate and N-acetylglycolylneuraminate occuring in the glycoconjugates of a tissue [23]) (Reversibility: ? [1, 2, 7, 8, 9, 14, 15, 16, 17, 23]) [1, 2, 7, 8, 9, 14, 15, 16, 17, 23] $ CMP-N-glycoloylneuraminate + NAD+ + H2 O      CMP-N-acetylneuraminate + 6,7-dimethyl-5,6,7,8-tetrahydrobiopterin + O2 (Reversibility: ? [1]) [1] $ CMP-N-glycoloylneuraminate + ?  CMP-N-acetylneuraminate + NADH + O2 (, NADPH and NADH are by far the most effective cofactors [1]) (Reversibility: ? [1, 2, 3, 7, 8, 9, 13, 15, 17, 19, 20, 21, 22]) [1, 2, 3, 7, 8, 9, 13, 15, 17, 19, 20, 21, 22] $ CMP-N-glycoloylneuraminate + NAD+ + H2 O

330

 CMP-N-acetylneuraminate + NADPH + O2 (, NADPH and NADH are by far the most effective cofactors [1]) (Reversibility: ? [1, 2, 3]) [1, 2, 3] $ CMP-N-glycoloylneuraminate + NADP+ + H2 O  CMP-N-acetylneuraminate + ascorbic acid + O2 (, ascorbate is ineffective [2]) (Reversibility: ? [1]) [1] $ CMP-N-glycoloylneuraminate + dehydroascorbate + H2 O  Additional information (, no activity towards free or a-glycosidically bound N-acetylneuraminic acid [4]) [4] $ ? 2  1,10-phenanthroline (, 2 mM, 88% inhibition [19]; , 5 mM, complete inhibition [22]) [13, 19, 20, 21, 22] 2,2'-dipyridyl (, 2 mM, 27% inhibition [19]) [19, 20] CHAPS (, 15 mM, complete inhibition [13]) [13] CMP-N-glycoloylneuraminate [22] Ca2+ (, 0.5 mM, 12% inhibition [13]) [13] Co2+ [2] Cu2+ [2] EDTA [2, 12] Hg2+ (, slight inhibition [13]) [13] KCN (, 2 mM, 80% inhibition [19]; , 5 mM, 49% inhibition [22]) [13, 19, 21, 22] Mg2+ [2] Mn2+ (, 0.5, slight inhibition [13]) [2, 13] Na2 HPO4 (, 2 mM, 86% inhibition [22]) [22] Na4 P2 O7 (, 2 mM, 54% loss of activity [22]) [22] Ni2+ [2] Tiron (, 5 mM, 82% inhibition [22]) [13, 21, 22] Zn2+ [2] Zwittergent 3-12 (, 5 mM, 40% inhibition [13]) [13] anti-(rat cytochrome b5 ) antiserum [7, 22] azide [13] cardiolipin (, 3 mM, 15% inhibition [22]) [22] cholic acid (, 10 mM, 89% inhibition [13]) [13] decylglucopyranoside (, 5 mM, 22% inhibition [13]) [13] ferrozine (, 2 mM, 83% inhibition [19]; , 0.7 mM, 82% inhibition [22]) [13, 19, 20, 21, 22] octylglucopyranoside (, 30 mM, 48% inhibition [13]) [13] phosphatidic acid (, 3 mM, 20% inhibition [22]) [22] phosphatidylinositol (, 3 mM, 68% inhibition [22]) [22] Additional information (, not inhibited by increased ionic strength, no inhibition by 1 M NaCl [13]) [13]
"%  NADH (, NADPH and NADH are by far the most effective cofactors [1]; , NADH is much more effective than NADPH [3]; , 331

most effective cofactor, optimal activity at 0.4 mM NADH, higher concentrations are slightly inhibitory [13]) [1, 2, 3, 7, 8, 9, 13, 15, 17, 19, 20, 21, 22] NADPH (, NADPH and NADH are by far the most effective cofactors [1]; , NADH is much more effective than NADPH [3]) [1, 2, 3] cytochrome b5 (, electron carrier, essential for activity [22]) [22] &

  Nonidet P-40 (, effective inhibitor [22]) [22] SDS (, 1 mM, modest activation [22]) [22] Triton X-100 (, effective inhibitor [22]) [22] ascorbate (, activates [13]) [13] decyl glucoside (, activation [22]) [22] dithiothreitol (, activates [13]) [13] glutathione (, activates [13]) [13] octanoic acid (, 1 mM, modest activation [22]) [22] octyl glucoside (, effective inhibitor [22]) [22] Additional information (, no activation by non-ionic detergents [13]; , highest activity in 50 mM Hepes buffer, significant inhibition at increasing concentrations [22]) [13, 22] '(   Ca2+ (, slight activation [2]) [2] Fe2+ (, absolute requirement [1]; , 0.2 mM, enhances activity [2]; , 0.5 mM FeSO4 enhances activity [13]; , 1 mM, activates [19]; , FeSO4 activates, optimal concentration is 0.5 mM [21]) [1, 2, 13, 19, 21] Fe3+ (,0.2 mM, enhances activity [2]; , enhances activity [13]) [2, 13] NaCl (, optimal activity in the presence of 100 mM NaCl [19]; , 100 mM, 3.5-fold increase in activity, optimal activity [20]) [19, 20] iron (, iron-sulfur protein of the Rieske type [6]; , contains non-heme iron as an electron acceptor [9]; , the enzyme contains a nonhaem iron cofactor [13]) [6, 9, 13] " & *-% , 0.00093 [20] 0.126 [15] 0.816 [21] 6.8 [9] . / *', 0.003 (CMP-N-acetylneuraminate) [21] 0.005 (CMP-N-acetylneuraminate, , amphiphilic system [15]) [9, 15] 0.0072 (CMP-N-acetylneuraminate) [19] 0.013 (CMP-N-acetylneuraminate, , soluble system [15]) [15] 0.018 (CMP-N-acetylneuraminate) [13] 0.6-2.6 (CMP-N-acetylneuraminate) [4] Additional information [2]

332

0  6-6.4 [13] 6-6.6 [19] 6.8-7.4 [22] 0 

 5.6-6.8 (, about 60% of maximal activity at pH 5.6 and pH 6.8 [13]) [13] )  *
, 22-27 [19] 25-33 [13] ) 

 *
, 15-43 (, 15 C: about 55% of maximal activity, 43 C: about 30% of maximal activity [13]) [13]

  #   ' 1  17000 (, 56000 (, 58000 (, 60000 (,

gel gel gel gel

filtration [22]) [22] filtration [7]) [7] filtration [9, 15]) [9, 15] filtration [21]) [21]

  monomer (, 1 * 64000, SDS-PAGE [9, 15]; , 1 * 65000, SDSPAGE [21]) [9, 15, 21]

 2 %$ %' %

% alimentary canal [13] body wall [13] brain (, the CMAH gene is inactivated shortly before the time when the brain expansion began in humankind's ancestry, 2.1-2.2 million years ago [11]) [11] gonad [13, 19, 20] heart (, weak activity [17]) [17] kidney (, weak activity [17]) [17] liver (, weak activity [17]) [2, 3, 7, 9, 10, 14, 15, 16, 17, 22, 23] lung [17] lymph node [17, 18] lymphocyte (, from thymus, spleen, lymph node and peripheral blood. Highest activity in peripheral blood lymphocytes [18]) [18] myeloma cell [4]

333

small intestine (, no significant temporal alterations in the activity in foetal and newborn small intestine. Birth is followed by a 2-8fold decrease in activity, depending on the region of the small intestine. Increase in activity from duodenum to ileum [24]) [17, 24] spleen [17] submandibular gland [1, 6, 7, 17, 21] thymus [17] 3#

cytosol (, full-length enzyme with normal enzymatic activity [16]; , in the vicinity of the nuclear membrane and the outer membrane of the mitochondria [18]) [3, 4, 6, 9, 10, 16, 18] endoplasmic reticulum (, naturally occuring truncated protein lacking 46 amino acids in the middle of the normal full-length protein [16]) [16] membrane (, bound to [8, 19]) [8, 19] microsome [15] soluble [5] $"

[21] [9, 10, 15, 17] [8, 20]
 

(the human CMP-N-acetylneuraminic acid hydroxylase is inactive because of a partial deletion in the hydroxylase gene [12]) [12] [6] (expression in COS-1 cells [10, 16]) [10, 12, 16] (expression in Escherichia coli [8]) [8] 

 

Additional information (, naturally occuring truncated protein, lacking 46 amino acids in the middle of the normal full-length protein, causes a change in intracellular distribution of the enzyme from cytosol to endoplasmic reticulum and a loss in activity [16]) [16]

4  )  4 (, 48 h, 10% loss of activity [20]) [20] 25 (, 2 h, 50% loss of activity [20]) [20] 5    "

, enzyme is greatly stabilized by CMP-N-acetylneuraminate [9] , after each cycle of freezing and thawing, 25% loss of activity [20]

334

   , -80 C, 0.2 mM CMP-N-acetylneuraminate, stable for at least 6 months [9] , -80 C, very stable [20] , 4 C, 48 h, 10% loss of activity [20]

"  [1] Shaw, L.; Schauer, R.: The biosynthesis of N-glycoloylneuraminic acid occurs by hydroxylation of the CMP-glycoside of N-acetylneuraminic acid. Biol. Chem. Hoppe-Seyler, 369, 477-486 (1988) [2] Shaw, L.; Schauer, R.: Detection of CMP-N-acetylneuraminic acid hydroxylase activity in fractionated mouse liver. Biochem. J., 263, 355-363 (1989) [3] Kozutsumi, Y.; Kawano, T.; Yamakawa, T.; Suzuki, A.: Participation of cytochrome b5 in CMP-N-acetylneuraminic acid hydroxylation in mouse liver cytosol. J. Biochem., 108, 704-706 (1990) [4] Muchmore, E.A.; Milewski, M.; Varki, A.; Diaz, S.: Biosynthesis of N-glycolyneuraminic acid. The primary site of hydroxylation of N-acetylneuraminic acid is the cytosolic sugar nucleotide pool. J. Biol. Chem., 264, 2021620223 (1989) [5] Bergwerff, A.A.; Hulleman, S.H.D.; Kamerling, J.P.; Vliegenthart, J.F.G.; Shaw, L.; Reuter, G.; Schauer, R.: Nature and biosynthesis of sialic acids in the starfish Asterias rubens. Identification of sialo-oligomers and detection of S-adenosyl-l-methionine: N-acylneuraminate 8-O-methyltransferase and CMP-N-acetylneuraminate monooxygenase activities. Biochimie, 74, 25-37 (1992) [6] Schlenzka, W.; Shaw, L.; Kelm, S.; Schmidt, C.L.; Bill, E.; Trautwein, A.X.; Lottspeich, F.; Schauer, R.: CMP-N-acetylneuraminic acid hydroxylase: the first cytosolic Rieske iron-sulfur protein to be described in Eukarya. FEBS Lett., 385, 197-200 (1996) [7] Shaw, L.; Schneckenburger, P.; Schlenzka, W.; Carlsen, J.; Christiansen, K.; Juergensen, D.; Schauer, R.: CMP-N-acetylneuraminic acid hydroxylase from mouse liver and pig submandibular glands. Interaction with membrane-bound and soluble cytochrome b5 -dependent electron transport chains. Eur. J. Biochem., 219, 1001-1011 (1994) [8] Martensen, I.; Schauer, R.; Shaw, L.: Cloning and expression of a membrane-bound CMP-N-acetylneuraminic acid hydroxylase from the starfish Asterias rubens. Eur. J. Biochem., 268, 5157-5166 (2001) [9] Kawano, T.; Kozutsumi, Y.; Kawasaki, T.; Suzuki, A.: Biosynthesis of N-glycolylneuraminic acid-containing glycoconjugates. Purification and characterization of the key enzyme of the cytidine monophospho-N-acetylneuraminic acid hydroxylation system. J. Biol. Chem., 269, 9024-9029 (1994) [10] Kawano, T.; Koyama, S.; Takematsu, H.; Kozutsumi, Y.; Kawasaki, H.; Kawashima, S.; Kawasaki, T.; Suzuki, A.: Molecular cloning of cytidine monophospho-N-acetylneuraminic acid hydroxylase. Regulation of species- and

335

[11]

[12] [13]

[14]

[15] [16]

[17] [18]

[19] [20] [21] [22]

336

tissue-specific expression of N-glycolylneuraminic acid. J. Biol. Chem., 270, 16458-16463 (1995) Chou, H.H.; Hayakawa, T.; Diaz, S.; Krings, M.; Indriati, E.; Leakey, M.; Paabo, S.; Satta, Y.; Takahata, N.; Varki, A.: Inactivation of CMP-N-acetylneuraminic acid hydroxylase occurred prior to brain expansion during human evolution. Proc. Natl. Acad. Sci. USA, 99, 11736-11741 (2002) Irie, A.; Suzuki, A.: CMP-N-acetylneuraminic acid hydroxylase is exclusively inactive in humans. Biochem. Biophys. Res. Commun., 248, 330-333 (1998) Schlenzka, W.; Shaw, L.; Schauer, R.: Catalytic properties of the CMP-Nacetylneuraminic acid hydroxylase from the starfish Asterias rubens: comparison with the mammalian enzyme. Biochim. Biophys. Acta, 1161, 131138 (1993) Takematsu, H.; Kawano, T.; Koyama, S.; Kozutsumi, Y.; Suzuki, A.; Kawasaki, T.: Reaction mechanism underlying CMP-N-acetylneuraminic acid hydroxylation in mouse liver: formation of a ternary complex of cytochrome b5, CMP-N-acetylneuraminic acid, and a hydroxylation enzyme. J. Biochem., 115, 381-386 (1994) Schneckenburger, P.; Shaw, L.; Schauer, R.: Purification, characterization and reconstitution of CMP-N-acetylneuraminate hydroxylase from mouse liver. Glycoconjugate J., 11, 194-203 (1994) Koyama, S.; Yamaji, T.; Takematsu, H.; Kawano, T.; Kozutsumi, Y.; Suzuki, A.; Kawasaki, T.: A naturally occurring 46-amino acid deletion of cytidine monophospho-N-acetylneuraminic acid hydroxylase leads to a change in the intracellular distribution of the protein. Glycoconjugate J., 13, 353-358 (1996) Malykh, Y.N.; Shaw, L.; Schauer, R.: The role of CMP-N-acetylneuraminic acid hydroxylase in determining the level of N-glycolylneuraminic acid in porcine tissues. Glycoconjugate J., 15, 885-893 (1998) Malykh, Y.N.; Krisch, B.; Shaw, L.; Warner, T.G.; Sinicrop, D.; Smith, R.; Chang, J.; Schauer, R.: Distribution and localization of CMP-N-acetylneuraminic acid hydroxylase and N-glycolylneuraminic acid-containing glycoconjugates in porcine lymph node and peripheral blood lymphocytes. Eur. J. Cell Biol., 80, 48-58 (2001) Gollub, M.; Schauer, R.; Shaw, L.: Cytidine monophosphate-N-acetylneuraminate hydroxylase in the starfish Asterias rubens and other echinoderms. Comp. Biochem. Physiol. B, 120, 605-615 (1998) Gollub, M.; Shaw, L.: Isolation and characterization of cytidine-5'-monophosphate-N-acetylneuraminate hydroxylase from the starfish Asterias rubens. Comp. Biochem. Physiol. B, 134, 89-101 (2003) Schlenzka, W.; Shaw, L.; Schneckenburger, P.; Schauer, R.: Purification and characterization of CMP-N-acetylneuraminic acid hydroxylase from pig submandibular glands. Glycobiology, 4, 675-683 (1994) Shaw, L.; Schneckenburger, P.; Carlsen, J.; Christiansen, K.; Schauer, R.: Mouse liver cytidine-5-monophosphate-N-acetylneuraminic acid hydroxylase. Catalytic function and regulation. Eur. J. Biochem., 206, 269-277 (1992)

[23] Lepers, A.; Shaw, L.; Schneckenburger, P.; Cacan, R.; Verbert, A.; Schauer, R.: A study on the regulation of N-glycoloylneuraminic acid biosynthesis and utilization in rat and mouse liver. Eur. J. Biochem., 193, 715-723 (1990) [24] Malykh, Y.N.; King, T.P.; Logan, E.; Kelly, D.; Schauer, R.; Shaw, L.: Regulation of N-glycolylneuraminic acid biosynthesis in developing pig small intestine. Biochem. J., 22, 2-29 (2002)

337

$    

:::

   
 1.14.99.19    O-1-alkyl-2-acyl-sn-glycero-3-phosphoethanolamine,hydrogen-donor:oxygen oxidoreductase    plasmanylethanolamine desaturase    1-O-alkyl-2-acyl-sn-glycero-3-phosphorylethanolamine desaturase alkylacylglycero-phosphorylethanolamine dehydrogenase alkylacylglycerophosphoethanolamine desaturase dehydrogenase, alkyl-acylglycerophosphorylethanolamine desaturase, alkylacylglycerophosphorylethanolamine plasmanyl D1 '-desaturase [7] plasmanylethanolamine D1 -desaturase [8]
    39391-13-4

     Rattus norvegicus (Charles River adult rats, strain CD [2]; Charles River male rats, strain CDF [3]; Sprague-Dawley strain, albino rats, 15 days old [5]) [2, 3, 4, 5, 6, 7, 8] Mesocricetus auratus (adult animals of either sex [1]) [1, 8] Mus musculus (NMRI, female, average weight 20 g [5]) [5, 8] Oryctolagus cuniculus [8]

!    "
 # 

O-1-alkyl-2-acyl-sn-glycero-3-phosphoethanolamine + AH2 + O2 = O-1-alk1-enyl-2-acyl-sn-glycero-3-phosphoethanolamine + A + 2 H2 O ( mixed function oxidase [1, 2])

338

1.14.99.19

Plasmanylethanolamine desaturase

   oxidation redox reaction reduction       O-1-alkyl-2-acyl-sn-glycero-3-phosphoethanolamine + electron donor + O2 (Reversibility: ? [1, 2, 5]) [1, 2, 5] $ O-1-alk-1-enyl-2-acyl-sn-glycero-3-phosphoethanolamine + oxidized electron donor + H2 O [1, 2, 5]      1-hexadecyl-2-acyl glycero-3-phosphoethanolamine + NADH + O2 ( in situ in the microsomes via acylation of 1-hexadecyl-glycero-3phosphoethanolamine [4]) (Reversibility: ? [4]) [4] $ ?  O-1-alkyl-2-acyl-sn-glycero-3-phosphoethanolamine + electron donor + O2 ( generated in situ by microsomal acylation of alkyllyso-glycero-3-phosphoethanolamine, better substrate than when it is added exogenously [8]) (Reversibility: ? [1, 2, 5, 6, 8]) [1, 2, 5, 6, 8] $ O-1-alk-1-enyl-2-acyl-sn-glycero-3-phosphoethanolamine + oxidized electron donor + H2 O [1, 2, 5, 6]  O-1-hexadecyl-rac-glycero-3-phosphoethanolamine + NADPH + O2 (Reversibility: ? [7]) [7] $ ?  Additional information ( no substrates: O-3-alkyl-2-acylsn-glycero-1-phosphoethanolamine, octadecylglycerol, octadecanal, octadecanol, stearic acid [1]; electron donors: NADPH or NADH [1, 8]; no selectivity of the desaturase for specific acyl chains [4]; highly specific with regard to chain length of saturated alkyl moieties of alkylacylglycerophosphoethanolamines [5]; high degree of substrate specificity, no substrate is O-1-alkyl-ethanediol-2-phosphoethanolamine [6]) [1, 4, 5, 6, 8] $ ? 2  1,10-phenanthroline [1] CN- ( be not inhibited by carbon monoxide [8]) [1, 2, 8] EDTA [1] NEM [1] NaN3 [1] cholate ( weak inhibition [1]) [1] menadione [1] oleate ( weak inhibition [1]) [1] p-chloromercuribenzoate [1] taurocholate [1]

339

Plasmanylethanolamine desaturase

1.14.99.19


"%  NADH [1, 2, 3, 4] NADPH ( or NADH [1, 2, 8]) [1, 2, 7, 8] cytochrome b5 ( could be involved [2]) [2, 8] &

  ATP ( stimulates the conversion by a factor of 4 [2]) [2, 3] Mg2+ ( stimulates the conversion by a factor of 2 [2]) [2] catalase [8] heat-lable factor of high molecular weight ( contained in the 100000 * g supernatant [1]) [1] '(   Mg2+ [1, 2, 3] " & *-% , 0.0000028 ( microsomes plus soluble fraction, enzyme requires a heat-labile factor of high molecular weight contained in the soluble fraction [1]) [1] 0.0000057-0.0000064 ( two-dimensional TLC method [7]) [7] 0.0000066 ( hydrolysis followed by solvent partition [7]) [7] 0.000025 ( tumor cells of rat on fat-free diet [3]) [3] 0.000029 ( tumor cells of rat on normal diet [3]) [3] 0  7-8 ( posphate buffer [1]) [1] )  *
, 37 ( enzyme assay [1, 2, 7, 8]) [1, 2, 7, 8]

 2 %$ %' %

% MDCK [8] P-388D1 [8] ascites ( leukemia 1210 and Sarcoma 180 cells [5]; Ehrlich cells [8]) [5, 8] brain [5, 6, 7, 8] sarcoma cell ( Fischer R-3259 sarcoma [2, 3, 4, 8]) [2, 3, 4, 8] small intestine mucosa [1, 8] 3#

microsome ( microsomal membrane, advisable to check the effect of the soluble fraction [8]) [1, 3, 4, 7, 8]

340

1.14.99.19

Plasmanylethanolamine desaturase

4  5    "

, activity loss at -23 C, should be used freshly [8]    , -23 C, postmitochondrial supernatant stable for two weeks [2] , -23 C, supernatant [3] , -70 C, TES buffer, 0.25 M sucrose, 1 mM EDTA [7]

"  [1] Paltauf, F.; Holasek, A.: Enzymatic synthesis of plasmalogens. Characterization of the 1-O-alkyl-2-acyl-8n-glycero-3-phosphorylethanolamine desaturase from mucosa of hamster small intestine. J. Biol. Chem., 248, 1609-1615 (1973) [2] Wykle, R.L.; Blank, M.L.; Malone, B.; Snyder, F.: Evidence for a mixed function oxidase in the biosynthesis of ethanolamine plasmalogens from 1-alkyl2-acyl-sn-glycero-3-phosphorylethanolamine. J. Biol. Chem., 247, 5442-5447 (1972) [3] Lee, F.C.; Wykle, R.L.; Blank, M.L.; Snyder, F.: Dietary control of stearyl CoA and alkylacylglycerophosphorylethanolamine desaturases in tumor. Biochem. Biophys. Res. Commun., 55, 574-579 (1973) [4] Wykle, R.L.; Schremmer, J.M.: Biosynthesis of plasmalogens by the microsomal fraction of Fischer R-3259 sarcoma. Influence of specific 2-acyl chains on the desaturation of 1-alkyl-2-acyl-sn-glycero-3-phosphoethanolamine. Biochemistry, 18, 3512-3517 (1979) [5] Weber, N.; Richter, J.: Formation of ether lipids and wax esters in mammalian cells. Specificity of enzymes with regard to carbon chains of substrates. Biochim. Biophys. Acta, 711, 197-207 (1982) [6] Baumann, W.J.; Madson, T.H.; Chang, N.; Bandi, P.C.; Schmidt, H.H.O.: On the substrate specificity of enol ether formation in rat brain. Metabolism of O-alkyl ethanediol phosphorylethanolamine. Biochem. Biophys. Res. Commun., 66, 717-724 (1975) [7] Das, A.K.; Hajra, A.K.: A novel chemical synthesis of 1-O-hexadecyl-rac-[23 H]glycero-3-phosphorylethanolamine and a simple assay for plasmanyl desaturase. J. Lipid Res., 37, 2706-2714 (1996) [8] Blank, M.L.; Snyder, F.: Plasmanylethanolamine D1 -desaturase. Methods Enzymol., 209, 390-396 (1992)

341

$ ?       *(! #

,

::=

   
 1.14.99.20    phylloquinone,hydrogen-donor:oxygen oxidoreductase (2,3-epoxidizing)    phylloquinone monooxygenase (2,3-epoxidizing)    epoxidase, phylloquinone oxygenase, phylloquinone mono- (2,3-epoxidizing) phylloquinone epoxidase vitamin K 2,3-epoxidase vitamin K epoxidase
    54596-37-1

     Rattus norvegicus (homozygous warfarin-resistent rats [1]; rats of a Sprague-Dawley-derived strain, 8-10 weeks old male and female rats [1]; Sprague-Dawley strain, fasted male rats, 250 - 350 g [2]; 7- to 10-day old rats [4]; Holtzman strain, male rats [5, 6]; 250-300 g [6]) [1, 2, 4, 5, 6, 7] Bos taurus [3, 8, 9, 10, 11] Homo sapiens (fetus, neonate, 1-18 years [12]) [12]

!    "
 # 

phylloquinone + AH2 + O2 = 2,3-epoxyphylloquinone + A + H2 O ( enzyme linked to the vitamin K dependent carboxylation system [2, 3]; enzyme has two distinct functions: vitamin K epoxidation and g-glutamyl carboxylation [9, 10]; vitamin K-dependent carboxylase activity and vitamin K epoxidase activity are properties of the same protein [8]; propeptide and glutamate-containing substrates bound to the vitamin K-dependent carboxylase convert its vitamin K epoxidase function from an inactive to an active state [11]) 342

1.14.99.20

Phylloquinone monooxygenase (2,3-epoxidizing)

   oxidation redox reaction reduction       phylloquinone + electron donor + O2 (Reversibility: ? [1-6, 9, 10, 11, 12]) [1-6, 9, 10, 11, 12] $ 2,3-epoxyphylloquinone + oxidized electron + H2 O [1-6, 9, 10, 11, 12]      phylloquinone + electron donor + O2 ( vitamin K1 , enzyme involves in vitamin K dependent prothrombin synthesis [2]) (Reversibility: ? [1-6, 9, 10, 11, 12]) [1-6, 9, 10, 11, 12] $ 2,3-epoxyphylloquinone + oxidized electron + H2 O [1, 2, 3, 4, 5, 12]  Additional information ( electron donor: NADPH [1, 4]; electron donor: NADH [2, 12]; NADH or NADPH, if reduced pyridine nucleotides are provided, no cytosolic component is required [2, 5]) [1, 2, 4, 5, 12] $ ? 2  2-chloro-3-phytyl-1,4-naphthoquinone [5] H2 O2 [4] glutathione peroxidase ( competitive inhibitor [4]) [4] tetrachloro-4-pyridinol [5] Additional information ( no inhibitors: CO and CN- [1]; CNno inhibitor [3]) [1, 3]
"%  NADH [2, 5, 12] NADPH [1, 2, 4, 5] &

  DTT [7] Mn2+ ( activating effect is limited to peptide substrate with vicinal glutamyl residues [7]) [7] glutamate-containing substrates ( bound to the vitamin K-dependent carboxylase converts its vitamin K epoxidase function from an inactive to an active state [11]) [11] propeptide ( bound to the vitamin K-dependent carboxylase converts its vitamin K epoxidase function from an inactive to an active state [11]) [11] '(   Mn2+ [7]

343

Phylloquinone monooxygenase (2,3-epoxidizing)

1.14.99.20

) &  * +, 0.4 (Phe-Leu-Glu-Glu-Leu) [8] " & *-% , 0.00077-0.00088 [3] 0.00466 [8] Additional information ( maximum enzyme activity requires both the microsomal and the soluble fraction [1]; high value in the early prenatal perod of 10-30 gestational weeks [12]) [1, 9, 10, 11, 12] . / *', 0.08 (O2 ) [7] )  *
, 17 ( enzyme assay 25 ( enzyme assay 27 ( enzyme assay 37 ( enzyme assay

[7]) [7] [9, 10, 11]) [9, 10, 11] [2, 5]) [2, 4, 5, 6] [1]) [1]

  #     ? ( x * 77000, SDS-PAGE [8]) [8] ? ( x * 94000, SDS-PAGE, wild type enzyme [9, 11]) [9, 11]

 2 %$ %' %

% liver ( obtained at autopsy [12]) [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12] 3#

cytosol [1] microsome ( solubilized microsomal recombinant protein preparation from baculovirus-infected Spodoptera frugiperda cells [9]; microsomal recombinant protein preparation from chinese hamster ovary cells [11]) [1, 2, 3, 4, 5, 6, 7, 8, 9, 11] $"

(partially [6]) [6] [11] (partially [8, 9]) [8, 9]
 

(wild-type and mutants of bovine enzyme species expressed in baculovirus-infected Spodoptera frugiperda cells [9]; wild-type and mutants of bovine enzyme species expressed in chinese hamster ovary cells CHO-Dukx-B11 [10]; expressed in chinese hamster ovary cells CHO-Dukx-B11 [11]) [9, 10, 11] 344

1.14.99.20

Phylloquinone monooxygenase (2,3-epoxidizing)

4  5    "

, preincubation for 15 min at 37 C results in an almost complete inhibition of the epoxidation [5] , stable for several days when left on ice, freezing and thawing destroys the activity [6]    , -20 C, lyophilized, stable for up to 1 month [7] , -80 C, 20% glycerol [10, 11] , -80 C, soluble enzyme preparation, at least 5% glycerol, stable to multiple freeze and thaw cycles [9]

"  [1] Willingham, A.K.; Matschiner, J.T.: Changes in phylloquinone epoxidase activity related to prothrombin synthesis and microsomal clotting activity in the rat. Biochem. J., 140, 435-441 (1974) [2] Suttie, J.W.; Geweke, L.O.; Martin, S.L.; Willingham, A.K.: Vitamin K epoxidase: dependence of epoxidase activity on substrates of the vitamin K-dependent carboxylation reaction. FEBS Lett., 109, 267-270 (1980) [3] DeMetz, M.; Soute, B.A.M.; Hemker, H.C.; Vermeer, C.: The inhibition of vitamin K-dependent carboxylase by cyanide. FEBS Lett., 137, 253-256 (1982) [4] Larson, A.E.; Suttie, J.W.: Vitamin K-dependent carboxylase: evidence for a hydroperoxide intermediate in the reaction. Proc. Natl. Acad. Sci. USA, 75, 5413-5416 (1978) [5] Sadowski, J.A.; Schnoes, H.K.; Suttie, J.W.: Vitamin K epoxidase: properties and relationship to prothrombin synthesis. Biochemistry, 16, 3856-3863 (1977) [6] Wallin, R.; Suttie, J.W.: Vitamin K-dependent carboxylase: evidence for cofractionation of carboxylase and epoxidase activities, and for carboxylation of a high-molecular-weight microsomal protein. Arch. Biochem. Biophys., 214, 155-163 (1982) [7] McTigue, J.J.; Suttie, J.W.: Oxygen dependence of vitamin K-dependent carboxylase and vitamin K epoxidase. FEBS Lett., 200, 71-75 (1986) [8] Hubbard, B.R.; Ulrich, M.M.W.; Jacobs, M.; Vermeer, C.; Walsh, C.; Furie, B.; Furie, B.C.: Vitamin K-dependent carboxylase: affinity purification from bovine liver by using a synthetic propeptide containing the g-carboxylation recognition site. Proc. Natl. Acad. Sci. USA, 86, 6893-6897 (1989) [9] Roth, D.A.; Whirl, M.L.; Velazquez-Estades, L.J.; Walsh, C.T.; Furie, B.; Furie, B.C.: Mutagenesis of vitamin K-dependent carboxylase demonstrates a carboxyl terminus-mediated interaction with vitamin K hydroquinone. J. Biol. Chem., 270, 5305-5311 (1995)

345

Phylloquinone monooxygenase (2,3-epoxidizing)

1.14.99.20

[10] Sugiura, I.; Furie, B.; Walsh, C.T.; Furie, B.C.: Profactor IX propeptide and glutamate substrate binding sites on the vitamin K-dependent carboxylase identified by site-directed mutagenesis. J. Biol. Chem., 271, 17837-17844 (1996) [11] Sugiura, I.; Furie, B.; Walsh, C.T.; Furie, B.C.: Propeptide and glutamatecontaining substrates bound to the vitamin K-dependent carboxylase convert its vitamin K epoxidase function from an inactive to an active state. Proc. Natl. Acad. Sci. USA, 94, 9069-9074 (1997) [12] Itoh, S.; Onishi, S.: Developmental changes of vitamin K epoxidase and reductase activities involved in the vitamin K cycle in human liver. Early Hum. Dev., 57, 15-23 (2000)

346

3 "     * 

,

::

   
 1.14.99.21    Latia-luciferin,hydrogen-donor:oxygen oxidoreductase (demethylating)    Latia-luciferin monooxygenase (demethylating)    Latia luciferin monooxygenase (demethylating) luciferase (Latia luciferin)
    62213-54-1

     Latia neritoides (freshwater limpet [1, 2]) [1, 2]

!    "
 # 

Latia luciferin + AH2 + 2 O2 = oxidized Latia luciferin + CO2 + formate + A + H2 O + hv ( possibly two enzyme components are involved in the reaction, an oxygenase followed by a monooxygenase for the actual light emitting step [1,2])    demethylation oxidation redox reaction reduction      (E)-2-methyl-4-(2,6,6-trimethyl-1-cyclohex-1-yl)-1-buten-1-ol-formate + NADH + O2 ( trivial name Latia luciferin [1]) (Reversibility: ? [1]) [1] $ oxidized Latia luciferin + NAD+ + CO2 + H2 O + formate + light [1]

347

Latia-luciferin monooxygenase (demethylating)

1.14.99.21


"%  FMN ( flavoprotein, reductase component [1]) [1] FMNH2 ( luciferase [1]) [1] NADH ( reductase component [1]) [1] ascorbate ( activation, especially together with optimal NADH [1]) [1] &

  dithiothreitol ( strong increase of activity [1]) [1] purple protein ( requirement [1,2]) [1, 2] 0  6.8 ( assay at [1]) [1] )  *
, 10 ( assay at [1]) [1] 25 ( assay at [2]) [2]

 2 %$ %' %

3#

soluble [2] $"

[1, 2]

"  [1] Shimomura, O.; Johnson, F.H.; Kohama, Y.: Reactions involved in bioluminescence systems of limpet (Latia neritoides) and luminous bacteria. Proc. Natl. Acad. Sci. USA, 69, 2086-2089 (1972) [2] Shimomura, O.; Johnson, F.H.: The structure of Latia luciferin. Biochemistry, 7, 1734-1738 (1968)

348

   =    

::

   
 1.14.99.22    ecdysone,hydrogen-donor:oxygen oxidoreductase (20-hydroxylating)    ecdysone 20-monooxygenase    a-ecdysone C-20 hydroxylase ecdysone 20-hydroxylase
    55071-97-1

     Schistocerca gregaria (desert locust [1, 4]) [1, 4] Manduca sexta (tobacco hornworm [2, 5, 8, 13]) [2, 5, 8, 13] Locusta migratoria migratorioides (L. migratoria migratorioides: african migration locust [3]; migration locust [10]) [3, 10] Drosophila melanogaster (canton S strain [2]) [2] Spinacia oleracea [6] Spodoptera littoralis (cotton leafworm [7]) [7] Neobellieria bullata (flesh-fly [9]) [9] Parasarcophaga argyrostoma (flesh-fly [9]) [9] Bombyx mori (silkworm [11, 12]) [11, 12] Orconectes limosus (crayfish [14]) [14]

!    "
 # 

ecdysone + AH2 + 2 O2 = 20-hydroxyecdysone + A + H2 O + hv ( NADPH-dependent monooxygenase containing cytochrome P-450, a hemethiolate protein [1-6, 11]; ecdysteroid biosynthesis regulation [6]; a cytochrome P-450-dependent steroid hydroxylase as part of the mitochondrial enzyme system with both ferredoxin and cytochrome P-450 components [7, 8]; microsomal enzyme system contains NADPH-dependent 349

Ecdysone 20-monooxygenase

1.14.99.22

cytochrome P-450 reductase without a second electron carrier type [8]; 2 different isozymes of mitochondria and microsomes may have different inducers, electron donors, endogenous inhibitors, and physiological optima [9])    oxidation redox reaction reduction       ecdysone + NADPH + O2 ( a-ecdysone, a secretory product of prothoracic glands [5]; last step in biosynthesis of insect molting hormone [5, 8]; serves as precursor of ecdysterone, the active moulting hormone in larval insects [1-5, 7, 8]; midgut microsomes show higher specific activity with 2fold higher Vmax, while total activity is 8-10fold higher in mitochondria [8]) (Reversibility: ? [1-13]) [1-13] $ 20-hydroxyecdysone + NADP+ + H2 O [1-13]      a-ecdysone + NADPH + O2 ( (22R)-2b,3b,14a,22,25-pentahydroxy-5b-cholest-7-en-6-one, the 5a-epimer of ecdysone, 2b-acetoxyecdysone, 3-hydroxyecdysone, 3-dehydro-ecdysone or ecdysterone are not hydroxylated [3]) (Reversibility: ? [1-14]) [1-14] $ 20-hydroxyecdysone + NADP+ + H2 O ( b-ecdysone, ecdysterone [3]) [1-14]  Additional information ( recombinant GST-fusion protein, expressed in E. coli, is also able to transfer electrons from NADPH to a microsomal steroid hydroxylase P450 and support steroidogenesis [12]) [12] $ ? 2  1,9-dideoxyforskolin ( dose dependent inhibition [2]) [2] 20-hydroxyecdysone ( competitive inhibition [3, 4]) [3, 4] 7,8-benzoflavone [3] 7-O-hemisuccinyl-7-deacetyl-forskolin [2] CO ( partially reversible by irradiation with monochromatic light [1,3,4,6]) [1, 3, 4, 6] Ca2+ [4] EDTA [4] KCN (25% inhibition) [1] KK-42 ( i.e. 1-benzyl-5[(E)-2,6-dimethyl-1,5-heptadienyl]imidazole [11]) [11] Mg2+ [4] NADP+ ( competitive inhibition [3]) [3] NEM [3] PCMB [3] SKF 525A [3] Tris/HCl-buffer [4] 350

1.14.99.22

Ecdysone 20-monooxygenase

Tween 80 ( microsomal isozyme [9]) [9] cytochrome c [3] diquat dibromide [9] ecdysterone ( competitive inhibition [3]) [3] forskolin ( 7b-acetoxy-8,13-epoxy-1a,6b,9a-dihydroxylabd-14en-11-one, adenylate cyclase activator [2]) [2] metyrapone ( 2-methyl-1,2-di-3'-pyridylpropan-1-one, up to 80% inhibition [1]) [1, 3, 4, 11] nicotinamide [3] non-ionic detergents ( Tween 20, Tween 80 and Triton X-100 [4]) [4] oxidized cytochrome c ( reversed by KCN [3]) [3] phospholipase A [3] phospholipase C [3] piperonylbutoxide [3] Additional information ( in pure N2 atmosphere, enzyme activity is 50% inhibited due to missing oxygen [6]) [6]
"%  NADH ( increase of activity, synergism together with NADPH, but cannot substitute NADPH as cofactor [3,4]) [3, 4, 7] NADPH ( ultimate hydrogen donor, cannot be substituted by NADH or ascorbate [3,6]; NADP+ is not effective together with either malate, isocitrate, succinate, but with glucose-6-phosphate 25% of the activity is restored [3]; dependent on [9]) [1-6, 8-12] cytochrome p450 ( essential [1-12]) [1-12] ferredoxin ( isozyme in mitochondria [7-9]) [7-9] &

  ATP ( increase of activity [4]) [4] " & *-% , 0.00000000617 ( fat body [9]) [9] 0.0000000103 ( fat body [9]) [9] 0.00000017 ( fat body [3]) [3] 0.00000041 ( mid- and hindgut [3]) [3] 0.00000075 ( microsomes, fat body [3]) [3] 0.00000115 ( Malphigian tubules [3]) [3] 0.000003 ( microsomes, Malphigian tubules [3]) [3] 0.00000433 [2] 0.00000562 ( Malphigian tubules, 10000 x g pellet [1]) [1] 0.0000183 [4] 0.0000197 ( fat body [2]) [2] 0.0000466 ( mitochondrial enzyme system [5]) [5] 0.0001721 ( midgut [2]) [2] 0.0021 ( microsomes [6]) [6] 27 ( recombinant GST-fusion protein, purified [12]) [12] Additional information [1, 13]

351

Ecdysone 20-monooxygenase

1.14.99.22

. / *', 0.000000367 (ecdysone, microsomal enzyme [8]) [8] 0.0000163 (ecdysone, mitochondrial enzyme [8]) [8] 0.00027 (ecdysone) [3] 0.00071 (ecdysone) [4] 0.00125 (a-ecdysone) [1] 0.00183 (ecdysone, mitochondrial enzyme system [5]) [5] 0.0105 (NADPH) [3] 0.025 (ecdysone) [6] 0  6.5 [1, 2] 6.6 [3] 6.8-8 ( potassium phosphate buffer [4]) [4] 7.2 ( microsomal isozyme [9]) [9] 7.5 [11] 0 

 6-8.5 [11] )  *
, 30 ( microsomal isozyme [9]) [9] 35 [1, 4, 11] 40 [3]

  #     ? ( ? * 60000, SDS-PAGE [10]; ? * 77700, sequence determination [12]; ? * 103000, SDS-PAGE, recombinant GST-fusion protein [12]) [10, 12]

 2 %$ %' %

% abdominal ganglion [14] antennal gland [14] body wall [9] epidermis [14] fat body ( larval [1-3, 13]; enzyme is located in mitochondrion [8]; third instar wandering stage larvae 67-68% of total activity, afterwhite prepuparial stage activity is inactivated in fat body [9]) [1-3, 7-9, 13] gonad [14] integument [8] leaf [6]

352

1.14.99.22

Ecdysone 20-monooxygenase

Malpighian tubule ( last instar larvae [10]) [1, 3, 4, 8-10] midgut ( larval, enzyme is located in mitochondrion and microsome [8]; larval [13]) [2, 8, 13, 14] ovary [14] ring gland [9] salivary gland [8] trachea ( 8-9% of total activity [9]) [9] Additional information ( enzyme activity measured in diapause and non-diapause egg subcellular preparations [11]; immunological analysis of enzyme content in diapause and non-diapause egg microsomes [12]; investigation of enzyme activity in fat body and midgut gland during developmental stages from egg to adult [13]) [11-13] 3#

microsome ( bound to sedimentable membrane [3]; microsomal isozyme [9]) [3, 6, 8-11] mitochondrion ( membrane bound [1, 4, 7]; mitochondrial isozyme [9]) [1, 4, 7-9] $"

[5] (solubilization of the enzyme from microsomes with detergent Synperonic NP10 [10]) [10] (not: purification of the enzyme from microsomes failed due to unstability of the enzyme [6]) [6] (recombinant GST-fusion protein [12]) [12]
 

(cloned CYP6H1 cDNA is probably identical with purified enzyme [10]) [10] (cDNA isolated from non-diapause eggs, expression in Escherichia coli BL21 as GST-fusion protein [12]) [12]

4  5    "

, bovine serum albumin stabilizes [5] , instable, linear activity under standard assay conditions for 10 min [6]    , -30 C, microsomal preparations with 30% v/v glycerol [3] , -80 C, quick frozen membranes, 15% glycerol v/v, at least 1 month [6] , 4 C to -20 C storage for prolonged periods not possible due to unstability of the enzyme, appreciable loss of activity [6]

353

Ecdysone 20-monooxygenase

1.14.99.22

"  [1] Johnson, P.; Rees, H.H: The mechanism of C-20 hydroxylation of a-ecdysone in the desert locust, Schistocerca gregaria. Biochem. J., 168, 513-520 (1977) [2] Keogh, D.P.; Mitchell, M.J.; Crooks, J.R.; Smith, S.L.: Effects of the adenylate cyclase activator forskolin and its inactive derivative 1,9-dideoxyforskolin on insect cytochrome P-450 dependent steroid hydroxylase activity. Experientia, 48, 39-41 (1991) [3] Feyereisen, R.; Durst, F.: Ecdysterone biosynthesis: a microsomal cytochrome-P-450-linked ecdysone 20-monooxygenase from tissues of the African migratory locust. Eur. J. Biochem., 88, 37-47 (1978) [4] Greenwood, D.R.; Rees, H.H.: Ecdysone 20-mono-oxygenase in the desert locust, Schistocerca gregaria. Biochem. J., 223, 837-847 (1984) [5] Weirich, G.F.: Ecdysone 20-monooxygenase. Methods Enzymol., 3, 454-458 (1985) [6] Grebenok, R.J.; Galbraith, D.W.; Benveniste, I.; Feyereisen, R.: Ecdysone 20monooxygenase , a cytochrome P-450 enzyme from spinach, Spinacia oleracea. Phytochemistry, 42, 927-933 (1996) [7] Shergill, J.K.; Cammack, R.; Chen, J.H.; Fisher, M.J.; Madden, S.; Rees, H.H.: EPR spectroscopic characterization of the iron-sulphur proteins and cytochrome P-450 in mitochondria from the insect Spodoptera littoralis (cotton leafworm). Biochem. J., 307, 719-728 (1995) [8] Weirich, G.F.; Williams, V.P.; Feldlaufer, M.F.: Ecdysone 20-hydroxylation in Manduca sexta midgut: kinetic parameters of mitochondrial and microsomal ecdysone 20-monooxygenases. Arch. Insect Biochem. Physiol., 31, 305312 (1996) [9] Darvas, B.; Rees, H.H.; Hoggard, N.: Ecdysone 20-monooxygenase systems in flesh-flies (diptera: sarcophagidae), Neobellieria bullata and Parasarcophaga argyrostoma. Comp. Biochem. Physiol. B, 105, 765-773 (1993) [10] Winter, J.; Eckerskorn, C.; Waditschatka, R.; Kayser, H.: A microsomal ecdysone-binding cytochrome P-450 from the insect Locusta migratoria purified by sequential use of type-II and type-I ligands. Biol. Chem., 382, 15411549 (2001) [11] Horike, N.; Sonobe, H.: Ecdysone 20-monooxygenase in eggs of the silkworm, Bombyx mori: enzymatic properties and developmental changes. Arch. Insect Biochem. Physiol., 41, 9-17 (1999) [12] Horike, N.; Takemori, H.; Nonaka, Y.; Sonobe, H.; Okamoto, M.: Molecular cloning of NADPH-cytochrome P-450 oxidoreductase from silkworm eggs. Eur. J. Biochem., 267, 6914-6920 (2000) [13] Mitchell, M.J.; Crooks, J.R.; Keogh, D.P.; Smith, S.L.: Ecdysone 20-monooxygenase activity during larval-pupal-adult development of the tobacco hornworm, Manduca sexta. Arch. Insect Biochem. Physiol., 41, 24-32 (1999) [14] Boecking, D.; Dauphin-Villemant, C.; Lafont, R.: Metabolism of 3-dehydroecdysone in the crayfish Orconectes limosus. Eur. J. Entomol., 92, 6374 (1995)

354

! 0   #     

::!

   
 1.14.99.23    3-hydroxybenzoate,hydrogen-donor:oxygen oxidoreductase (2-hydroxylating)    3-hydroxybenzoate 2-monooxygenase    3-HBA-2-hydroxylase 3-hydroxybenzoate 2-hydroxylase oxygenase, 3-hydroxybenzoate 2-mono
    73507-96-7

     Pseudomonas testosteroni (mutant which is unable to grow on m-hydroxybenzoate as sole source of carbon and energy) [1, 2] Pseudomonas putida (BS893) [3]

!    "
 # 

3-hydroxybenzoate + AH2 + O2 = 2,3-dihydroxybenzoate + A + H2 O    oxidation redox reaction reduction       3-hydroxybenzoate + electron donor + O2 (Reversibility: ? [1-3]) [1-3] $ 2,3-dihydroxybenzoate + oxidized electron donor + H2 O

355

3-Hydroxybenzoate 2-monooxygenase

1.14.99.23

     3-hydroxybenzoate + electron donor + O2 (Reversibility: ? [1-3]) [1-3] $ 2,3-dihydroxybenzoate + oxidized electron donor + H2 O

"  [1] Daumy, G.O.; McColl, A.S.: Induction of 3-hydroxybenzoate 2-hydroxylase in a Pseudomonas testosteroni mutant. J. Bacteriol., 149, 384-385 (1982) [2] Daumy, G.O.; McColl, A.S.; Andrews, G.C.: Bioconversion of m-hydroxybenzoate to 2,3-dihydroxybenzoate by mutants of Pseudomonas testosteroni. J. Bacteriol., 141, 293-296 (1980) [3] Starovoytov, I.I.; Selifonov, S.A.; Nefedova, M.Y.; Adanin, V.M.: A new pathway for bacterial catabolism of 3-hydroxybenzoic acid. FEMS Microbiol. Lett., 28, 183-186 (1985)

356

 :a    

::

   
 1.14.99.24    steroid,hydrogen-donor:oxygen oxidoreductase (9-epoxidizing)    steroid 9a-monooxygenase    steroid 9a-hydroxylase
    82869-33-8

     Arthrobacter sp. [1] Corynebacterium sp. (synonym Nocardia restrictus, ATCC 14887 (MCI 1354) [2-4, 6]) [1-4, 6] Mycobacterium fortuitum (KCTC 1122, ATCC 6842 [3, 5]) [3, 5] Mycobacterium sp. [1, 3] Nocardia sp. (strain M117 [1, 3]) [1, 3, 5] Rhodococcus erythropolis (strain KCTC 1062, synonym Nocardia erythropolis ATCC 25544 [3]) [3] Rhodococcus rhodochorus (strain KCTC 1061, synonym Nocardia erythropolis ATCC 17895 [3]) [3] Rhodococcus sp. (strain IOC-77 [6]) [3, 6]

!    "
 # 

pregna-4,9(11)-diene-3,20-dione + AH2 + O2 = 9,11a-epoxypregn-4-ene3,20-dione + A + H2 O (An enzyme system involving a flavoprotein (FMN) and two iron-sulfur proteins)    oxidation redox reaction reduction 357

Steroid 9a-monooxygenase

1.14.99.24

      4,9(11)-pregnadiene-3,20-dione + NADH + O2 ( key-enzyme in the steroid-ring-B-splitting pathway [1]; key enzyme system in steroid nucleus degradation in company with d-dehydrogenase [4]; key enzyme system controlling the microbial 9a-hydroxylation of 4-androstene-3,17-dione [6]) (Reversibility: ? [1-6]) [1-6] $ 9,11a-epoxypregn-4-ene-3,20-dione + NAD+ + H2 O [1]      4,9(11)-pregnadiene-3,20-dione + NADH + O2 ( key-enzyme in the steroid-ring-B-splitting pathway [1]) (Reversibility: ? [1-6]) [1-6] $ 9,11a-epoxypregn-4-ene-3,20-dione + NAD+ + H2 O [1]  4-androstene-3,17-dione + NADH + O2 (Reversibility: ? [2, 6]) [2, 6] $ 9a-hydroxy-4-androstene-3,17-dione + NAD+ + H2 O [2]  9(11)-dehydro-17a-methyl-testosterone + NADH + O2 (Reversibility: ? [3-5]) [3-5] $ 9a,11a-oxido-17b-hydroxy-17a-methyl-4-androstene-3-one + 9a,11aoxido-17b-hydroxy-17a-methyl-1,4-androstadiene-3-one + NAD+ + H2 O 2  2,2'-bipyridine ( 52% inhibition [1]) [1, 3] 8-hydroxyquinoline [3] Cd(CH3 COO)2 ( 80% inhibition [1]) [1] CuSO4 ( 100% inhibition [1]) [1] Hg(CH3 COO)2 ( 33% inhibition [1]) [1] metyrapone [3] o-phenanthroline ( 72% inhibition [1]) [1, 3] potassium cyanide ( 60% inhibition [1]) [1] sodium azide ( 100% inhibition [1]) [1] sodium cyanide [3]
"%  FAD ( flavoprotein [1]) [1, 5] NADH [1, 3, 5] NADPH ( conversion rate higher with NADH [1]) [1, 3] cytochrome P450 [3] &

  N,N-dimethylformamide ( reaction rate 111% [2]) [2] dimethyl sulfoxide ( reaction rate 111% [2]) [2] ethanol ( presence of ethanol enhances the hydroxylation by resting non-induced cells [6]) [6] methanol ( reaction rate 111% [2]) [2] '(   Fe2+ ( iron-sulfur protein [1]; iron-sulfur group [5]) [1, 5] Mg2+ ( higher hydroxylation obtained by increasing magnesium ion concentration with a maximum of stimulation at 20 mM or more [1]) [1]

358

1.14.99.24

Steroid 9a-monooxygenase

" & *-% , 6.18 ( NADH reductase component [5]) [5] . / *', 0.068 (NADH) [5] 0  8 ( entrapped cells [2]) [2] 9 ( free cells [2]) [2] 9.5 [1] 0 

 5.5-10 [1] 6-9.5 ( entrapped cells [2]) [2] ) 

 *
, 20-30 ( entrapped cells [2]) [2]

  #   ' 1  60000 ( NADH reductase component, SDS-PAGE [5]) [5] 120000 ( hydroxylase system composed of 3 proteins, protein III, comparative gel filtration [1]) [1] 214000 ( hydroxylase system composed of 3 proteins, protein II, comparative gel filtration [1]) [1]   Additional information ( enzyme system involves a flavoprotein reductase and 2 iron-sulfur proteins [1]) [1]

 2 %$ %' %

3#

cytoplasm [3] cytosol [3, 5] membrane [3] $"

(NADH reductase component [5]) [5] (partially [1]) [1, 3] 

medicine ( enzyme catalyzes the production of 9a-hydroxy4-androstene-3,17-dione, an important intermediate for the semi-synthesis of potent anti-inflammatory drugs such as 9a-fluorocorticoids from 4-androstene-3,17-dione [4, 5]; 9a-steroid hydroxylating activity is used for con-

359

Steroid 9a-monooxygenase

1.14.99.24

version of different steroid compounds into valuable 9a-hydroxy derivatives used in the pharmaceutical industry [6]) [4-6] synthesis ( enzyme occurs in many bacterial genera used in industrial processes [1, 3]; steroid 9a-hydroxylase in company with d-dehydrogenase is used in industrial processes [4]) [1, 3, 4]

4    , protein II and protein III are oxygen-labile [1]    , -20 C, free cells retain the original hydroxylation activity for at least 1 month [2] , -100 C, enzyme activity in clear supernatant frozen in liquid nitrogen is stable for more than 1 year [1]

"  [1] Strijewski A.: The steroid-9 a-hydroxylation system from Nocardia species. Eur. J. Biochem., 128, 125-135 (1982) [2] Sonomoto K.; Usui, N.; Tanaka, A.; Fukui, S.: 9a-Hydroxylation of 4-androstene-3,17-dione by gel-entrapped Corynebacterium sp. cells. Eur. J. Appl. Microbiol. Biotechnol., 17, 203-210 (1983) [3] Kang, H.K.; Lee, S.S.: Heterogeneous natures of the microbial steroid 9a-hydroxylase in nocardioforms. Arch. Pharmacol. Res., 20, 519-524 (1997) [4] Kang, H.K.; Lee, S.S.: Microbial 9a-hydroxylase: epoxidation of 9(11)-dehydro-17a-methyl-testosterone. Arch. Pharmacol. Res., 20, 525-528 (1997) [5] Kang, H.K.; Lee, S.S.: Purification of the NADH reductase component of the steroid 9a-hydroxylase from Mycobacterium fortuitum. Arch. Pharmacol. Res., 20, 590-596 (1997) [6] Mutafov, S.; Angelova, B.; Avramova, T.; Boyadjieva, L.; Dimova, I.: The inducibility of 9a-steroid hydroxylating activity in resting Rhodococcus sp. cells. Process Biotechnol., 32, 585-589 (1997)

360

3  
 

::

   
 1.14.99.25 (transferred to EC 1.14.19.3)    linoleoyl-CoA desaturase

361

 0         

::4

   
 1.14.99.26    2-hydroxypyridine,hydrogen-donor:oxygen oxidoreductase (5-hydroxylating)    2-hydroxypyridine 5-monooxygenase    2-hydroxypyridine oxygenase
    96779-45-2

     Bacillus brevis (isolated by enrichment on isonicotinic acid [1]) [1]

!    "
 # 

2-hydroxypyridine + AH2 + O2 = 2,5-dihydroxypyridine + A + H2 O    hydroxylation oxidation redox reaction reduction       2-hydroxypyridine + NADH + O2 (Reversibility: ? [1]) [1] $ 2,5-dihydroxypyridine + NAD+ + H2 O      2-hydroxypyridine + NADH + O2 (Reversibility: ? [1]) [1] $ 2,5-dihydroxypyridine + NAD+ + H2 O 2  FMN ( 85% inhibition at 1.7 mM, 2 min preincubation [1]) [1] Na2 As2 O3 ( partial inhibition [1]) [1] 362

1.14.99.26

2-Hydroxypyridine 5-monooxygenase

NaF ( 53% inhibition at 1.7 mM, 5 min preincubation [1]) [1] NaN3 ( 53% inhibition at 1.7 mM, 5 min preincubation [1]) [1] NiCl2 ( 47% inhibition at 1.7 mM [1]) [1]
"%  NAD+ [1] NADH [1] NADPH ( 2% activity of NADH [1]) [1] 0  7-7.2 [1] 0 

 6.3-8.5 ( half-maximal activity at pH 6.3 and pH 8.5 [1]) [1]

 2 %$ %' %

3#

cytoplasm [1]

4  5    "

, 45 mM dithiothreitol affords partial protection against denaturation [1] , 50% glycerol affords partial protection against denaturation [1] , dialysis of extracts in cold, 4 C, against phosphate buffer, pH 7.0, 0.05 M, for 4 h, 90% activity loss [1] , enzyme is completely blocked by anaerobic conditions [1]    , -10 C or 0 C, overnight, complete loss of activity [1] , -20 C, Tris HCl buffer, pH 7.4, 0.05 M, 10% ethanol, 50% glycerol, 0.045 M dithiothreitol, 1 mM 2-mercaptoethanol, stable for 16 d [1]

"  [1] Sharma, M.L.; Kaul, S.M.; Shukla, O.P: Metabolism of 2-hydroxypyridine by Bacillus brevis (INA). Biol. Mem., 9, 43-52 (1984)

363

@   !    

::8

   
 1.14.99.27    5-hydroxy-1,4-naphthoquinone,hydrogen-donor:oxygen oxidoreductase (3hydroxylating)    juglone 3-monooxygenase    juglone hydroxylase naphthoquinone hydroxylase naphthoquinone-hydroxylase
    98865-54-4

     Pseudomonas putida (strains J1 and J2 [1]; strain L2 [2]; strain J1, two isofunctional enzymes named enzyme 1, present in induced and non-induced cells and enzyme 2, present only in juglone-induced cells [3]) [1-3]

!    "
 # 

5-hydroxy-1,4-naphthoquinone + AH2 + O2 = 3,5-dihydroxy-1,4-naphthoquinone + A + H2 O    hydroxylation oxidation redox reaction reduction       5-hydroxy-1,4-naphthoquinone + O2 (Reversibility: ? [1-3]) [13] $ 3,5-dihydroxy-1,4-naphthoquinone + H2 O 364

1.14.99.27

Juglone 3-monooxygenase

     1,4-naphthoquinone + O2 (Reversibility: ? [1-3]) [1-3] $ 2-hydroxy-1,4-naphthoquinone + H2 O  2-chloro-1,4-naphthoquinone + O2 (Reversibility: ? [1-3]) [1-3] $ 2-chloro-3-hydroxy-1,4-naphthoquinone + H2 O  5,8-dihydroxy-1,4-naphthoquinone + O2 (Reversibility: ? [1, 3]) [1, 3] $ 2,5,8-trihydroxy-1,4-naphthoquinone + H2 O  5-hydroxy-1,4-naphthoquinone + O2 (Reversibility: ? [1-3]) [13] $ 3,5-dihydroxy-1,4-naphthoquinone + H2 O  5-hydroxy-2-methyl-1,4-naphthoquinone + O2 (Reversibility: ? [1, 2]) [1, 2] $ 3,5-dihydroxy-2-methyl-1,4-naphthoquinone + H2 O  Additional information ( hydroxyl substituent at C-5 is indispensable for induction of juglone hydroxylase [1]) [1] $ ? 2  3,3'-dithiobis(6-nitrobenzoate) ( weak inhibition [3]) [3] HgCl2 ( at 0.5 mM complete inhibition of enzyme 1 and enzyme 2 [3]) [3] p-chloromercuribenzoate ( complete inhibition of enzyme 1 and enzyme 2 [3]) [3] '(   Fe ( 0.39 atoms per subunit for enzyme 1, 0.04 atoms per subunit for enzyme 2 [3]) [3] " & *-% , 242 [3] . / *', 0.0042 (5-hydroxy-1,4-naphthoquinone, enzyme 1 [3]) [3] 0.0185 (5-hydroxy-1,4-naphthoquinone, enzyme 2 [3]) [3] 0  7.5-8.5 ( enzyme 1 and enzyme 2 [3]) [3]

  #   ' 1  56000 ( enzyme 2, gel filtration [3]) [3] 59000 ( enzyme 1, gel filtration [3]) [3]   ? ( 2 * 25000, enzyme 1, SDS-PAGE [3]) [3] dimer ( 2 * 23500, enzyme 2, SDS-PAGE [3]) [3]

365

Juglone 3-monooxygenase

1.14.99.27

 2 %$ %' %

$"

[3]

4  5    "

, enzyme 1 is not stable in potassium phosphate buffer pH 7.5, 10 vol% methanol, ethanol or propanol stabilizes [3] , enzyme 2 is most stable in imidazole/HCl buffer, 10 vol% acetone stabilizes [3]

"  [1] Muller, U.; Lingens, F.: Degradation of 1,4-naphthoquinones by Pseudomonas putida. Biol. Chem. Hoppe-Seyler, 369, 1031-1043 (1988) [2] Wessendorf, J.; Rettenmaier, H.; Lingens, F.: Degradation of lawsone by Pseudomonas putida L2. Biol. Chem. Hoppe-Seyler, 366, 945-951 (1985) [3] Rettenmaier, H.; Lingens, F.: Purification and some properties of two isofunctional juglone hydroxylases from Pseudomonas putida J1. Biol. Chem. Hoppe-Seyler, 366, 637-646 (1985)

366

3  7    

::7

   
 1.14.99.28    3,7-dimethylocta-1,6-dien-3-ol,hydrogen-donor:oxygen oxidoreductase (8hydroxylating)    linalool 8-monooxygenase    cytochrome P450 111 cytochrome P450lin linalool-8-monooxygenase oxygenase, linalool 8-mono
    95329-13-8

     Pseudomonas putida (PpG777 [1]) [1, 2, 3]

!    "
 # 

3,7-dimethylocta-1,6-dien-3-ol + AH2 + O2 = (E)-3,7-dimethylocta-1,6-dien3,8-diol + A + H2 O ( mixed function monooxygenase consisting of LINreductase, Fe2 S2 -redoxin and cytochrome LIN P-450 [2])    oxidation redox reaction reduction       3,7-dimethylocta-1,6-dien-3-ol + NADH + O2 ( linalool, initial reaction in linalool catabolism [2]) (Reversibility: ? [2]) [2] $ ?

367

Linalool 8-monooxygenase

1.14.99.28

     (E)-3,7-dimethylocta-1,6-dien-3,8-diol + NADH + O2 ( 8-hydroxylinalool [2]) (Reversibility: ? [2]) [2] $ 8-oxolinalool + NAD+ + H2 O [2]  3,7-dimethylocta-1,6-dien-3-ol + NADH + O2 ( both isomers of linalool, strict substrate specificity: geraniol, nerol or citronellol are not hydroxylated [1]) (Reversibility: ? [1, 2]) [1, 2] $ (E)-3,7-dimethylocta-1,6-dien-3,8-diol + NAD+ + H2 O ( 8-hydroxylinalool [2]) [1]  6-methyl-hex-5-en-2-ol + NAD+ (Reversibility: ir [1]) [1] $ 6-methyl-hex-5-en-2-one + NADH  Additional information ( overview: synthetic analogues of linalool [1]) [1] $ ? 2  CO ( complete inhibition [2]) [2]
"%  NADH ( b-NADH [1]) [1, 2] '(   Fe2+ ( requirement of two enzyme components, iron-heme protein and iron-sulfur protein [2]) [2] ) &  * +, 120 (NADH, substrate-stimulated NADH oxidation using excess amounts of P-450 and FAD component of the enzyme [2]) [2] 1920 (NADH, substrate-stimulated NADH oxidation using excess amounts of iron-sulfur and FAD component of the enzyme [2]) [2] 2580 (NADH, substrate-stimulated NADH oxidation using excess amounts of iron sulfur and P-450 component of the enzyme [2]) [2] Additional information ( overview: values for cytochrome LIN P-450 with various synthetic substrates [1]) [1] " & *-% , 0.0042 ( reductase [2]) [2] 0  7 ( assay at [1]) [1] )  *
, 25 ( assay at [1]) [1]

  #   ' 1  Additional information ( multi-component enzyme consisting of LIN-reductase, Fe2 S2 -redoxin and cytochrome LIN-P450. MW of the reductase: 43700, analytical data from amino acid composition and prosthetic

368

1.14.99.28

Linalool 8-monooxygenase

group quantification, 45000, gel filtration. MW of Fe2 S2 -redoxin: 11000, gel filtration, 12800, analytical data from amino acid composition and prosthetic group quantification. MW of cytochrome LIN-P450: 44800, analytical data from amino acid composition and prosthetic group quantification, 45000, gel filtration [2]) [2]   Additional information ( enzyme consists of LIN-reductase, MW 44000, and Fe2S2- redoxin, MW 10700, and cytochrome LIN-P450, MW 47000, SDS-PAGE [2]) [2]

 2 %$ %' %

3#

cytoplasm [2] $"

(purification of 3 enzyme components: iron-sulphur, FAD and P-450 [2]) [2]

4     , -196 C, LIN-redoxin reductase and cytochrome LINP-450 in solution stable over a long period after ultrafiltration or dialysis and repeated freeze/ thaw-cycles [2] , 0 C, LIN-redoxin loses its prosthetic group, 5 mM DTT retards apoprotein formation [2]

"  [1] Bhattacharyya, P.K.; Samanta, T.B.; Ullah, A.H.J.; Gunsalus, I.C.: Chemical probes into the active centre of a heme thiolate monoxygenase. Proc. Indian Acad. Sci. Chem. Sci., 93, 1289-1304 (1984) [2] Ullah, A.H.J.; Murray, R.I.; Bhattacharyya, P.K.; Wagner, G.C.; Gunsalus, I.C.: Protein components of a cytochrome P-450 linalool 8-methyl hydroxylase. J. Biol. Chem., 265, 1345-1351 (1990) [3] Bernhardt, R.; Gunsalus I.C.: Reconstitution of cytochrome P4502B4 (LM2) activity with camphor and linalool monooxygenase electron donors. Biochem. Biophys. Res. Commun., 187, 310-317 (1992)

369

9       

:::

   
 1.14.99.29    deoxyhypusine,hydrogen-donor:oxygen oxidoreductase (2-hydroxylating)    deoxyhypusine monooxygenase    DOHH deoxyhypusine hydroxylase deoxyhypusyl hydroxylase oxygenase, deoxyhypusine di
    101920-83-6 102576-87-4

     Rattus norvegicus [1, 4, 7, 9, 10] Cricetulus griseus [2, 5, 9] Homo sapiens [3, 5, 6, 8]

!    "
 # 

protein N6 -(4-aminobutyl)-l-lysine + AH2 + O2 = protein N6 -[(R)-4-amino2-hydroxybutyl]-l-lysine + A + H2 O    oxidation redox reaction reduction      N'-(4-aminobutyl)lysine + electron donor + O2 (i.e. deoxyhypusine) (Reversibility: ? [1-10]) [1, 2, 3, 4, 5, 6, 7, 8, 9, 10] $ N'-(4-amino-2-hydroxybutyl)lysine + oxidized electron donor + H2 O (i.e. hypusine) [1, 2, 3, 4, 5, 6, 7, 8, 9, 10]

370

1.14.99.29

Deoxyhypusine monooxygenase

2  1,10-diaminodecane (, 94% of initial activity at 2 mM [9]) [9] 1,10-phenanthroline (, complete inhibition at 0.01 mM [1,10]) [1, 10] 1,3-diaminopropane (, 86% of initial activity at 0.5 mM [9]) [9] 1,6-diaminohexane (, 97% of initial activity at 2 mM [9]) [9] 1,7-diaminoheptane (, 91% of initial activity at 2 mM [9]) [9] 1,8-diaminooctane (, 93% of initial activity at 2 mM [9]) [9] 2,2'-dipyridyl (, IC50 0.026 mM [3]; , IC50 0.029 mM [6]) [2, 3, 4, 6] 2,3-dihydroxybenzoic acid (, slight inhibition at 2 mM [7]) [7] 2-(2-hydroxy-5-methylphenyl)-1,3-thiazole-4-carboxylic acid (, inhibition in vitro and in cells [5]) [5] 2-(2-hydroxy-5-methylphenyl)-4,5-dihydro-1,3-thiazole-4-carboxylic acid ( inhibition in vitro and in cells [5]) [5] 3,4-dihydroxybenzoic acid (, above 50% inhibition at 2 mM [7]) [7] 4,6-diphenyl-1-hydroxy-pyridine-2-one (, IC50 0.0007 mM [6]) [6] CaCl2 (, 98% of initial activity at 0.005 mM [1]) [1] Co(C2 H3 O2 )2 (, above 0.01 mM [6]; , 94% of initial activity at 0.005 mM [1]) [1, 6] CuCl2 (, above 0.01 mM [6]) [6] EDTA (, high ionic strength EDTA, Tris concentrations above 30 mM [1]; IC50 0.0003 mM [6]) [1, 4, 6] FeCl3 (, 65% of initial activity at 0.005 mM [1]) [1] FeSO4 (, 13% of initial activity at 0.005 mM [1,10]) [1, 10] Lys-Thr-Gly-deoxyhypusine-His-Gly-His-Ala-Lys (, competitive inhibition [9]) [9] Mn(C2 H3 O2 )2 (, above 0.001 mM [6]) [6] MnCl2 (, 64% of initial activity at 0.005 mM [1]) [1] N-(2-cyanoethyl)butane-1,4-diamine (, 80% of initial activity at 2 mM [9]) [9] N-(3-cyanopropyl)propane-1,3-diamine (, 79% of initial activity at 2 mM [9]) [9] N1 -acetyl-l-Orn-l-Pro-Gly (, above 2 mM [7]) [7] N1 -acetyl-N4 -(2,3-dihydroxybenzoyl)-l-Orn-l-Pro-Gly (, IC50 0.2 mM [7]) [7] N1 -acetyl-N4 -(3,4-dihydroxybenzoyl)-l-Orn-l-Pro-Gly (, IC50 0.03 mM [7]) [7] Ni(C2 H3 O2 )2 (, above 0.001 mM [6]) [6] NiSO4 (, 72% of initial activity at 0.005 mM [1]) [1] Zn(C2 H3 O2 )2 (, above 0.01 mM [6]) [6] ZnCl2 (, 93% of initial activity at 0.005 mM [1]) [1] cadaverine (, 87% of initial activity at 0.5 mM [9]) [9] caldine (, 47% of initial activity at 0.5 mM [9]) [9] ciclopirox (, IC50 0.005 mM, complete inhibition above 0.01 mM [3]; IC50 0.0006 mM [6]) [3, 6] deferiprone (, IC50 0.117 mM [3]; , 0.2 mM in cells [8]) [3, 8] desferrioxamine B (, IC50 0.016 mM [3]) [3] 371

Deoxyhypusine monooxygenase

1.14.99.29

desferrioxamine mesylate [2] ethyl 3,4-dihydroxybenzoate (, IC50 0.5 mM [7]) [7] methyl 2,3-dihydroxybenzoate (, IC50 1.6 mM [7]) [7] metipirox (, IC50 0.0028 mM [6]) [6] mimosine (, IC50 0.191 mM [3]; , S-isomer, complete inhibition at 0.2 mM in LAZ463 cells [5]; reversible inhibition [5]; , IC50 0.0033 mM [6]; , 0.2 mM in cells [8]) [3, 5, 6, 8] picolinic acid [2, 4, 10] putrescine (, 85% of initial activity at 0.5 mM [9]) [9] pyridine 2,3-dicarboxylate ( inhibitory effect of pyridine depends on the carboxyl group position [4]) [1, 4] pyridine 2,4-dicarboxylate ( inhibitory effect of pyridine depends on the carboxyl group position [4]) [1] pyridine 2,5-dicarboxylate ( inhibitory effect of pyridine depends on the carboxyl group position [4]) [1, 4] pyridine 3,4-dicarboxylate ( inhibitory effect of pyridine depends on the carboxyl group position [4]) [1, 4] pyridine 3,5-dicarboxylate ( inhibitory effect of pyridine depends on the carboxyl group position [4]) [1, 4] spermidine (, 58% of initial activity at 0.5 mM [9]) [9] spermine (, 41% of initial activity at 0.5 mM [9]) [9] thermine (, 35% of initial activity at 0.5 mM [9]) [9] Additional information (, no inhibition by CuSO4, HgCl2 , MgCl2 , CdSO4 at 0.005 mM [1]; , no inhibition by 2-oxoglutarate, 2-oxoadipinate, 2-oxosuccinate, 3-oxoglutarate, glutarate, malonate, pyruvate [4]; , no inhibition at 2 mM N-(2-cyanopropyl)-3-cyanopropylamine [9]; , partial reconstitution of 0.01 mM 1,10-phenanthroline inhibited activity with Co(C2 H3 O2 )2 at 0.03 mM or FeSO4 at 0.005 mM [1, 10]) [1, 4, 9, 10] Additional information (, no inhibition by S-isomers of kojic acid, 3pyridylalanine, 4-pyridylalanine at 0.3 mM in vitro and at 0.2 mM in cells, no inhibition by 2-(2-aminophenyl)-1,3-thiazole-4-carboxylic acid at 0.4 mM in cells [5]) [5] Additional information (, partial reconstitution of chelator inhibited activity with CuCl2 or Zn(C2 H3 O2 )2 [6]) [6]
"%  Additional information (, no activity with NADPH, ascorbic acid, mercaptoethylamine, mercaptoacetic acid [1]) [1] &

  sulfhydryl compound (, absolute requirement of partially purified enzyme, not necessary for crude extract [1]) [1, 10] '(   Fe2+ (, 1.5-fold activation at 0.001 mM [6]) [6] Fe3+ (, 1.5-fold activation at 0.001 mM [6]) [6]

372

1.14.99.29

Deoxyhypusine monooxygenase

" & *-% , 416 [1, 7, 10] Additional information ( corresponds to 2.8 pmol/min/mg for partially purified enzyme [1, 7, 10]) [1, 7, 10] . / *', 0.000052 (deoxyhypusine) [1, 10] . / *', 0.032 (N1 -acetyl-N4 -(3,4-dihydroxybenzoyl)-l-Orn-l-Pro-Gly, competitive inhibition [7]) [7] 0.25 (spermine, , competitive inhibition [9]) [9] 0.44 (Lys-Thr-Gly-deoxyhypusine-His-Gly-His-Ala-Lys, , competitive inhibition [9]) [9] 0  7-7.5 (, in 20 mM Tris buffer [1, 7, 9, 10]) [1, 7, 9, 10] 7.5 (, assay in 50 mM sodium phosphate buffer [6]) [6] )  *
, 37 (, assay at [1, 7, 9]) [1, 7, 9] 37 (, assay at [6]) [6]

 2 %$ %' %

% HeLa-S3 cell [6] T-lymphocyte [8] ovary [2, 9] testis [1, 4, 7, 9] umbilical vein endothelium [3] Additional information (, distribution in mammals [1, 10]) [1, 10] $"

(partial [1]) [1] 

medicine (, ciclopirox inhibits cell proliferation and angiogenesis in vitro [3]) [3]

4  5    "

, 20 mM Tris buffer stabilizes [1] , freezing/thawing, stable for numerous times [1] , sulfhydryl compound necessary [1]

373

Deoxyhypusine monooxygenase

1.14.99.29

   , -20 C, stable for at least 6 months [1]

"  [1] Abbruzzese, A.; Park, M.H.; Folk J.E.: Deoxyhypusine hydroxylase from rat testis. Partial purification and characterization. J. Biol. Chem., 261, 30853089 (1986) [2] Park, M.H.; Cooper, H.L.; Folk, J.E.: The biosynthesis of protein-bound hypusine (N -(4-amino-2-hydroxybutyl)lysine). Lysine as the amino acid precursor and the intermediate role of deoxyhypusine (N e-(4-aminobutyl)lysine). J. Biol. Chem., 257, 7217-7222 (1982) [3] Clement, P.M.J.; Hanauske-Abel, H.M.; Wolff, E.C.; Kleinman, H.K.; Park, M.H.: The antifungal drug ciclopirox inhibits deoxyhypusine and proline hydroxylation, endothelial cell growth and angiogenesis in vitro. Int. J. Cancer, 100, 491-498 (2002) [4] Beninati, S.; Ferraro, G.; Abbruzzese, A.: Catalytic properties of deoxyhypusine hydroxylase. Ital. J. Biochem., 39, 183A-185A (1990) [5] Hanauske-Abel, H.M.; Park, M.H.; Hanauske, A.R.; Popowicz, A.M.; Lalande, M.; Folk, J.E.: Inhibition of the G1-S transition of the cell cycle by inhibitors of deoxyhypusine hydroxylation. Biochim. Biophys. Acta, 1221, 115-124 (1994) [6] Csonga, R.; Ettmayer, P.; Auer, M.; Eckerskorn, C.; Eder, J.; Klier, H.: Evaluation of the metal ion requirement of the human deoxyhypusine hydroxylase from HeLa cells using a novel enzyme assay. FEBS Lett., 380, 209-214 (1996) [7] Abbruzzese, A.; Hanauske-Abel, H.M.; Park, M.H.; Henke, S.; Folk, J.E.: The active site of deoxyhypusyl hydroxylase: use of catecholpeptides and their component chelator and peptide moieties as molecular probes. Biochim. Biophys. Acta, 1077, 159-166 (1991) [8] Andrus, L.; Szabo, P.; Grady, R.W.; Hanauske, A.R.; Huima-Byron, T.; Slowinska, B.; Zagulska, S.; Hanauske-Abel, H.M.: Antiretroviral effects of deoxyhypusyl hydroxylase inhibitors. A hypusine-dependent host cell mechanism for replication of human immunodeficiency virus type 1 (HIV-1). Biochem. Pharmacol., 55, 1807-1818 (1998) [9] Abbruzzese, A.; Park, M.H.; Beninati, S.; Folk, J.E.: Inhibition of deoxyhypusine hydroxylase by polyamines and by a deoxyhypusine peptide. Biochim. Biophys. Acta, 997, 248-255 (1989) [10] Abbruzzese, A.; Liguori, V.; Park, M.H.: Deoxyhypusine hydroxylase. Adv. Exp. Med. Biol., 250, 459-466 (1988)

374


  8(7 

::!=

   
 1.14.99.30    carotene, hydrogen-donor:oxygen oxidoreductase    carotene 7,8-desaturase    carotene 7,8-desaturase desaturase, z-carotene desaturase, z-carotene (Capsicum annuum clone pCapZDS precursor reduced) desaturase, z-carotene (Nostoc muscorum clone pZDS1A reduced) desaturase, z-carotene (Synechocystis strain PCC 6803 clone cs0223/cs0128/ ps0014/cs0681/cs0294 gene ctrQ-2 reduced) protein (Synechocystis strain PCC 6803 clone cs0223/cs0128/ps0014/cs0681/ cs0294 open reading frame slr0940 reduced) GenBank D90914-derived protein GI 1653487 z-Carotene desaturase z-Carotene desaturase (Anabaena strain PCC 7120 clone pZDS1A) z-Carotene desaturase (Capsicum annuum clone pCapZDS precursor reduced) z-Carotene desaturase (Synechocystis strain PCC 6803 gene crtQ-2)
    115300-02-2 154768-69-1 (desaturase, z-carotene (Nostoc muscorum clone pZDS1A reduced) /z-carotene desaturase (Anabaena strain PCC 7120 clone pZDS1A)) 171716-20-4 (desaturase, z-carotene (Capsicum annuum clone pCapZDS precursor reduced) /z-carotene desaturase (Capsicum annuum clone pCapZDS precursor reduced)) 184853-38-1 (protein (Synechocystis strain PCC 6803 clone cs0223/cs0128/ ps0014/cs0681/cs0294 open reading frame slr0940 reduced) /zeta-carotene desaturase (Synechocystis strain PCC 6803 gene crtQ-2) /desaturase, zetacarotene (Synechocystis strain PCC 6803 clone cs0223/cs0128/ps0014/ cs0681/cs0294 gene ctrQ-2 reduced) /GenBank D90914-derived protein GI 1653487)

375

Carotene 7,8-desaturase

1.14.99.30

     Anabaena sp. (strain PCC 7129 [1]) [1, 4, 5] Capsicum annuum [2] Narcissus pseudonarcissus [3]

!    "
 # 

neurosporene + AH2 + O2 = lycopene + A + 2 H2 O    oxidation reduction       z-carotene + O2 (, the enzyme catalyzes the last two steps in a series of desaturations [1]) [1, 2] $ ?      9,9'-di-cis pro-zeta-carotene + O2 [1] $ 7,7',9,9'-tetra-cis-prolycopene [1]  b-zeacarotene + O2 [1] $ g-carotene [1]  neurosporene + O2 [1, 2] $ lycopene [1, 2]  z-carotene + O2 (, the enzyme employs O2 as the terminal electron acceptor. The artificial quinones: 2,3,5,6-tetramethyl-1,4-benzoquinone-2,5-dibromo-3-methyl-6-isopropyl-1,4-benzoquinone, 2,3-dimethyl5,6-methylenedioxy-1,4-benzoquinone dichloroindophenol or 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole are able to replace oxygen. Oxygen does not act in a mixed-function oxygenase-like mechanism at the desaturase itself [3]) [1-3] $ lycopene [1, 2] 2  Diphenylamine [1] herbicides (, J852 and LS80707) [1] " & *-% , Additional information [1] . / *', 0.0097 (z-carotene, ) [1] 0.0103 (neorosporene, ) [1]

376

1.14.99.30

Carotene 7,8-desaturase

  #     ? (, x * 53000, SDS-PAGE [1]; , x * 56740, calculation from nucleotide sequence [5]; , x * 59400, calculation from nucleotide sequence [2]) [1, 2, 5]

 2 %$ %' %

$"

(recombinant enzyme) [1]
 

(overexpression in Escherichia coli [1, 4, 5]; discussion of the phylogenetic relationship between different types of phytoene desaturase and the carotene 7,8-desaturase [4]) [1, 4, 5] (expression in Escherichia coli) [2]

"  [1] Albrecht, M.; Linden, H.; Sandmann, G.: Biochemical characterization of purified z-carotene desaturase from Anabaena PCC 7120 after expression in Escherichia coli. Eur. J. Biochem., 236, 115-120 (1996) [2] Albrecht, M.; Klein, A.; Hugueney, P.; Sandmann, G.; Kuntz, M.: Molecular cloning and functional expression in E. coli of a novel plant enzyme mediating z-carotene desaturation. FEBS Lett., 372, 199-202 (1995) [3] Mayer, M.P.; Beyer, P.; Kleinig, H.: Quinone compounds are able to replace molecular oxygen as terminal electron acceptor in phytoene desaturation in chromoplasts of Narcissus pseudonarcissus L.. Eur. J. Biochem., 191, 359-363 (1990) [4] Sandmann, G.: Phytoene desaturase: genes, enzymes and phylogenetic aspects. J. Plant Physiol., 143, 444-447 (1994) [5] Linden, H.; Misawa, N.; Saito, T.; Sandmann, G.: A novel carotenoid biosynthesis gene coding for zeta-carotene desaturase: functional expression, sequence and phylogenetic origin. Plant Mol. Biol., 24, 369-379 (1994)

377

'  
  *, 

::!

   
 1.14.99.31    n-tetradecanoyl-CoA,NADPH:O2 oxidoreductase [11-(E)-desaturating]    myristoyl-CoA 11-(E) desaturase    (E)-11 myristoyl CoA desaturase desaturase, myristoly coenzyme A (E)-11
    199543-17-4

     Spodoptera littoralis [1, 2]

!    "
 # 

myristoyl-CoA + NAD(P)H + H+ + O2 = (E)-11-tetradecenoyl-CoA + NAD(P)+ + 2 H2 O (, reaction involves a first slow, isotope-sensitive C11 H cleavage, with probable formation of an unstable intermediate, followed by a second fast C12 -H bond removal [2]; , a single enzyme may be responsible for the formation of both (Z)- and (E)-11-tetradecanoic acids. It is also possible that two mechanistically identical discrete enzymes are involved in each desaturation [2])    oxidation redox reaction reduction       myristoyl-CoA + NAD(P)H + O2 (, enzyme is involved in the biosynthesis of Spodoptera littoralis sex pheromone [1, 2]; , a single enzyme may be responsible for the formation of both (Z)- and (E)-11378

1.14.99.31

Myristoyl-CoA 11-(E) desaturase

tetradecanoic acids. It is also possible that two mechanistically identical discrete enzymes are involved in each desaturation [2]) (Reversibility: ? [1]) [1, 2] $ (E)-11-tetradecenoyl-CoA + NAD(P)+ + 2 H2 O [1, 2]      myristoyl-CoA + NAD(P)H + O2 (, a single enzyme may be responsible for the formation of both (Z)- and (E)-11-tetradecanoic acids. It is also possible that two mechanistically identical discrete enzymes are involved in each desaturation [2]) (Reversibility: ? [1, 2]) [1, 2] $ (E)-11-tetradecenoyl-CoA + NAD(P)+ + 2 H2 O [1, 2]

 2 %$ %' %

3#

membrane (, bound to [2]) [2]

"  [1] Navarro, I.; Font, I.; Fabrias, G.; Camps, F.: Stereospecificity of the (E)- and (Z)-11 myristoyl CoA desaturases in the biosynthesis of Spodoptera littoralis sex pheromone. J. Am. Chem. Soc., 119, 11335-11336 (1997) [2] Pinilla, A.; Camps, F.; Fabrias, G.: Cryptoregiochemistry of the D1 1-myristoyl-CoA desaturase involved in the biosynthesis of Spodoptera littoralis sex pheromone. Biochemistry, 38, 15272-15277 (1999)

379

'  
  *A, 

::!

   
 1.14.99.32    n-tetradecanoyl-CoA,NADPH:O2 oxidoreductase [11-(Z)-desaturating]    myristoyl-CoA 11-(Z) desaturase    (Z)-11 myristoyl CoA desaturase desaturase, myristoly coenzyme A (E)-11
    199543-17-4

     Spodoptera littoralis [1, 2]

!    "
 # 

myristoyl-CoA + NAD(P)H + H+ + O2 = (Z)-11-tetradecenoyl-CoA + NAD(P)+ + 2 H2 O (, reaction involves a first slow, isotope-sensitive C11 H cleavage, with probable formation of an unstable intermediate, followed by a second fast C12 -H bond removal [2]; , a single enzyme may be responsible for the formation of both (Z)- and (E)-11-tetradecanoic acids. It is also possible that two mechanistically identical discrete enzymes are involved in each desaturation [2])    oxidation redox reaction reduction       myristoyl-CoA + NAD(P)H + O2 (, enzyme is involved in the biosynthesis of Spodoptera littoralis sex pheromone [1, 2]; , a single enzyme may be responsible for the formation of both (Z)- and (E)-11380

1.14.99.32

Myristoyl-CoA 11-(Z) desaturase

tetradecanoic acids. It is also possible that two mechanistically identical discrete enzymes are involved in each desaturation [2]) (Reversibility: ? [1]) [1, 2] $ (Z)-11-tetradecenoyl-CoA + NAD(P)+ + 2 H2 O [1, 2]      myristoyl-CoA + NAD(P)H + O2 (, a single enzyme may be responsible for the formation of both (Z)- and (E)-11-tetradecanoic acids. It is also possible that two mechanistically identical discrete enzymes are involved in each desaturation [2]) (Reversibility: ? [1, 2]) [1, 2] $ (Z)-11-tetradecenoyl-CoA + NAD(P)+ + H2 O [1, 2]

 2 %$ %' %

3#

membrane (, bound to [2]) [2]

"  [1] Navarro, I.; Font, I.; Fabrias, G.; Camps, F.: Stereospecificity of the (E)- and (Z)-11 myristoyl CoA desaturases in the biosynthesis of Spodoptera littoralis sex pheromone. J. Am. Chem. Soc., 119, 11335-11336 (1997) [2] Pinilla, A.; Camps, F.; Fabrias, G.: Cryptoregiochemistry of the D1 1-myristoyl-CoA desaturase involved in the biosynthesis of Spodoptera littoralis sex pheromone. Biochemistry, 38, 15272-15277 (1999)

381

D "     

::!!

   
 1.14.99.33    linoleate, hydrogen-donor:oxygen oxidoreductase (D12-unsaturating)    D12 -fatty acid dehydrogenase    Crepenynate synthase D12 linoleate acetylenase D12 fatty acid acetylenase crepenylate synthase linoleate D12 -fatty acid acetylenase (desaturase)
    197025-40-4

     Crepis alpina [1]

!    "
 # 

linoleate + AH2 + O2 = crepenynate + A + H2 O    oxidation redox reaction reduction       linoleate + NAD(P)H + O2 (, enzyme is involved in biosynthesis of crepenynic acid [1]) (Reversibility: ? [1]) [1] $ crepenynate + NAD(P)+ + H2 O [1]      linoleate + NADH + O2 (Reversibility: ? [1]) [1] $ crepenynate + NAD+ + H2 O [1]

382

1.14.99.33

D12-fatty acid dehydrogenase

 linoleate + NADPH + O2 (Reversibility: ? [1]) [1] $ crepenynate + NADP+ + H2 O [1] 2  CN- [1]

 2 %$ %' %

% seed [1] 3#

endoplasmic reticulum [1] microsome [1]
 

(gene Crep1 expressed in Saccharomyces cerevisiae YN94-1 strain [1]) [1]

"  [1] Lee, M.; Lenman, M.; Banas, A.; Bafor, M.; Singh, S.; Schweizer, M.; Nilsson, R.; Liljenberg, C.; Dahlqvist, A.; Gummeson, P.O.; Sjoedahl, S.; Green, A.; Stymne, S.: Identification of non-heme diiron proteins that catalyze triple bond and epoxy group formation. Science, 280, 915-918 (1998)

383

'   "&  

::!

   
 1.14.99.34    7-O-methylluteone,NADPH:O2 oxidoreductase    monoprenyl isoflavone epoxidase    epoxidase, monoprenyl isoflavone monoprenyl isoflavone monooxygenase
    198496-86-5

     Botrytis cinerea [1]

!    "
 # 

7-O-methylluteone + NADPH + H+ + O2 = dihydrofurano derivatives + NADP+ + H2 O    oxidation redox reaction reduction       7-O-methylluteone + NADPH + O2 (, key enzyme in metabolism of prenylated flavonoids. Enzyme is induced by the substrate analogue 6prenylnaringenin [1]) (Reversibility: ? [1]) [1] $ dihydrofurano derivatives + NADP+ + H2 O [1]      2'-hydroxylupalbigenin + NADPH + O2 (Reversibility: ? [1]) [1] $ ?

384

1.14.99.34

Monoprenyl isoflavone epoxidase

 2,3-dihydrokievitone + NADPH + O2 (Reversibility: ? [1]) [1] $ ?  7-O-methyl-2,3-dehydrokievitone + NADPH + O2 (Reversibility: ? [1]) [1] $ ?  7-O-methylluteone + NADH + O2 (Reversibility: ? [1]) [1] $ dihydrofurano derivative of 7-O-methylluteone + NADP+ + H2 O [1]  7-O-methylluteone + NADPH + O2 (Reversibility: ? [1]) [1] $ dihydrofurano derivative of 7-O-methylluteone + NADP+ + H2 O [1]  licoisoflavone + NADPH + O2 (Reversibility: ? [1]) [1] $ ?  luteone + NADPH + O2 (Reversibility: ? [1]) [1] $ ?  wighteone + NADPH + O2 (Reversibility: ? [1]) [1] $ ?
"%  FAD (, required for maximal activity [1]) [1] NADPH [1]

 2 %$ %' %

% mycelium [1]

"  [1] Tanaka, M.; Tahara, S.: FAD-dependent epoxidase as a key enzyme in fungal metabolism of prenylated flavonoids. Phytochemistry, 46, 433-439 (1997)

385

)    
    

::!

   
 1.14.99.35    thiophene-2-carbonyl-CoA, hydrogen-donor:oxygen oxidoreductase    thiophene-2-carbonyl-CoA monooxygenase    dehydrogenase, thiophene-2-carbonyl coenzyme A thiophene-2-carbonyl-CoA dehydrogenase thiophene-2-carboxyl-CoA monooxygenase thiphene-2-carboxyl-CoA hydroxylase
    208540-44-7

     Aquamicrobium defluvii (gen. nov. sp. nov. [1]) [1]

!    "
 # 

thiophene-2-carbonyl-CoA + AH2 + O2 = 5-hydroxythiophene-2-carbonylCoA + A + H2 O    oxidation redox reaction reduction       thiophene-2-carbonyl-CoA + AH2 + O2 (, enzyme is involved in metabolism of thiophene 2-carboxylate [1]) (Reversibility: ? [1]) [1] $ 5-hydroxythiophene-2-carbonyl-CoA + A + H2 O

386

1.14.99.35

Thiophene-2-carbonyl-CoA monooxygenase

     thiophene-2-carbonyl-CoA + AH2 + O2 (, tetrazolium salts, e.g. thiazolyl blue, iodo-nitro tetrazolium chloride or nitro blue tetrazolium chloride are active as artificial electron acceptor with phenazine ethosulfate as a mediator [1]) (Reversibility: ? [1]) [1] $ 5-hydroxythiophene-2-carbonyl-CoA + A + H2 O '(   molybdenum (, enzyme contains molybdenum [1]) [1] 0  9 [1]

  #   ' 1  220000 (, gel filtration [1]) [1]

"  [1] Bambauer, A.; Rainey, F.A.; Stackebrandt, E.; Winter, J.: Characterization of Aquamicrobium defluvii gen. nov. sp. nov., a thiophene-2-carboxylate-metabolizing bacterium from activated sludge. Arch. Microbiol., 169, 293-302 (1998)

387

b
  (B    

   
 1.14.99.36    b-carotene:oxygen 15,15'-oxidoreductase (bond-cleaving)    b-carotene 15,15'-monooxygenase    BCO [7] CDO EC 1.13.11.21 (formerly) b-CD [8] b-carotene 15,15'-dioxygenase b-carotene 15,15'-monooxygenase [7] bCDIOX [13] carotene 15,15'-dioxygenase carotene dioxygenase oxygenase, b-carotene 15,15'-di
    37256-60-3

     no activity in Felis catus (intestine [1]) [1] Oryctolagus cuniculus [1, 3, 4, 14, 18] Gallus gallus [1, 9, 12, 21] Domania subtryug [1] Clarias batrachus [1] Rattus norvegicus [2, 3, 5, 6, 13, 14, 20] Cavia porcellus [4, 12] Homo sapiens [7, 11, 15, 17] Mus musculus [8, 10] Sus scrofa [12, 16] Ovis aries [19] Bos taurus (cattle [19]) [19] Capra hircus [19]

388

::!4

1.14.99.36

b-Carotene 15,15'-monooxygenase

!    "
 # 

b-carotene + O2 = 2 retinal (, mechanism [12])    oxidation redox reaction reduction       b-carotene + O2 (, conversion of the plant product bcarotene into a product necessary for the growth and life of the animal organism [5]; , the enzyme catalyzes the first step in the synthesis of vitamin A from dietary carotenoids. May also play a role in peripheral vitamin A synthesis from plasma-borne provitamin A carotenoids [7]; , crucial enzyme in development and metabolism that governs the de novo entry of vitamin A from plant-derived precursors, enzyme may play a critical role in gastrulation [8] [8]; , key enzyme in the metabolism of b,b-carotene to vitamin A [9]; , enzyme may play a critical role in gastrulation [10]; , enzyme plays an important role in retinoid synthesis. BCDO may also be a candidate gene for retinal degenerative disease [15]; , the enzyme is responsible for providing vertebrates with vitamin A by catalyzing oxidative cleavage of b-carotene at its central double bond to two molecules of retinal in intestinal cells [16]) (Reversibility: ? [5, 7, 8, 9, 10, 15, 16]) [5, 7, 8, 9, 10, 15, 16] $ retinal [5]      13-cis-b-carotene + O2 (, 11.4% of the activity with all-trans-bcarotene [13]) (Reversibility: ? [13]) [13] $ retinal  a-carotene + O2 (, 8.2% of the activity with all-trans-b-carotene [13]) (Reversibility: ? [13]) [13] $ retinal  b-carotene + O2 (Reversibility: ? [8, 10, 11, 12]) [8, 10, 11, 12] $ retinal [10, 11, 12] 2  1,1'-biphenyl [16] 1,10-phenanthroline [1, 4, 6, 14, 16] 15,15'-dehydro-b-apo-10'-carotenol (, inhibits reaction with b-apo10'-carotenol [1]) [1] 2,2'-dipyridyl [1, 4, 6, 7] 2,6-di-tert-butyl-4-methylphenol (, 0.001 mM, strong mixed-type inhibition [16]) [16] Ag+ [5]

389

b-Carotene 15,15'-monooxygenase

1.14.99.36

EDTA (, 0.02 mM, very slight inhibition [6]) [6] Fe2+ (, 4 mM, 60% inhibition [4]) [4] NEM [1, 4, 5, 6] PCMB (, reversed by glutathione [1]) [1, 4, 7] SDS (, required for maximal activity [14]) [4, 14] astaxanthin (, competitive [18]) [18] butylated hydroxyanisole (, moderate inhibition [16]) [16] canthaxanthin (, mixed inhibition [13]) [13] curcumin (, moderate inhibition [16]) [16] desferrioxamine (, noncompetitive inhibitor [17]) [17] iodoacetamide [1, 5, 6] iodoacetate [4] lutein (, competitive [18]) [18] luteolin (, remarkable noncompetitive inhibition [16]) [16] lycopene (, competitive [18]) [18] n-propyl gallate (, moderate inhibition [16]) [16] nordihydroguaiaretic acid (, moderate inhibition [16]) [16] p-hydroxymercuribenzoate [5, 6] phenanthrene [16] phloretin (, remarkable noncompetitive inhibition [16]) [16] quercetin (, remarkable noncompetitive inhibition [16]) [16] rhamnetin (, remarkable noncompetitive inhibition [16]) [16] sodium arsenide [5, 6] sodium glycocholate [19] zeaxanthin (, non-competitive [13]) [13] Additional information (, catechol structure of the ring B and a planar flavone structure are essential for inhibition [16]) [16] &

  1-S-octyl-b-d-thioglucopyranoside (, detergent required, maximal activity at 1% w/v [7]) [7] GSH (, activates [4]; , stimulates [5]; , thiol-dependent enzyme [14]) [4, 5, 14] SDS (, 2-6 mg, stimulates [5]; , stimulates [6]; , detergent required, low activation [7]) [5, 6, 7] Triton X-100 (, detergent required [7]) [7] Tween 20 (, stimulates [6]) [6] Tween 40 (, stimulates [6]) [6] Tween 80 (, stimulates [6]) [6] glycocholate (, plus lecithin, stimulates [5]) [5] hexadecyl trimethyl ammonium bromide (, stimulates [6]) [6] lecithin (, plus glycocholate, stimulates [5]; , egg or plant lecithin stimulates [6]) [5, 6] linoleic acid (, stimulates [6]) [6] lysolecithin (, lysolecithin, stimulates [6]) [6] monoolein (, significant stimulation [6]) [6] octyl b-glucoside (, detergent required [7]) [7]

390

1.14.99.36

b-Carotene 15,15'-monooxygenase

oleyl acid phosphate (, stimulates [6]) [6] palmitic acid (, stimulates [6]) [6] sodium cholate (, detergent required [7]) [7] sodium dodecyl phosphate (, stimulates [6]) [6] sodium glycocholate (, optimum concentration is 6 mM [12]) [12] sphingomyelin (, stimulates [6]) [6] Additional information (, the enzyme is completely inactive in absence of any added bile salt or detergent [6]; , maximal reaction by addition of an appropriate combination of detergent and bile salt, SDS, and egg lecithin [19]) [6, 19] '(   Fe2+ (, activates [4]; , maximal activation, 5.8fold, at 1 mM [4]) [4] iron (, the iron-dependent enzyme is sensitive to copper status in vivo [20]) [20] ) &  * +, 0.006 (b-carotene) [8, 10] 0.66 (b-carotene) [7] 40.06 (b-cryptoxanthin) [7] " & *-% , 0.000035 [1] 0.000036 [4] . / *', 0.0016 (b-carotene) [11] 0.0033 (b-carotene, , intestinal enzyme [5]) [5, 6] 0.0035 (13-cis-b-carotene) [13] 0.0057 (all-trans-b-carotene) [13] 0.0062 (a-carotene) [13] 0.0067 (b-cryptoxanthin) [13] 0.0071 (b-carotene) [7] 0.0077 (8'-apo-b-carotenal) [13] 0.0085 (8'-apo-b-carotenal) [13] 0.0092 (10'-apo-b-carotenal) [13] 0.0095 (b,b-carotene) [4] 0.03 (b-cryptoxanthin) [7] 0.067 (b-apo-10'-carotenol) [1] . / *', 0.00079 (2,6-di-tert-butyl-4-methylcatechol) [16] 0.0016 (canthaxanthin) [13] 0.0058 (rhamnetin) [16] 0.0078 (zeaxanthin) [13] 0.0099 (phloretin) [16] 0.0133 (luteolin) [16] 0.0169 (curcumin) [16]

391

b-Carotene 15,15'-monooxygenase

1.14.99.36

0.0474 (butylated hydroxyanisole) [16] 0.1187 (3,5-di-tert-butyltoluene) [16] 0  7.5-8 [6] 7.7 [5] 7.8 (, reaction with b-apo-10'-carotenol [1]) [1, 4] 8 [14] 8.6 [4] 0 

 7-8.5 (, pH 7.0: about 60% of maximal activity, pH 8.5: about 75% of maximal activity [14]) [14] 7-9 (, pH 7.0: about 50% of maximal activity, pH 9.0: about 35% of maximal activity, reaction with b-apo-10'-carotenol [1]) [1]

  #   ' 1  50000 (, gel filtration [9]) [9] 230000 (, gel filtration [7]) [7]   ? (, x * 63859, calculation from nucleotide sequence [8]) [8] tetramer [7]

 2 %$ %' %

% CACO-2 cell (, intestinal cell line Caco-2/subclone TC7 [11, 17]; , when TC7 cells are maintained in serum-free medium, activity is increased 8.2fold after 19 days of postconfluence [11]) [11, 17] brain [15] embryo (, mRNA is abundant at embryonic day 7, with lower expression at embryonic days 11, 13 and 15 [8]) [8] intestine (, mucosa [1, 3, 6, 12, 14, 19, 21]; , pylorus to the lower ileum [1]; , activity is 20-30% greater in vitamin-A deficient animals than in the controls [2]; , high levels of BCO mRNA, highest level in jejunum [7]; , highest expression in duodenum, mRNA level in ileum is markedly low [9]; , activity detected in two subclones of Caco-2, PF11 and TC7. When TC7 cells are maintained in serum-free medium, activity is increased 8.2fold after 19 days of postconfluence. No activity detected in IPEC-1, HepG2, HL60, Wurzburg, or parent Caco-2, PF11 and TC7 [11]) [1, 2, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 16, 19, 21]

392

1.14.99.36

b-Carotene 15,15'-monooxygenase

kidney (, high levels of BCO mRNA [7]; , only marginal activity [9]) [7, 9, 10, 15] liver (, high levels of BCO mRNA [7]) [5, 7, 8, 9, 10, 15, 17] ovary (, low levels of BCO mRNA [7]) [7] prostate gland (, low levels of BCO mRNA [7]) [7] retina (, enzyme is highly expressed in retinal pigment epithelium [15]) [15] skeletal muscle (, low levels of BCO mRNA [7]) [7] small intestine (, mucosa [17]) [10, 17, 20] stomach [15] testis (, low levels of BCO mRNA [7]) [7, 8, 9, 10, 15] Additional information (, no expression in lung [9]; , no activity is detected in adult stomach tissue [17]) [9, 17] 3#

cytosol [3, 8, 10] soluble [12] $"

[1, 4] [9] (partial [6]) [6] (partial [12]) [4, 12] [7]
 

(expression in Escherichia coli and in CHO cells [9]) [9] (expression by an baculovirus/Spodoptera frugiperda 9 insect cell system [7]; baculovirus expressed [15]) [7, 15] (expression in Escherichia coli [8, 10]) [8, 10, 15]

4  )  52 (, 3 min, no effect [14]) [14] 64 (, 55 seconds, complete inactivation of intestinal enzyme [5]) [5] 5    "

, carotenoids stabilize the enzyme during the isolation from small intestinal mucosa [18] , liver enzyme may be frozen and thawed repeatedly without loss of activity [5] , loss of activity during concentration by ultrafiltration or (NH4 )2 SO4 precipitation as well as during dialysis [12]    , -20 C, as (NH4 )2 SO4 precipitate, 25-55%, for one month without considerable loss in activity [4]

393

b-Carotene 15,15'-monooxygenase

1.14.99.36

"  [1] Lakshamanan, M.R.; Chansang, H.; Olson, J.A.: Purification and properties of carotene 15,15-dioxygenase of rabbit intestine. J. Lipid Res., 13, 477-482 (1972) [2] Villard, L.; Bates, C.J.: Carotene dioxygenase (EC 1.13.11.21) activity in rat intestine: effects of vitamin A deficiency and of pregnancy. Br. J. Nutr., 56, 115-122 (1986) [3] Lakshman, M.R.; Mychkovsky, I.; Attlesey, M.: Enzymatic conversion of alltrans-b-carotene to retinal by a cytosolic enzyme from rabbit and rat intestinal mucosa. Proc. Natl. Acad. Sci. USA, 86, 9124-9128 (1989) [4] Singh, H.; Cama, H.R.: Enzymatic cleavage of carotenoids. Biochim. Biophys. Acta, 370, 49-61 (1974) [5] Goodman, D.S.; Olson, J.A.: The conversion of all-trans b-carotene into retinal. Methods Enzymol., 15, 462-475 (1969) [6] Goodman, D.S.; Huang, H.S.; Kanai, M.; Shiratori, T.: The enzymatic conversion of all-trans b-carotene into retinal. J. Biol. Chem., 242, 3543-3554 (1967) [7] Lindqvist, A.; Andersson, S.: Biochemical properties of purified recombinant human b-carotene 15,15'-monooxygenase. J. Biol. Chem., 277, 2394223948 (2002) [8] Redmond, T.M.; Gentleman, S.; Duncan, T.; Yu, S.; Wiggert, B.; Gantt, E.; Cunningham, F.X.: Identification, expression, and substrate specificity of a mammalian b-carotene 15,15'-dioxygenase. J. Biol. Chem., 276, 6560-6565 (2001) [9] Wyss, A.; Wirtz, G.M.; Woggon, W.D.; Brugger, R.; Wyss, M.; Friedlein, A.; Riss, G.; Bachmann, H.; Hunziker, W.: Expression pattern and localization of b,b-carotene 15,15'-dioxygenase in different tissues. Biochem. J., 354, 521-529 (2001) [10] Redmond, T.M.; Gentleman, S.; Duncan, T.; Yu, S.; Wiggert, B.; Gantt, E.; Cunningham, F.X.: Identification, expression, and substrate specificity of a mammalian b-carotene 15,15'-dioxygenase. J. Biol. Chem., 276, 6560-6565 (2001) [11] During, A.; Albaugh, G.; Smith, J.C., Jr.: Characterization of b-carotene 15,15'-dioxygenase activity in TC7 clone of human intestinal cell line Caco-2. Biochem. Biophys. Res. Commun., 249, 467-474 (1998) [12] Devery, J.; Milborrow, B.V.: b-Carotene-15,15'-dioxygenase (EC 1.13.11.21) isolation reaction mechanism and an improved assay procedure. Br. J. Nutr., 72, 397-344 (1994) [13] Grolier, P.; Duszka, C.; Borel, P.; Alexandre-Gouabau, M.C.; Azais-Braesco, V.: In vitro and in vivo inhibition of b-carotene dioxygenase activity by canthaxanthin in rat intestine. Arch. Biochem. Biophys., 348, 233-238 (1997) [14] Dmitrovskii, A.A.; Gessler, N.N.; Gomboeva, S.B.; Ershov, Y.V.; Bykhovsky, V.Y.: Enzymic oxidation of b-apo-8'-carotenol to b-apo-14'-carotenal by an

394

1.14.99.36

[15]

[16] [17] [18] [19] [20] [21]

b-Carotene 15,15'-monooxygenase

enzyme different from b-carotene-15,15'-dioxygenase. Biochemistry (Moscow), 62, 787-792 (1997) Yan, W.; Jang, G.F.; Haeseleer, F.; Esumi, N.; Chang, J.; Kerrigan, M.; Campochiaro, M.; Campochiaro, P.; Palczewski, K.; Zack, D.J.: Cloning and characterization of a human b,b-carotene-15,15'-dioxygenase that is highly expressed in the retinal pigment epithelium. Genomics, 72, 193-202 (2001) Nagao, A.; Maeda, M.; Lim, B.P.; Kobayashi, H.; Terao, J.: Inhibition of bcarotene-15,15'-dioxygenase activity by dietary flavonoids. J. Nutr. Biochem., 11, 348-355 (2000) During, A.; Smith, M.K.; Piper, J.B.; Smith, J.C.: b-Carotene 15,15'-dioxygenase activity in human tissues and cells: evidence of an iron dependency. J. Nutr. Biochem., 12, 640-647 (2001) Ershov Yu, V.; Bykhovsky, V.; Dmitrovskii, A.A.: Stabilization and competitive inhibition of b-carotene 15,15'-dioxygenase by carotenoids. Biochem. Mol. Biol. Int., 34, 755-763 (1994) Yang, A.; Tume, R.K.: A comparison of b-carotene-splitting activity isolated from intestinal mucosa of pasture-grazed sheep, goats and cattle. Biochem. Mol. Biol. Int., 30, 209-217 (1993) During, A.; Fields, M.; Lewis, C.G.; Smith, J.C.: b-Carotene 15,15'-dioxygenase activity is responsive to copper and iron concentrations in rat small intestine. J. Am. Coll. Nutr., 18, 309-315 (1999) Wirtz, G.M.; Bornemann, C.; Giger, A.; Muller, R.K.; Schneider, H.; Schlotterbeck, G.; Schiefer, G.; Woggon, W.D.: The substrate specificity of b,b-carotene 15,15'-monooxygenase. Helv. Chim. Acta, 84, 2301-2315 (2001)

395

)  a   

::!8

   
 1.14.99.37    taxa-4,11-diene,hydrogen-donor:oxygen oxidoreductase (5a-hydroxylating)    taxadiene 5a-hydroxylase    oxygenase, taxadiene 5-monotaxadiene 5-hydroxylase taxadiene 5-monooxygenase

     Taxus brevifolia [1, 3] Taxus cuspidata [1, 2, 3]

!    "
 # 

taxa-4,11-diene + AH2 + O2 = taxa-4(20),11-dien-5a-ol + A + H2 O ( requires P450, reaction includes rearrangement of the 4(5)-double bound to a 4(20)-double bond, possibly through allylic oxidation, proposed mechanism [1])    oxidation redox reaction reduction       taxa-4,11-diene + NADPH + O2 ( reaction is completely dependent on NADPH, enzyme catalyzes the first oxygenating step in taxol biosynthesis pathway [1]) (Reversibility: ? [1]) [1, 2, 3] $ taxa-4(20),11-dien-5a-ol + NADP+ + H2 O [1, 2, 3]

396

1.14.99.37

Taxadiene 5a-hydroxylase

     taxa-4,11-diene + NADPH + O2 ( reaction is completely dependent on NADPH [1]) (Reversibility: ? [1]) [1, 2, 3] $ taxa-4(20),11-dien-5a-ol + NADP+ + H2 O [1, 2, 3] 2  CO ( inhibits taxadiene hydroxylation in the dark, inhihibition is partially reversed by blue light at 450 nm [1]) [1] clotrimazole ( 0.003 mM, complete inhibition [1]) [1] cytochrome c ( 0.4 mM, 50% inhibition [1]) [1] miconazole ( 0.003 mM, complete inhibition [1]) [1]
"%  NADPH ( absolutely required [1]) [1] &

  FAD ( saturating amounts increase activity [1]) [1] FMN ( saturating amounts increase activity [1]) [1] . / *', 0.006 (taxa-4,11-diene, approx. value [1]) [1] 0  7.2 ( activity in extracts, approx. 50% of maximal activity at pH 6.2 and pH 8.2 [1]) [1]

 2 %$ %' %

% cell suspension culture [1, 2] stem [1] 3#

microsome ( 70% activity resides in light membrane fraction, approx. 25% in dense membrane fraction [1]) [1, 2] 

medicine ( enzyme may be useful in the biological production of the potent antimitotic drug taxol which shows activity against a range of cancers [1, 3]) [1, 3]

"  [1] Hefner, J.; Rubenstein, S.M.; Ketchum, R.E.B.; Gibson, D.M.; Williams, R.M.; Croteau, R.: Cytochrome P450 -catalyzed hydroxylation of taxa-4(5),11(12)diene to taxa-4(20),11(12)-dien-5a-ol: the first oxygenation step in taxol biosynthesis. Chem. Biol., 3, 479-489 (1996)

397

Taxadiene 5a-hydroxylase

1.14.99.37

[2] Wheeler, A.L.; Long, R.M.; Ketchum, R.E.B.; Rithner, C.D.; Williams, R.M.; Croteau, R.: Taxol biosynthesis: differential transformations of taxadien-5aol and its acetate ester by cytochrome P450 hydroxylases from Taxus suspension cells. Arch. Biochem. Biophys., 390, 265-278 (2001) [3] Walker, K.; Croteau, R.: Taxol biosynthetic genes. Phytochemistry, 58, 1-7 (2001)

398

 



   
 1.15.1.1    superoxide:superoxide oxidoreductase    superoxide dismutase    Cu,Zn-SOD Cu-Zn superoxide dismutase EC-SOD ( extracellular superoxide dismutase [42, 92]) [42, 92] Fe-SOD Mn-SOD SOD SOD-1 SOD-2 SOD-3 SOD-4 SODF SODS copper-zinc superoxide dismutase cuprein cytocuprein dismutase, superoxide erythrocuprein ferrisuperoxide dismutase hemocuprein hepatocuprein nectarin I [103] superoxidase dismutase superoxide dismutase I superoxide dismutase II
    9054-89-1

399

Superoxide dismutase

1.15.1.1

     Bos taurus (Cu,Zn-SOD [9, 34, 36, 37, 60, 66, 77]) [1, 9, 11, 22, 34-37, 60, 66, 77] Streptococcus mutans (Mn-SOD and Fe-SOD [8]; 2 isoenzymes I and II [2]) [2, 8] Pisum sativum (Mn-SOD [25, 47, 96]) [3, 25, 47, 96] Bacteroides gingivalis (structural intermediate between Mn-SOD and FeSOD [4,24]; metal content depends on O2 -concentration [24]) [4, 24] Homo sapiens (EC-SOD with Cu,Zn-SOD activity [92]; Cu,Zn-SOD [10, 11, 33, 40-42, 86, 87, 90, 98]; Mn-SOD [5, 32, 42]) [5, 8, 10, 11, 32, 33, 40-42, 86, 87, 90, 92, 98] Photobacterium leiognathi (strain ATCC25521 [6]; Cu,Zn-SOD [6, 8, 9]) [6, 8-10] Caulobacter crescentis (strain CB15 [9]; Cu,Zn-SOD [8,9]) [8, 9] Bacteroides thetaiotaomicron [8] Rhodococcus bronchialis [8] Mycobacterium phlei [8] Thermoplasma acidophilum (Fe-SOD [8]) [8] Escherichia coli (strain B [49]; Mn-SOD [8,9]; Fe-SOD [8,9,49]) [8, 9, 22, 49] yeast (Mn-SOD [9]; Cu,Zn-SOD [7,9]) [7, 9] Geobacillus stearothermophilus (Mn-SOD [8, 9, 38, 39]) [8, 9, 38, 39] Rattus norvegicus (Mn-SOD [30,43,45]; Cu,Zn-SOD [43, 44]) [8, 12, 30, 43-45] Aerobacter aerogenes (strain IFO 3317 [13]) [13] Arachis hypogaea (5 isozymes: SOD-II-SOD-V are Cu,Zn-SODs, SOD-I is a Mn-SOD [14]) [14] Bacillus halodenitrificans (Mn-SOD [15]) [15] Brassica oleracea (var. bullata sub var. gemmifera [16]; Cu,Zn-SOD [16]) [16] Cyprinus carpio (Mn-SOD, and 2 isozymes of Cu,Zn-SOD [17]) [17] Ascaris suum (3 isozymes: Mn-SOD, Cu,Zn-SOD I and II [18]) [18] Methanobacterium thermoautotrophicum (Fe-SOD, but features are similar to Mn-SOD [19]) [19] Pinus sylvestris (4 isozymes: SOD-I, SOD-II, SOD-III, SOD-IV [20]) [20] Dirofilaria immitis (Cu,Zn-SOD [21]) [21] Triticum aestivum [22] Saccharomyces cerevisiae (Mn-SOD [65,102]; Cu,Zn-SOD [46,66]) [8, 22, 46, 65, 66, 102] Citrullus vulgaris (Cu,Zn-SOD: SOD-II [23]) [23] Drosophila melanogaster (Cu,Zn-SOD [26,72]) [26, 72] Nuphar luteum (sub spec. macrophyllum [27]; Fe-SOD [27]) [27] Acholeplasma laidlawii (Mn-SOD [28]) [28] Lycopersicon esculentum (Fe-SOD [69]; Cu,Zn-SOD: 2 isozymes I and II [29]) [29, 69]

400

1.15.1.1



























Superoxide dismutase

Flavobacterium marinolyticum (NCMB 559 [31]) [31] Flavobacterium oceanosedimentum (ATCC 31317 [31]) [31] Flavobacterium okeanokoides (NCMB 561 [31]) [31] Flavobacterium odoratum (CCM 2873 [31]) [31] Flavobacterium breve (CCM 2867 [31]) [31] Flavobacterium ferrugineum [31] Flavobacterium halmephilum (CCM 2833 [31]; Fe-SOD and Mn-SOD [31]) [31] Flavobacterium sp. (A-101, A-217, A-364 [31]) [31] Lens esculenta (Cu,Zn-SOD [50]) [50] Gluconobacter cerinus (IFO 3268, Mn-SOD [51]) [51] Bacillus circulans (IFO 3329 [52]; Mn-SOD [52]) [52] Halobacterium halobium (strain NRL, enzyme is oxygen-inducible [53]) [8, 53] Xenopus laevis (Cu,Zn-SOD: 3 isozymes AA, AB, BB [54]) [54] Paracoccus denitrificans (Cu,Zn-SOD [8,55]; Mn-SOD [56]) [8, 55, 56] Spinacia oleracea (Mn-SOD [57]) [48, 57] Azotobacter chroococcum (Mn-SOD [58]) [58] Brassica campestris (Fe-SOD [59]) [59] Zea mays (SOD-I [61]; SOD-II, SOD-IV both belonging to Cu,Zn-SODs [62]; SOD-III [62]) [8, 61, 62] Neurospora crassa (Cu,Zn-SOD [63,64]; Mn-SOD [64]) [63, 64] Thermus aquaticus (Mn-SOD [67]) [67] Porphyridium cruentum (Mn-SOD [68]) [68] Nocardia asteroides (strain GUH-2 [70]; structural intermediate between Mn-SOD and Fe-SOD, Zn-containing [70]) [8, 70] Crithidia fasciculata (Fe-SOD [71]; 3 isozymes [71]) [71] Trypanosoma brucei [71] Trypanosoma cruzi [71] Leishmania tropica [71] Bacteroides fragilis (Fe-SOD, when grown anaerobically [8]; Mn-SOD, O2 inducible [73]) [8, 73] Ginkgo biloba (Mn-SOD, Cu,Zn-SOD [74]; Fe-SOD: 2 isozymes, contains also Zn2+ and Cu2+ [74]) [8, 74] Plectonema boryanum (Fe-SOD [75]) [8, 75] Thermus thermophilus (strain HB8 [76]; Mn-SOD [8,76]) [8, 76] Rhodopseudomonas sphaeroides (aerobically grown [78]; Mn-SOD [78]) [78] Spirulina platensis (Fe-SOD [78]) [78] Pseudomonas ovalis (Fe-SOD [8,79]) [8, 79] Methanobacterium bryantii (Fe-SOD [80]) [8, 80] Heterometrus fulvipes (Mn-SOD [81]) [81] Rhodococcus fragilis [8] Anacystis nidulans [8] Propionibacterium shermanii (structural intermediate between Mn-SOD and Fe-SOD [8,9]) [8, 9] Mus musculus [8] 401

Superoxide dismutase















1.15.1.1

Pseudomonas putida [10] Bacillus subtilis [22] Marchantia paleacea (var. diptera [82]; Cu,Zn-SOD [82]) [82] Candida albicans (SOD-3 gene, Mn-SOD [83]) [83] Aspergillus fumigatus (Cu,Zn-SOD [84]) [84] Photobacterium damselae (subsp. piscicida, formerly Pasteurelle piscicida [85]; Fe-SOD [85]) [85] Gallus gallus (Cu,Zn-SOD [88]) [88] Pseudomonas carboxydohydrogena (strain DSM 1083 [89]; Mn-SOD [89]) [89] Anas platyrhynchos domestica (Peking duck [91]; Cu,Zn-SOD [91]) [91] Canis familiaris (Cu,Zn-SOD [93]) [93] Aspergillus flavus (Cu,Zn-SOD [94]) [94] Aspergillus niger (Cu,Zn-SOD [94]) [94] Aspergillus nidulans (Cu,Zn-SOD [94]) [94] Aspergillus terreus (Cu,Zn-SOD [94]) [94] Cinnamomum camphora (Fe-SOD [95]) [95] Sulfolobus solfataricus (oxygen-inducible, extracellular Fe-SOD [97]) [97] Desulfovibrio gigas (Fe-SOD [99]) [99] Sinorhizobium meliloti (strain Rm5000 [100]; structural intermediate between Mn-SOD and Fe-SOD [100]) [100] Citrus lemon (Cu,Zn-SOD [101]) [101] Nicotiana sp. (nectarin I with Mn-SOD activity [103]) [103]

!    "
 # 

2 O-2 + 2 H+ = O2 + H2 O2 (A metalloprotein. Enzymes from most eukaryotes contain both copper and zinc, those from mitochondria and most prokaryotes contain manganese or iron. ligand binding site and structure [98, 101, 102]; Cu2+ -binding [35]; mechanism [8, 9, 34, 37, 38]; active site, manganese-binding site and contact site between monomers [8]; amino acid composition, comparison [25, 28, 40, 41, 46, 54, 57, 59, 65, 69, 74, 78, 79, 89, 94, 96]; amino acid sequence alignment and comparison [8, 53, 83, 85, 91, 99-102]; active site is not conserved, differing from others of Mn-SOD and Fe-SOD [8]; three-dimensional structure [9])    oxidation redox reaction reduction       O-2 + H+ ( Mn-SOD, expression is strongly stimulated during stationary phase in cell culture, 402

1.15.1.1

Superoxide dismutase

enzyme is atypical and plays an important role in cell protection against reactive oxygen in the cytosol in the stationary phase [83]; defense against oxidants [21, 97]) (Reversibility: ? [8, 9, 21, 22, 83, 97]) [8, 9, 21, 22, 83, 97] $ O2 + H2 O2 [8, 9, 21, 22, 97]      O-2 + H+ (Reversibility: r [16, 87]; ? [1-15, 17-85, 88-103]) [1-103] $ O2 + H2 O2 ( Mn-SOD is unaffected by H2 O2 [38, 39]; Fe-SODs are inhibited by H2 O2, but Mn-SODs are not [8]) [1-103]  Additional information ( dismutation of superoxide anions is promoted by reduction of Cu2+ to Cu+ [44]; addition of hexacyanoferrate results in reduction of Cu(II) to Cu(I) [37]; enzyme can reduce ferrocyanide to ferricyanide at pH 5.0-8.7 [36]) [36, 37, 44] $ ? 2  2,4,6-trinitrobenzenesulfonate ( 0.5 M, pH 9.0, 25 C, native wildtype enzyme: half-life 3.5 min, recombinant wild-type enzyme: half-life: 5.1 min, recombinant mutant H30A: half-life 5.5 min, recombinant mutant K170R half-life 101 min [102]) [102] 5,5'-dithiobis(2-nitrobenzoate) (Mn-SOD [32, 41]) [32, 41] CN- ( no inhibition: FeSOD [99]; no inhibition [13, 19, 25, 27, 30, 57, 58, 71, 74, 85, 89]; no inhibition Mn-SOD [45, 64, 68, 81, 103]; Cu,Zn-SOD [34, 45, 64, 72, 88]; extracellular enzyme [42]; slight inhibition, Mn-SOD [39, 83]; at 1-3 mM, complete inhibition [72]; SOD-2 and SOD-4 inhibited, SOD3 not inhibited [62]; contains a cyanide-sensitive enzyme in cytosol and mitochondrial intermembrane space and one cyanide-insensitive enzyme in mitochondrial matrix [64]) [16, 20, 34, 39, 42, 45, 55, 62, 64, 72, 83, 88, 94, 99] EDTA ( Mn-SOD [25, 81]; slightly [25]; no inhibition [79, 84]) [25, 81, 94] H2 O2 ( no inhibition: Mn-SOD [8, 15, 25, 28, 39, 57, 68, 78, 81, 83, 89, 100, 103]; no inhibition: Fe-SOD [19, 100]; Cu,Zn-SOD [8, 29, 84, 88]; complete inhibition [84]; extracellular enzyme, rapidly [42]; Fe-SOD [27, 7880, 85]; SOD-2 and SOD-4 inhibited, SOD-3 not [62]) [8, 13, 20, 27, 29, 42, 57, 62, 69-71, 74, 78-80, 84, 85, 88, 99, 100] N3 - ( no inhibition, Fe-SOD [19, 39]; no inhibition: Zn,Cu-SOD [64]; binds to Fe3+ , but has no effect on activity [49]; MnSOD [25, 68, 81, 89]; extracellular enzyme [42, 81]; Fe-SOD, 403

Superoxide dismutase

1.15.1.1

slightly [27, 72]; SOD-2 and SOD-4 inhibited, SOD-3 not inhibited [62]; Mn-SOD is inhibited by 50%, enzyme reconstituted by Fe3+ shows increased inhibition [73]) [8, 13, 15, 25, 27, 29, 42, 62, 64, 68-73, 81, 89, 94, 99] O2 ( repressed expression [85]) [85] OH- ( Cu,Zn-SOD, competitively [34]) [34] chloroform ( Mn-SOD [81]) [81] concanavalin A ( inhibition in vivo and in vitro, essentially dependent on calcium chloride [22]) [22] diethyldithiocarbamate ( inhibits recombinant Cu,Zn-SOD, at 0.05-0.1 mM inactivation occurs gradually within 1 h [90]; copper-chelator, wild-type and mutant Cu,Zn-SOD [87]; complete inhibition, Cu,Zn-SOD [84]; Mn-SOD [81]; strong inhibition, extracellular enzyme [42]; slightly [25, 81]) [25, 42, 81, 84, 87, 90, 94] ethanol ( Mn-SOD [81]) [81] fluoride ( Fe-SOD [49]) [49] guanidinium chloride ( Mn-SOD, 70% inhibition at 1 mM [81]) [81] iodoacetamide ( Cu,Zn-SOD [88]; Mn-SOD [32]) [32, 88] nitroprusside ( Mn-SOD [81]) [81] o-phenanthroline ( Mn-SOD [81]; slightly [25]; Fe-SOD [79]; depending on assay method [79]) [25, 79, 81, 94] p-hydroxymercuribenzoate ( Mn-SOD [25, 81]; completely inhibited at 1 mM [25, 81]) [25, 81] penicillamine ( copper-chelator, wild-type and mutant Cu,Zn-SOD [87]) [87] perchlorate ( competitive [9]) [9] phenyl mercuric acetate (Cu,Zn-SOD [41]) [41] phenylglyoxal ( 25% activity remaining after 3 h for native and recombinant wild-type and recombinant mutant H30A, complete inactivation of recombinant mutant K179R after 7 min [102]) [102] sodium dodecyl sulfate ( 1%, complete inhibition [81]; 2% w/v, Cu,Zn-SOD and EC-SOD [42]) [42, 81] urea ( Mn-SOD, 90% inhibition at 6 M [81]) [81] Additional information ( no inhibition by dithiothreitol and b-mercaptoethanol [88]; no inhibition by N-ethylmaleide [79]; insensitive to fluoride [13]) [13, 79, 88]
"%  Additional information ( a liganding water molecule is evident [8]) [8] &

  8-hydroxyquinoline-5-sulfonate ( Fe-SOD, slightly stimulating, depending on the assay method [79]) [79] o-phenanthroline ( Fe-SOD, slightly stimulating [79]; depending on assay method [79]) [79] sulfhydryl compounds ( e.g. reduced glutathione, cysteine, 2-mercaptopropionylglycine activate [44]) [44] 404

1.15.1.1

Superoxide dismutase

'(   Co2+ ( Co2+ binds at zinc site [98]; Co(II) can substitute for zinc in erythrocytes [77]) [77, 98] Cu2+ ( no copper [2]; 2 mol of Cu per mol of enzyme [3, 9, 37, 46, 50, 63, 72, 74]; heart: 1.64 mol of Cu per mol of enzyme, erythrocyte: 1.84 mol of Cu per mol of enzyme [1]; 1.7 mol per mol of enzyme [41]; extracellular EC-SOD with Cu,Zn-SOD activity [92]; Cu,Zn-SOD mutant H63C [98]; 1.1 mol per mol of Cu,Zn-SOD [16]; 1 gatom per mol of enzyme [23, 88]; 1.631.78 mol per mol of isoenzyme I [29]; 1.86-1.97 mol per mol of isoenzyme II [29]; SOD-1, SOD-2 and SOD-4 [62]; mitochondrial cyanide-sensitive enzyme [64]) [1, 3, 8, 9, 14, 16-18, 20, 21, 23, 26, 29, 34-37, 4044, 46, 48, 50, 54, 55, 60, 62-64, 66, 72, 74, 82, 84, 86-88, 91, 92, 94, 98, 101] Mn2+ ( contains no manganese [1, 23]; 1.22 mol per mol of enzyme [8, 15, 70]; isoform I, 1.85 atoms manganese per mol of enzyme [2]; accepts iron and/or manganese as cofactor [4, 24, 100]; 0.05 mol per mol of enzyme [8]; 2.2 mol per mol of enzyme [8]; 1.7 mol per mol of enzyme [8]; 1.2-1.8 mol per mol of Mn-SOD [8, 30]; 4 mol per mol of enzyme [9]; 3.69 mol per mol of enzyme [45]; 1 atom per subunit [65, 78]; 0.5 mol per mol of subunit [28, 103]; 1.3 gatoms per mol of enzyme [31]; 1.5 mol per mol of enzyme [8]; 1.1 mol per mol of enzyme [8, 73, 89]; 0.22 mol per mol of enzyme [8]; 2 atoms of manganese per molecule [67, 76]; less than 0.2 mol per mol of enzyme [80]; 0.9 Mn per mol of enzyme [83]; 0.89 mol per mol of liver Mn-SOD [93]; 0.75 mol per mol of subunit [100]; specific for [103]) [2, 4, 8, 9, 14, 15, 18, 24, 25, 28, 30-32, 38, 43, 45, 47, 51-53, 56, 57, 65, 67, 68, 70, 73, 76, 78, 80, 81, 83, 89, 93, 100, 102, 103] Zn2+ ( 2 mol of Zn2+ per mol of enzyme [3, 9, 37, 48, 50, 63, 64, 66, 72, 74]; 1.8 mol of Zn per mol of enzyme [1, 8, 46, 80]; 1.0 gatom per mol of enzyme [8, 23]; no zinc [2]; 1.6 mol per mol of enzyme [41]; extracellular EC-SOD with Cu,Zn-SOD activity [92]; 0.6-0.7 mol per mol of enzyme [8]; 1.3 mol per mol of Cu,Zn-SOD [16]; 1.34-1.81 mol per mol of isoenzyme I [29]; 1.9-20.0 mol per mol of isoenzyme II [29]; 1.5 mol per mol of enzyme [8]; 0.5 mol per mol of enzyme [53]; SOD-1, SOD-2 and SOD-4 [62]; mitochondrial cyanide-sensitive enzyme [64]; 1.2 mol per mol of enzyme [8]; 0.2 mol of Cu per mol of enzyme [8, 73, 75]; 2.2 mol per mol of enzyme [8]; 0.85 mol per mol of enzyme [88]) [1, 3, 8, 9, 14, 16-18, 20, 21, 23, 26, 29, 3437, 40-44, 46, 48, 50, 53-55, 60, 62-64, 66, 72, 74, 80, 82, 84, 86-88, 91, 92, 94, 101] iron ( contains no iron [23]; 1.0405

Superoxide dismutase

1.15.1.1

1.45 mol per mol of enzyme [8, 27, 69, 70, 74, 75, 78, 79]; 2.0 mol per mol of enzyme [8, 70]; 1.8-1.9 mol (gatoms) per mol of enzyme [8, 31, 49]; accepts iron and/or manganese as cofactor [4, 24]; 0.9 mol per mol of enzyme [8]; each Fe3+ ion has 2 coordination positions available for interaction with solute molecules but only 1 is necessary for catalysis [49]; 1.6 mol per mol of enzyme [59]; 2.7-2.8 mol per mol of enzyme [71, 80]; 0.4 mol per mol of Mn-SOD [83]; 0.5-1.0 atom Fe2+ per subunit [95]; 0.24 mol Fe2+ per mol of subunit [100]) [4, 8, 9, 13, 19, 24, 27, 31, 49, 59, 69-71, 74, 75, 78-80, 83, 85, 95, 99, 100] Additional information ( Mn-SOD contains as well Fe3+ , but is only active with manganese [83]; Cu2+ and Zn2+ binding sites are very close to each other [77]; Cu2+ is not necessarily required [74]; Cu2+ -binding site [35]; metal content of the enzyme depends on the growth condition: anaerobic culture condition promote a higher Fe-content, aerobic conditions promote a higher Mn-content [24]; role of copper and zinc in protein conformation and activity [60]; relevance of the zinc imidazolate bond to the redox properties [36]; overview: metal content [8, 9]; enzyme from eukaryotes contains both copper and zinc, enzymes from most prokaryotes contain manganese or iron [8, 9]) [8, 9, 24, 35, 36, 60, 74, 77, 83] ) &  * +, 6000000 (O-2 ) [34] " & *-% , Additional information ( Mn-SOD, native and recombinant wild-type and mutants [102]; dimeric form shows 65% higher activity than monomeric form [101]; native and recombinant, wild-type and restored enzyme, with Fe2+ and Mn2+ [100]; recombinant EC-SOD [92]; Fe-SOD and Mn-SOD activity in Flavobacteria [31]; overview: superoxide dismutase assays [11]; modeling of kinetics [38]) [1-3, 8, 11, 13, 14, 17, 18-24, 26-32, 38, 41-47, 49-52, 57-59, 61-65, 67-70, 72-76, 79-81, 83, 88, 89, 92-103] . / *', 0.355 (O-2 ) [34] . / *', 20 (perchlorate) [9] 0  6.5 ( Mn-SOD [39]) [39] 7 ( isoenzyme I [29]) [29] 7-11 ( native and recombinant Cu,Zn-SOD, activity is pHindependent [84]) [84, 94] 7.4 ( assay at [85]) [85]

406

1.15.1.1

Superoxide dismutase

7.8 ( assay at [1, 27]; Mn-SOD [14, 78]; Fe-SOD [78]) [1, 14, 27, 78] 8.5 ( Mn-SOD [81]) [81] 8.6 ( Mn-SOD [25]) [25] 8.9 ( Cu,Zn-SOD [88]; assay at [38]) [38, 88] 9 ( assay at [102]; Mn-SOD [89]) [89, 102] 9.5 ( Mn-SOD [51]) [51] 10.1 ( Mn-SOD [18]) [18] 10.2 ( Cu,Zn-SOD I and II [18]) [18] Additional information ( pH-dependence, overview [9]) [9] 0 

 3-9 ( Fe-SOD [99]) [99] 6-10.6 ( Mn-SOD [25,81]) [25, 81] 6-11 ( Mn-SOD [51]) [51] 6.5-10.2 ( activity decreases as pH increases [39]) [39] 7-11 ( native and recombinant Cu,Zn-SOD, activity is pH-independent [84]) [84] 7.6-10.5 [35] )  *
, 25 ( assay at [1, 25, 38, 39, 61, 85, 89, 97, 102]) [1, 25, 38, 39, 61, 85, 89, 97, 102] 37 ( native and recombinant Cu,Zn-SOD [84]) [84] 45 ( Mn-SOD [81]) [81]

  #   ' 1  19500 ( native Cu,Zn-SOD [84]; amino acid sequence determination, SDS-PAGE [84]) [84] 21500 ( recombinant Cu,Zn-SOD [84]; amino acid sequence determination [4,84]; SDS-PAGE [84]) [4, 84] 30000 ( Cu,Zn-SOD isoenzyme I, gel filtration, sedimentation equilibrium centrifugation [29]) [29] 30500 ( PAGE [66]) [66] 30800-31600 ( mitochondrial cyanide-sensitive enzyme, gel filtration, sedimentation equilibrium analysis [64]) [64] 31000 ( enzyme from cytoplasm [9]; about, Cu,ZnSOD and Mn-SOD, gel filtration [64]; Cu,Zn-SOD, sedimentation equilibrium [63]; gel filtration, sedimentation equilibrium [3]; gel filtration [16]) [9, 16, 63, 64] 31000-31500 ( gel filtration [3]) [3] 31000-32200 ( gel filtration, sedimentation equilibrium analysis [48]) [48] 31000-33000 ( gel filtration, SOD-1 [61]; SOD-2, SOD-4 [62]) [61, 62] 407

Superoxide dismutase

1.15.1.1

31200 ( Cu,Zn-SOD, sedimentation equilibrium centrifugation analysis [66]) [1, 2, 66] 32000 ( gel filtration [41,88,91]; Cu,Zn-SOD [41,88,91]) [41, 88, 91] 32000-32500 ( gel filtration [72]; Cu,Zn-SOD [9]) [9, 72] 32400 ( Cu,Zn-SOD, gel filtration [17]) [17] 32500 ( Cu,Zn-SOD [66]; sedimentation equilibrium centrifugation analysis [66]) [66] 32600 ( Cu,Zn-SOD [82]) [82] 32700 ( Cu,Zn-SOD, sedimentation equilibrium analysis [46]) [46] 33000 ( gel filtration [55,58]; Fe-SOD, gel filtration [50]; Cu,Zn-SOD isoenzyme II, gel filtration, sedimentation equilibrium analysis [29]; Cu,Zn-SOD-II, gel filtration [23]) [23, 29, 50, 55, 58] 35000 ( gel filtration [1,20]) [1, 20] 36000 ( gel filtration [21]; Fe-SOD, gel filtration [31]) [21, 31] 36500 ( Fe-SOD, sedimentation equilibrium [75]) [75] 37400 ( gel filtration [78]; Mn-SOD [78]; FeSOD [78]) [78] 38500 ( gel filtration [53]) [8, 53] 39000 ( isoenzyme 3, gel filtration [71]) [71] 39800 ( Fe-SOD [85]; Cu,Zn-SOD II, gel filtration [18]) [18, 85] 40000 ( Fe-SOD, gel filtration, sedimentation analysis [79]; Mn-SOD [9,68]; Fe-SOD [9]; sedimentation equilibrium analysis [68]) [9, 68, 79] 40250 ( superoxide dismutase I, sedimentation equilibrium analysis [2]) [2] 41000 ( Fe-SOD, gel filtration [59]) [59] 41000-43000 ( gel filtration, sedimentation equilibrium [69]) [69] 41400 ( sedimentation equilibrium analysis [15]) [15] 41500 ( gel filtration [28]; Mn-SOD [28,56]) [28, 56] 42000 ( Cu,Zn-SOD I, gel filtration [18]) [18] 42000-43000 [8] 42500 ( Mn-SOD, gel filtration [89]) [89] 43000 ( Fe-/Mn-SOD, gel filtration [100]; isoenzyme 2, gel filtration [71]; Mn-SOD, gel filtration [73]) [71, 73, 100] 44000 ( Mn-SOD, gel filtration [93]) [93] 45000 ( gel filtration [13]) [9, 13] 46000 ( gel filtration [27,52]; Mn-SOD [52]) [8, 27, 52] 47000 ( Fe-SOD, gel filtration [74]) [8, 74] 48000 ( gel filtration [51]) [51] 52000 ( Mn-SOD, gel filtration [57]) [57] 55000 ( Cu,Zn-SOD, gel filtration [94]) [94] 408

1.15.1.1

Superoxide dismutase

56000 ( Mn-SOD, gel filtration [31]) [31] 60000 ( gel filtration [30]) [30] 63000 ( Fe-SOD, gel filtration [97]) [97] 68500 ( Cu,Zn-SOD, gel filtration [94]) [94] 69000 ( Cu,Zn-SOD, gel filtration [94]) [94] 73000 ( Mn-SOD, gel filtration [18]) [18] 80000 ( Mn-SOD [67] gel filtration [67]) [8, 67] 82000 [8] 82000-84000 ( Mn-SOD, gel filtration, sedimentation equilibrium [76]) [76] 85000 ( recombinant His-tagged Mn-SOD, gel filtration [83]; sedimentation equilibrium, SOD-3 [62]) [62, 83] 88000 ( gel filtration [32]) [32] 89000 ( sedimentation equilibrium analysis [45]) [45] 90000 ( SOD-III, gel filtration [62]) [62] 91000 ( Fe-SOD, sedimentation equilibrium analysis [80]) [8, 80] 92000 ( Mn-SOD, gel filtration [96]) [96] 94000 ( Mn-SOD, gel filtration [47]) [47] 96000 ( Mn-SOD gel filtration [65]; enzyme from mitochondria [9]) [9, 65] 100000 ( gel filtration [70,81]; Mn-SOD [81]) [8, 70, 81] 105000 ( gel filtration [19]) [19] 123000 ( Cu,Zn-SOD, gel filtration [94]) [94] 165000 ( nectarin I: Mn-SOD, native PAGE [103]) [103] 186000 ( Fe-SOD, gel filtration [99]) [99] Additional information ( primary structure of human erythrocyte enzyme [40]) [40]   ? ( x * 15764-15809, Cu,Zn-SOD wild-type and mutant D90A, electro spray mass spectroscopy [87]; x * 22000, amino acid sequence determination [5]; x * 25000, mitochondria, SDS-PAGE [30]; x * 32000 [8]; x * 25000, SDS-PAGE [85]; x * 185000, Cu,Zn-SOD, SDS-PAGE [94]; x * 18000, Cu,Zn-SOD, SDS-PAGE [94]; x * 19250, Cu,Zn-SOD, SDS-PAGE [94]; x * 175000, Cu,Zn-SOD, SDS-PAGE [94]; x * 22931, Fe-SOD, amino acid sequence determination [95]; x * 22500-24000, Fe-SOD, SDS-PAGE [97]; x * 22000, FeSOD, SDS-PAGE [99]; x * 22500-29000, nectarin I: Mn-SOD, SDS-PAGE and mass spectroscopy [103]) [5, 8, 30, 85, 87, 94, 95, 97, 99, 103] dimer ( 2 * 16300, SDS-PAGE [1]; 2 * 18500, isozyme I, SDS-PAGE [2]; 2 * 19500, isozyme II, SDS-PAGE [2]; 2 * 32000 [6]; 2 * 22900, Mn-SOD [8]; 2 * 21000, Fe-SOD [8]; cytoplasmic enzyme [9]; 2 * 17000, SDS-PAGE [43]; 2 * 22000, SDS-PAGE [13]; 2 * 26000, SDS-PAGE [15,52]; 2 * 16000, SDS-PAGE, isoenzyme B [16]; 2 * 20400, SOD-3, SDS-PAGE [20]; 2 * 16500, SOD-1, SDS-

409

Superoxide dismutase

1.15.1.1

PAGE [20]; 2 * 18000, SDS-PAGE [21]; 2 * 16000, Cu,ZnSOD, SDS-PAGE [66,88,91,93]; 2 * 18300, Cu,Zn-SOD, SDS-PAGE [46]; 2 * 16500, Cu,Zn-SOD-II, SDS-PAGE [23]; 2 * 21600, SDS-PAGE after denaturation in boiling SDS [28]; 2 * 22500, Fe-SOD, SDS-PAGE [69]; 2 * 15100, isoenzyme II, SDS-PAGE [29]; isoenzyme I, SDSPAGE [29]; 2 * 23500, Mn-SOD, SDS-PAGE [51]; 2 * 23500, SDSPAGE [56]; 2 * 26000, SDS-PAGE [57]; 2 * 20000, Fe-SOD, SDSPAGE [59]; 2 * 15900, SOD-4, SDS-PAGE [62]; 2 * 14500, SOD-1, SDS-PAGE [61]; 2 * 17000, SOD-2, SDS-PAGE [62]; 2 * 16800, SDS-PAGE [63]; 2 * 19000, SDS-PAGE [68]; 2 * 25000, SDS-PAGE [70]; 2 * 23000, gel filtration, isoenzyme 2 and 3 [71]; 2 * 20000, SDS-PAGE [8,73]; 2 * 18500, Fe-SOD, SDS-PAGE [75]; 2 * 18300, Mn-SOD, SDS-PAGE [78]; 2 * 18100, Fe-SOD, SDS-PAGE [78]; 2 * 19500, Fe-SOD, SDS-PAGE [79]; 2 * 17500, Cu,Zn-SOD [82]; 2 * 21700, Mn-SOD, SDS-PAGE [89]; 2 * 22000, Mn-SOD, SDS-PAGE [93]; 2 * 23000, Fe-/Mn-SOD, SDS-PAGE [100]) [1, 2, 6-9, 13, 15, 16, 20, 23, 27-29, 43, 46, 50-53, 56, 57, 59, 61-63, 66, 68-76, 78, 79, 82, 88, 89, 91, 93, 100, 101] monomer ( 1 * 33000, SDS-PAGE after treatment with urea and 2-mercaptoethanol [55]; 1 * 100000, Mn-SOD, reducing SDS-PAGE [81]) [55, 81, 101] tetramer ( 4 * 27000, Mn-SOD, SDS-PAGE [96]; 4 * 28000, recombinant EC-SOD, SDS-PAGE [92]; 4 * 21300, SDS-PAGE [32]; mitochondrial enzyme [9]; 4 * 22000, Mn-SOD, SDS-PAGE [43]; 4 * 22400, Mn-SOD, SDS-PAGE [45]; 4 * 24096, amino acid sequence determination [19]; 4 * 25000, Mn-SOD, reducing SDS-PAGE [65]; 4 * 24000, SOD-III, SDSPAGE [62]; 4 * 21000, Mn-SOD, SDS-PAGE [67]; 4 * 25000, SDSPAGE [70]; 4 * 21000, Mn-SOD, SDS-PAGE [76]; 4 * 26000, FeSOD, SDS-PAGE [80]; 4 * 25400, Mn-SOD, SDS-PAGE [83]) [8, 9, 19, 32, 43, 45, 62, 65, 67, 70, 76, 80, 83, 92, 96] Additional information ( three-dimensional structure of Mn-SOD monomer [8]; Cu,Zn-SOD exists as 70% dimeric form and 30% monomeric form [101]; enzyme is only active as tetramer or pentamer [103]) [8, 101, 103] $   "

glycoprotein ( no carbohydrate-containing enzyme [46,75]; SOD-II, SOD-III, SOD-IV: little or no detectable carbohydrate [62]; Cu,Zn-SOD in mammalian extracellular fluids [8]; nectarin I: Mn-SOD; biantennary nonasaccharide with composition (GlnNAc)4 :Man3 :Xyl:Fuc [103]) [8, 62, 46, 75, 92, 103]

410

1.15.1.1

Superoxide dismutase

 2 %$ %' %

% cell culture [82] cotyledon [23, 50] erythrocyte ( Cu,Zn-SOD [77,86,93]; EC-SOD [42]) [1, 9, 10, 34, 37, 40, 41, 77, 86, 93] fruit [3, 101] germ [22] heart (Cu,Zn-SOD, in vessels, including endothelium [93]; Mn-SOD, endothelium [93]) [1, 93] kernel [61] leaf [25, 27, 29, 47, 48, 57, 59, 69, 74, 96] liver ( Mn-SOD [42,45,93]; Cu,ZnSOD [44,66,88]) [10, 12, 17, 30, 32, 42, 44, 45, 66, 88, 93] lung [42] milk [22] mycelium [63] nectar ( nectarin I [103]) [103] seed ( Fe-SOD [95]; Cu,Zn-SOD [74]) [74, 95] seedling ( etiolated, Mn-SOD and Cu,Zn-SOD [74]) [14, 74] shoot [50] sprout [16] venom [81] Additional information ( overview: content of Cu,Zn-SOD and Mn-SOD in rat tissues [43]) [43] 3#

cell wall ( extracellular Fe-SOD, associated with cell-surface [97]; intermediate form between Fe- and Mn-SOD, contains Zn2+ , associated with outer cell wall and selectively secreted into the medium [70]) [70, 97] chloroplast ( SOD-I, associated [61]; SOD-III [20]; SOD-II-V: Cu,Zn-SODs [14]; thylakoid-bound, MnSOD [57]; Fe-SOD, stroma [74]) [14, 20, 29, 48, 57, 61, 74] cytoplasm ( SOD-II, SOD-IV [62]; Mn-SOD [58]; SOD-I [20]; SOD-I: Mn-SOD [14,83]; Cu,Zn-SOD [9,21,63,82]; Fe-SOD [71,75]) [8-10, 14, 20, 21, 29, 55, 58, 62-64, 71, 75, 82, 83] extracellular ( Mn-SOD, nectar [103]; Fe-SOD in culture fluid [97]; EC-SOD with Cu,Zn-SOD activity [92]; Mn-SOD in scorpion venom [81]; Cu,ZnSOD in mammalian extracellular fluids [8]) [8, 22, 42, 81, 92, 97, 103] glyoxysome [23] lysosome ( Cu,Zn-SOD [12]) [12]

411

Superoxide dismutase

1.15.1.1

mitochondrion ( SOD-I [62]; Mn-SOD [9, 12, 14, 18, 30, 64, 74]; Cu,Zn-SOD [74]) [9, 12, 14, 18, 30, 62, 64, 65, 74] peroxisome ( Mn-SOD [96]) [96] Additional information ( wide-spread distribution [1]; overview: subcellular distribution in rat liver [12]; cytosolic Mn-SOD resembles the chloroplast enzyme of higher plants [82]) [1, 12, 82] $"

(from milk [22]; Cu,Zn-SOD [66]) [1, 22, 66] (2 isoenzymes: superoxide dismutase I and II [2]) [2] (Mn-SOD [25,47,96]) [3, 25, 47, 96] (3 isoenzymes of anaero-SOD and 3 isoenzymes of aero-SOD [24]) [24] (EC-SOD recombinant from Escherichia coli as His-tagged protein and partially from insect cells [92]; Cu,Zn-SOD wild-type and mutant recombinant from Escherichia coli [87,90]; Cu,Zn-SOD, wild-type and mutants recombinant from Spodoptera frugiperda cells [86]; Cu,Zn-SOD [42]; recombinant Cu,Zn-SOD [33]; Cu,Zn-SOD from erythrocytes [10,40]; Cu,Zn-SOD from liver [10]; Mn-SOD from liver [10,32,42]; extracellular [42]) [10, 32, 33, 41, 42, 86, 87, 90, 92] [6] (extracellular enzyme [22]; Fe-SOD [49]) [22, 49] (partially [12]; Mn-SOD [30,43,45]; Cu,Zn-SOD [43,44]) [30, 43-45] [13] (5 isoenzymes [14]) [14] (Mn-SOD [15]) [15] (var. gemmifera, 3 isoenzymes [16]) [16] (Cu,Zn-SOD, amino acid analysis [17]) [17] [18] (recombinant from Escherichia coli [19]) [19] (2, SOD-I and SOD-III, of 4 isoenzymes [20]) [20] [21] [22] (Mn-SOD [65]; Cu,Zn-SOD [46,66]) [22, 46, 65, 66] (Cu,Zn-SOD [23]) [23] (Cu,Zn-SOD [72]; allozyme variants: DSDS and DSDF [26]) [26, 72] (Fe-SOD [27]) [27] (Mn-SOD [28]) [28] (Fe-SOD [69]; isoenzymes I and II [29]) [29, 69] (Fe-SOD and Mn-SOD [31]) [31] (Cu,Zn-SOD from shoots and cotyledons [50]) [50] [51] (Mn-SOD [52]) [52] (3 electromorphs: AA, BB, AB [54]) [54] (partially [55]) [55] (Mn-SOD [57]) [48, 57] (Mn-SOD [58]) [58]

412

1.15.1.1

Superoxide dismutase

(Fe-SOD [59]) [59] (SOD-I [61]; SOD-II, SOD-III, SOD-IV [62]) [61, 62] (Cu,Zn-SOD [63]; Cu, Zn-SOD and Mn-SOD [64]) [63, 64] (Mn-SOD [67]) [67] (Mn-SOD [68]) [68] (intermediate between Fe- and Mn-SOD, contains Zn2+ as well [70]) [70] (3 isoenzymes: 1, 2 and 3 [71]) [71] (Mn-SOD [73]) [73] (Fe-SOD, 2 isozymes [74]) [74] (Fe-SOD [75]) [75] (Mn-SOD [76]) [76] (Mn-SOD [78]) [78] (Fe-SOD [78]) [78] (Fe-SOD [79]) [79] (Fe-SOD [80]) [80] (Mn-SOD [81]) [81] (Fe-SOD [10]) [10] (extracellular enzyme [22]) [22] (Cu,Zn-SOD [82]) [82] (Mn-SOD recombinant from Escherichia coli [83]) [83] (Cu,Zn-SOD, native and recombinant from Pichia pastoris [84]) [84] (Fe-SOD [85]) [85] (Cu,Zn-SOD [88]) [88] (Mn-SOD [89]) [89] (Cu,Zn-SOD, recombinant from Escherichia coli and native enzyme [91]) [91] (Mn-SOD from liver [93]) [93] (Fe-SOD and reconstructed Fe-/Mn-SOD [95]) [95] (Fe-SOD [97]) [97] (Fe-SOD [99]) [99] (structural intermediate between Mn-SOD and Fe-SOD [100]) [100] (recombinant His-tagged Cu,Zn-SOD from Escherichia coli [101]; 2 forms, 1 dimer and 1 monomer [101]) [101] (nectarin I: Mn-SOD from nectar [103]) [103] (overview: purification of extracellular superoxide dismutases [22]) [22] (Cu,Zn-SOD [94]) [94]  

(apoprotein expressed in insect cells can be restored by addition of Cu2+ , fully active [86]; recombinant EC-SOD refolds from inclusion bodies in E. coli after denaturing [92]) [86, 92] (reconstitution of active enzyme after withdrawal of Mn2+ by addition of Mn2+ to apoprotein in 8 M urea at acid pH [67]) [67]

413

Superoxide dismutase

1.15.1.1

(after reconstitution with Fe3+ instead of Mn2+ , the enzyme shows properties similar to Fe-SODs [73]; Zn2+ inhibits reconstitution with Mn2+ or Fe3+ [73]) [73] (reconstitution of active enzyme after withdrawal of metal either with the native metal or with cadmium, chromium or iron [8]) [8] (reconstructed Fe-/Mn-SOD is almost equal to native Fe-SOD [95]) [95] (activity cannot be restored by Fe2+ , Cu2+ , Zn2+ , and Cu2+ /Zn2+ , but by Mn2+ [103]) [103] (reconstitution of active enzyme after withdrawal of metal by either Mn or Fe yielding an active enzyme irrespective of the metal ion initially present [9,24]) [9, 24] (reconstitution of active protein after withdrawal of metal, higher activity with Mn2+ , lower activity with Fe3+ [83,100]) [83, 100]
 #

(recombinant human Cu,Zn-SOD expressed in yeast, hanging drop method by vapour diffusion from 50 mM phosphate, pH 7.7, resulting in 3 different crystal forms [33]; enzyme 10 mg per ml in Tris/HCl 50 mM, pH 8.2 by dialysis against ammonium sulfate 2.8 M, pH 8.2, 4 C [32]; from recombinant Mn-SOD, asymmetric unit, hanging drop technique, room temperature, equilibration of 3-4 mg/ml enzyme in ammonium phosphate, pH 5.9, plus 10% 2-methyl-2,4-pentanediol against 32% 2-methyl-2,4-pentanediol, X-ray analysis [5]) [5, 32, 33] (from Cu,Zn-SOD, always twinned, hexagonal crystals with asymmetric units, from 2-methyl-2,4-pentanediol in potassium phosphate buffer, pH 6.5, hanging drop technique by vapour diffusion, X-ray analysis [6]) [6] [9] (asymmetric unit, from Cu,Zn-SOD, sitting drop technique by vapour diffusion, 25 mM citrate, 10 mM phosphate buffer, pH 6.5, 6% w/v polyethylene glycol, stabilization by 35% polyethylene glycol, X-ray analysis, modeling of three-dimensional structure [7]) [7] (dialysis against 0.1 mM EDTA than against water, Mn-SOD [65]) [65] [48] (Mn-SOD, from ammonium sulfate solution, octahedral crystals [76]) [76] (Fe-SOD, dialysis against 55% saturated ammonium sulfate solution, pH 4.5, 1 week at 2 C under reduced pressure [79]) [9, 79] (extracellular enzyme, tetraborate crystallization of ethanolic enzyme extract, then recrystallization from buffer than from water [22]) [22]
 

(expression of H63C mutant in Escherichia coli [98]; EC-SOD, overexpression in Escherichia coli as His-tagged protein and in Tn-5B1-4 cells of Trichoplusia ni via baculovirus infection [92]; Cu,Zn-SOD, overexpression in Escherichia coli [90]; Cu,Zn-SOD, expression of wild-type and mutant in Escherichia coli [87]; Cu,Zn-SOD, overexpression of wild-type and mutants in Spodoptera frugiperda cells Sf21 via baculovirus infection [86]; expression 414

1.15.1.1

Superoxide dismutase

in yeast [33]; Mn-SOD, expression in Escherichia coli [5]) [5, 33, 86, 87, 92, 98] (expression in Escherichia coli [19]) [19] (Mn-SOD, expression of wild-type and mutants in Escherichia coli [102]) [102] [62] (Mn-SOD, expression in Escherichia coli as His-tagged protein, DNA sequence analysis [83]; overexpression in Saccharomyces cerevisiae mutant lacking Cu,Zn-SOD, restores activity of the mutant [83]) [83] (expression in Pichia pastoris, DNA and amino acid sequence determination and comparison [84]) [84] (Cu,Zn-SOD, overexpression in Escherichia coli [91]) [91] (Fe-SOD, expression in Escherichia coli, DNA and amino acid sequence determination [95]) [95] (atypical SOD, functional and structural intermediate between Fe-SOD and Mn-SOD, expression in Escherichia coli [100]) [100] (Cu,Zn-SOD, expression in Escherichia coli as His-tagged protein, DNA and amino acid sequence analysis [101]) [101] 

 

D90A ( Cu,Zn-SOD, mutant found in familial amyotrophic lateral sclerosis, activity similar compared to native and recombinant wild-type, but enhanced OH-generating activity, mutant is more sensitive to inhibition by copper-chelators [87]) [87] G41N ( Cu,Zn-SOD, site-directed mutagenesis, analogous to mutant found in familial amyotrophic lateral sclerosis, 47% activity compared to the wild-type [86]) [86] G85R ( Cu,Zn-SOD, site-directed mutagenesis, analogous to mutant found in familial amyotrophic lateral sclerosis, 99% activity compared to the wild-type [86]) [86] H30A ( active site mutant, site-directed mutagenesis, activity, sensitivity to heat and inhibitors unchanged compared to wild-type [102]) [102] H43R ( Cu,Zn-SOD, site-directed mutagenesis, analogous to mutant found in familial amyotrophic lateral sclerosis, 66% activity compared to the wild-type [86]) [86] H63C ( Cu,Zn-SOD, mutant with exchange of metal-bridging proton-donor His63 for Cys, binds Cu2+ , but not Zn2+ , 1% remaining activity compared to wild-type [98]) [98] K170R ( active site mutant, site-directed mutagenesis, unchanged activity, decreased thermal stability, more stable to 2,4,6-trinotrobenzenesulfonate than the wild-type, completely inactivated by phenylglyoxal [102]) [102] Additional information ( naturally occuring hybrids between Fe-SOD and Mn-SOD, altered metal content [8,9]; naturally occuring exchange of Arg-189 for Lys in the active site of Mn-SOD, sensitivity to lysine-modifying agents [8]) [8, 9]

415

Superoxide dismutase

1.15.1.1



medicine ( recombinant protein is useful as serodiagnostic marker for identification of Aspergillus fumigatus infections, crossreactive antibodies [84]; investigations with help of mutants to elucidate structure-function relation in familial amyotrophic lateral sclerosis [86]; potential target for drug design [5]) [5, 84, 86]

4  0  2.2-11 ( Cu,Zn-SOD, stable [101]) [101] 3-10.8 ( 30 min, pH 3.0: about 30% loss of activity, pH 10.8: rapid inactivation above [78]) [78] 3-11 [31] 4-10.5 ( 12 h, 23 C, stable [67]) [67] 5-8 ( rapid inactivation above pH 8.0 and below pH 5.0 [52]) [52] 5-10.8 ( 30 min, unstable below pH 5.5 and above pH 10.8 [78]) [78] 5.5 ( 5 C, 1 day, 35% loss of activity [32]) [32] 6-7.5 ( 25 C, 2 h stable [88]) [88] 6-11 ( 37 C, 30 min, stable [51]) [51] 7 ( rapid inactivation below [13]; 20 C, 36 h, stable [52]) [13, 52] 7-11 ( 25 C, 36 h, stable [13]) [13] 7.2 ( 4 C, 1 day, 2% loss of activity [32]) [32] 8-9.3 ( 4 C, 1 day, 10-20% loss of activity [32]) [32] Additional information ( highest stability at alkaline pH [25]) [25] )  4 ( Mn-SOD, complete loss of activity after 7 days [18]; 25% loss of activity after 4 months [25]) [18, 25] 35 ( pH 7.0, stable below, inactivation above [13]) [13] 37 ( wild-type, purified stable for at least 1 week [86]; 60 min, stable [31]) [31, 86] 40 ( 1 h, about 20% loss of activity [28]; pH 7.0, stable up to, rapidly inactivated above [52]; pH 7.8, half-life: 70 min, isoenzyme I, 177 min, isoenzyme II [29]; 60 min, 40% loss of activity [31]) [28, 29, 31, 52] 40-60 ( 10 min, stable [58]) [58] 45 ( pH 7.4, 1 h stable [88]) [88] 50 ( 1 h, stable [94,99]; 88.5% remaining activity after 1 h [89]; loss of 40% activity after 60 min [31]; several h, stable [26]; 1 h, about 55% loss of activity [28]; 40 min, 50% loss of activity [47]) [26, 28, 31, 47, 89, 94, 99] 55 ( 70% loss of activity after preexposure [81]; 10 min, about 45% loss of activity, 30 min, 75% loss of activity [28]) [28, 81]

416

1.15.1.1

Superoxide dismutase

60 ( pH 7. 8, half-life: 5 min, isoenzyme I, 34 min, isoenzyme II [29]; 5 min, complete inactivation of isoenzyme B, 50% loss of isoenzyme A activity [16]; 10 min, about 75% loss of activity [28]; 5 min, 50% loss of activity [47]; half-life: 22 min [69]) [16, 28, 29, 47, 69] 70 ( loss of activity after 30 min, Cu,Zn-SOD [94]; recombinant Cu,Zn-SOD, after 2 h 20% activity remaining, after 3 h all activity is lost [91]; complete loss of activity after 40 min [89]; 5 min, complete loss of activity [26]; 5 min, stable [51]; 40 min, 40% loss of activity [51]; half-life: 4 min [69]; half-life: 12.75 min [78]; half-life: 6.5 min [78]) [26, 51, 69, 78, 89, 91, 94] 75 ( native wild-type enzyme: half-life 4.7 h, recombinant wild-type enzyme: half-life: 2.8 h, recombinant mutant H30A: half-life 2.7 h, recombinant mutant K170R half-life 0.36 h [102]; loss of 66% activity [88]; total loss of activity [81]) [81, 88, 102] 90 ( nectarin I: Mn-SOD, 85% remaining activity after 1 h [103]; dimer: half-life 99 min, dimeric form is more stable than monomeric form [101]; complete loss of activity after 5 min [89]) [89, 101, 103] 95 ( 10 min, stable up to [67]) [67] 100 ( complete loss of activity [103]; 60 min, complete loss of activity [31]) [31, 103] Additional information ( thermal stability of Mn-SOD at several temperatures, enzyme is more labile at higher temperatures [47]; comparison of thermostability of various Gluconobacter strains [51]) [47, 51]    &   acetone ( Fe-SOD [74]) [74] chloroform ( Fe-SOD [74, 85]; Cu,Zn-SOD [74]) [74, 85] dimethyl sulfoxide ( stable up to 70% v/v [78]; stable up to 55% v/v [78]) [78] ethanol ( Fe-SOD, Cu,Zn-SOD [74]) [74] Additional information ( chloroform/ethanol is used during purification of Fe-SOD and for detection of Cu,Zn-SOD, acetone precipitation [74]; Mn-SOD and Fe-SOD: not stable to organic solvents, Cu,Zn-SOD: stable to organic solvents [10]) [10, 74] 5    "

, SDS, 1%, complete loss of activity after 6 h [32] , urea: 8 M, stable [32] , 1 cycle of freezing and thawing causes 30% loss of activity [45] , 3 cycles of freezing and thawing cause less than 20% loss of activity, Mn-SOD [43] , freezing causes rapid deterioation [45] , lyophilization: less than 10% loss of activity [28] , quite stable to freezing [69] 417

Superoxide dismutase

1.15.1.1

, urea: 8 M, 0 C, isoenzyme I unfolds immediately, isoenzyme II stays folded [29] , polyethylene glycol stabilizes [51] , due to high salt requirement for enzyme stability purification is performed in presence of 2 M NaCl [53] , guanidinium chloride: 6 M, pH 7.5, 16 h, 23 C, stable [67] , urea: 8 M, pH 7.5, 16 h, 23 C, stable [67] , dimeric form is more stable against proteolysis than monomeric form [101] , dimethyl sulfoxide: Spirulina enzyme is stable up to 55%, Rhodopseudomonas enzyme up to 70% v/v [78]    , 5 C, crystals, active in water, 2 years [22] , -20 C, 1 month, 18% loss of activity [47] , -20 C, 4 months, 20% loss of activity [25] , 4 C, 1 month, 24% loss of activity [47] , -35 C, protein concentration 45 mg/ml, 50% glycerol [32] , -80 C, for at least 1 year [15] , 4 C, complete loss of activity within 7 days [18] , -80 C [20] , 4 C, purified, stable [27] , 4 C, 10 mM phosphate buffer, pH 7.2, 4 weeks [28] , -70 C, at least 1 month [31] , 4 C, loss of activity within approximately 7 days [31] , 4 C, 4 days, 50% loss of activity [74] , -70 C, 2 months [80]

"  [1] Keele, B.B.; McCord, J.M.; Fridovich, I.: Further characterization of bovine superoxide dismutase and its isolation from bovine heart. J. Biol. Chem., 246, 2875-2880 (1971) [2] Vance, P.G.; Keele, B.B.; Rajagopalan, K.V.: Superoxide dismutase from Streptococcus mutans. Isolation and characterization of two forms of the enzyme. J. Biol. Chem., 247, 4782-4786 (1972) [3] Sawada, Y.; Ohyama, T.; Yamazaki, I.: Preparation and physicochemical properties of green pea superoxide dismutase. Biochim. Biophys. Acta, 268, 305-312 (1972) [4] Amano, A.; Shizukuishi, S.; Tsunemitsu, A.; Maekawa, K.; Tsunasawa, S.: The primary structure of superoxide dismutase purified from anaerobically maintained Bacteroides gingivalis. FEBS Lett., 272, 217-220 (1990) [5] Wagner, U.G.; Werber, M.M.; Beck, Y.; Hartman, J.R.; Frolow, F.; Sussman, J.L.: Characterization of crystals of genetically engineered human manganese superoxide dismutase. J. Mol. Biol., 206, 787-788 (1989)

418

1.15.1.1

Superoxide dismutase

[6] Redford, S.M.; McRee, D.E.; Getzoff, E.D.; Steinman, H.M.; Tainer, J.A.: Crystallographic characterization of a Cu,Zn superoxide dismutase from Photobacterium leiognathi. J. Mol. Biol., 212, 449-451 (1990) [7] Frigerio, F.; Falconi, M.; Gatti, G.; Bolognesi, M.; Desideri, A.; Marmocchi, F.; Rotilio, G.: Crystallographic characterization and three-dimensional model of yeast Cu,Zn superoxide dismutase. Biochem. Biophys. Res. Commun., 160, 677-681 (1989) [8] Beyer, W.; Imlay, J.; Fridovich, I.: SODs: varieties and distributions. X-ray crystallography of Mn-SODs and Fe-SODs. Prog. Nucleic Acid Res. Mol. Biol., 40, 221-253 (1991) [9] Cass, A.E.G.: Superoxide dismutases. Top. Mol. Struct. Biol., 6, 121-156 (1985) [10] Bannister, J.V.; Bannister, W.H.: Isolation and characterization of superoxide dismutase. Methods Enzymol., 105, 88-93 (1984) [11] Flohe, L.; Ýtting, F.: Superoxide dismutase assays. Methods Enzymol., 105, 93-104 (1984) [12] Geller, B.L.; Winge, D.R.: Subcellular distribution of superoxide dismutases in rat liver. Methods Enzymol., 105, 105-121 (1984) [13] Kim, S.W.; Lee, S.O.; Lee, T.H.: Purification and characterization of superoxide dismutase from Aerobacter aerogenes. Agric. Biol. Chem., 55, 101108 (1991) [14] Sulochana, K.N.; Venkaiah, B.: Purification of isozymes of superoxide dismutase from groundnut (Arachis hypogea) seedlings. Biochem. Int., 22, 133-140 (1990) [15] Denariaz, G.; Payne, W.J.; LeGall, J.: Characterization of the superoxide dismutase of the denitrifying bacterium, Bacillus halodenitrificans. Biol. Met., 3, 14-18 (1990) [16] Walker, J.L.; McLellan, K.M.; Robinson, D.S.: Isolation and purification of superoxide dismutase purified from brussels sprouts (Brassica oleracea L. var. bullata sub var. gemmifera). Food Chem., 41, 1-9 (1991) [17] Vig, E.; Gabrielak, T.; Leyko, W.; Nemcsok, J.; Matkovics, B.: Purification and characterization of Cu,Zn-superoxide dismutase from common carp liver. Comp. Biochem. Physiol. B, 94, 395-397 (1989) [18] Sanchez-Moreno, M.; Garcia-Ruiz, M.A.; Sanchez-Navas, A.; Monteoliva, M.: Physico-chemical characteristics of superoxide dismutase in Ascaris suum. Comp. Biochem. Physiol. B, 92, 737-740 (1989) [19] Takao, M.; Yasui, A.; Oikawa, A.: Unique characteristics of superoxide dismutase of a strictly anaerobic archaebacterium Methanobacterium thermoautotrophicum. J. Biol. Chem., 266, 14151-14154 (1991) [20] Wingsle, G.; Gardeström, P.; Hällgren, J.E.; Karpinski, S.: Isolation, purification, and subcellular localization of isozymes of superoxide dismutase from scots pine (Pinus sylvestris L.) needles. Plant Physiol., 95, 21-28 (1991) [21] Callahan, H.L.; Crouch, R.K.; James, E.R.: Dirofilaria immitis superoxide dismutase: purification and characterization. Mol. Biochem. Parasitol., 49, 245-252 (1991)

419

Superoxide dismutase

1.15.1.1

[22] Munkres, K.D.: Purification of exocellular superoxide dismutases. Methods Enzymol., 186, 249-260 (1990) [23] Bueno, P.; del Rio, L.A.: Purification and properties of glyoxysomal cuprozinc superoxide dismutase from watermelon cotyledons (Citrullus vulgaris Schrad). Plant Physiol., 98, 331-336 (1992) [24] Amano, A.; Shizukuishi, S.; Tamagawa, H.; Iwakura, K.; Tsunasawa, S.; Tsunemitsu, A.: Characterization of superoxide dismutases purified from either anaerobically maintained or aerated Bacteroides gingivalis. J. Bacteriol., 172, 1457-1463 (1990) [25] Sevilla, F.; Lopez-Gorge, J.; del Rio, L.A.: Characterization of a manganese superoxide dismutase from the higher plant Pisum sativum. Plant Physiol., 70, 1321-1326 (1982) [26] Lee, Y.M.; Misra, H.P.; Ayala, F.J.: Superoxide dismutase in Drosophila melanogaster: biochemical and structural characterization of allozyme variants. Proc. Natl. Acad. Sci. USA, 78, 7052-7055 (1981) [27] Salin, M.L.; Bridges, S.M.: Isolation and characterization of an iron-containing superoxide dismutase from water lily, Nuphar luteum. Plant Physiol., 69, 161-165 (1982) [28] Reinards, R.; Altdorf, R.; Ohlenbusch, H.D.: Purification and properties of a manganese-containing superoxide dismutase from Acholeplasma laidlawii. Hoppe-Seyler's Z. Physiol. Chem., 365, 577-585 (1984) [29] Kwiatowski, J.; Kaniuga, Z.: Isolation and characterization of cytosolic and chloroplast isoenzymes of Cu,Zn-superoxide dismutase from tomato leaves and their relationship to other Cu,Zn-superoxide dismutases. Biochim. Biophys. Acta, 874, 99-115 (1986) [30] Ishikawa, T.; Hunaiti, A.R.; Piechot, G.; Wolf, B.: Isolation and characterization of basic superoxide dismutase consisting of Mr-25,000 subunits in rat liver. Eur. J. Biochem., 170, 317-323 (1987) [31] Sanchez-Moreno, M.; Monteoliva-Sanchez, M.; Ramoz-Cormenzana, A.; Monteoliva, M.: Superoxide dismutase in strains of the genus Flavobacterium: isolation and characterization. Arch. Microbiol., 152, 407-410 (1989) [32] Matsuda, Y.; Hagashiyama, S.; Kijima, Y.; Suzuki, K.; Kawano, K.; Akiyama, M.; Kawata, S.; Tarui, S.; Deutsch, H.F.; Taniguchi, N.: Human liver manganese superoxide dismutase. Purification and crystallization, subunit association and sulfhydryl reactivity. Eur. J. Biochem., 194, 713-720 (1990) [33] Parge, H.E.; Getzoff, E.D.; Scandella, C.S.; Hallewell, R.A.; Tainer, J.A.: Crystallographic characterization of recombinant human CuZn superoxide dismutase. J. Biol. Chem., 261, 16215-16218 (1986) [34] Rigo, A.; Viglino, P.; Rotilio, G.: Kinetic study of O2 -dismutation by bovine superoxide dismutase. Evidence for saturation of the catalytic sites by O-2. Biochem. Biophys. Res. Commun., 63, 1013-1018 (1975) [35] Rigo, A.; Terenzi, M.; Viglino, P.; Calabrese, L.; Cocco, D.; Rotilio, G.: The binding of copper ions to copper-free bovine superoxide dismutase. Properties of the protein recombined with increasing amounts of copper ions. Biochem. J., 161, 31-35 (1977)

420

1.15.1.1

Superoxide dismutase

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421

Superoxide dismutase

1.15.1.1

[51] Tsukuda, K.; Kido, T.; Ueda, S.; Terakawa, M.; Shimasue, Y.; Soda, K.: Isolation and characterization of manganese-containing superoxide dismutase from Gluconobacter cerinus. Agric. Biol. Chem., 51, 3323-3329 (1987) [52] Lee, T.H.; Lee, S.O.: Purification and properties of superoxide dismutase from Bacillus circulans. Agric. Biol. Chem., 52, 1361-1367 (1988) [53] Salin, M.L.; Oesterhelt, D.: Purification of a manganese-containing superoxide dismutase from Halobacterium halobium. Arch. Biochem. Biophys., 260, 806-810 (1988) [54] Capo, C.R.; Polticelli, F.; Calabrese, L.; Schinina, M.E.; Carri, M.T.; Rotilio, G.: The Cu,Zn superoxide dismutase isoenzymes of Xenopus laevis: purification, identification of a heterodimer and differential heat sensitivity. Biochem. Biophys. Res. Commun., 173, 1186-1193 (1990) [55] Vignais, P.M.; Terech, A.; Meyer, C.M.; Henry, M.F.: Isolation and characterization of a protein with cyanide-sensitive superoxide dismutase activity from the prokaryote, Paracoccus denitrificans. Biochim. Biophys. Acta, 701, 305-317 (1982) [56] Terech, A.; Vignais, P.M.: A manganese-containing superoxide dismutase from Paracoccus denitrificans. Biochim. Biophys. Acta, 657, 411-424 (1981) [57] Hayakawa, T.; Kanematsu, S.; Asada, K.: Purification and characterization of thylakoid-bound Mn-superoxide dismutase in spinach chloroplasts. Planta, 166, 111-116 (1985) [58] Buchanan, A.G.; Lees, H.: Superoxide dismutase from nitrogen-fixing Azotobacter chroococcum: purification, characterization, and intracellular location. Can. J. Microbiol., 26, 441-447 (1980) [59] Salin, M.L.; Bridges, S.M.: Isolation and characterization of an iron-containing superoxide dismutase from a eucaryote, Brassica campestris. Arch. Biochem. Biophys., 201, 369-374 (1980) [60] Rotilio, G.; Calabrese, L.; Bossa, F.; Barra, D.; Agro, A.F.; Mondovi, B.: Properties of the apoprotein and role of copper and zinc in protein conformation and enzyme activity of bovine superoxide dismutase. Biochemistry, 11, 2182-2187 (1972) [61] Baum, J.A.; Chandlee, J.M.; Scandalios, J.G.: Purification and partial characterization of a genetically-defined superoxide dismutase (SOD-1) associated with maize chloroplasts. Plant Physiol., 73, 31-35 (1983) [62] Baum, J.A.; Scandalios, J.G.: Isolation and characterization of the cytosolic and mitochondrial superoxide dismutases of maize. Arch. Biochem. Biophys., 206, 249-264 (1981) [63] Misra, H.P.; Fridovich, I.: The purification and properties of superoxide dismutase from Neurospora crassa. J. Biol. Chem., 247, 3410-3414 (1972) [64] Henry, L.E.A.; Cammack, R.; Schwitzguebel, J.P.; Palmer, J.M.; Hall, D.O.: Intracellular localization, isolation and characterization of two distinct varieties of superoxide dismutase from Neurospora crassa. Biochem. J., 187, 321-328 (1980) [65] Ravindranath, S.D.; Fridovich, I.: Isolation and characterization of a manganese-containing superoxide dismutase from yeast. J. Biol. Chem., 250, 6107-6112 (1975) 422

1.15.1.1

Superoxide dismutase

[66] Weser, U.; Prinz, R.; Schallies, A.; Fretzdorff, A.; Krauss, P.; Voelter, W.; Voetsch, W.: Microbial and hepatic cuprein (superoxide dismutase). Isolation and characterisation of cuprein (superoxide dismutase) from Saccharomyces cerevisiae and bovine liver. Hoppe-Seyler's Z. Physiol. Chem., 353, 1821-1831 (1972) [67] Sato, S.; Harris, J.I.: Superoxide dismutase from Thermus aquaticus. Isolation and characterisation of manganese and apo enzymes. Eur. J. Biochem., 73, 373-381 (1977) [68] Misra, H.P.; Fridovich, I.: Purification and properties of superoxide dismutase from a red alga, Porphyridium cruentum. J. Biol. Chem., 252, 6421-6423 (1977) [69] Kwiatowski, J.; Safianowska, A.; Kaniuga, Z.: Isolation and characterization of an iron-containing superoxide dismutase from tomato leaves, Lycopersicon esculentum. Eur. J. Biochem., 146, 459-466 (1985) [70] Beaman, B.L.; Scates, S.M.; Moring, S.E.; Deem, R.; Misra, H.P.: Purification and properties of a unique superoxide dismutase from Nocardia asteroides. J. Biol. Chem., 258, 91-96 (1983) [71] Le Trant, N.; Meshnick, S.R.; Kitchener, K.; Eaton, J.W.; Cerami, A.: Ironcontaining superoxide dismutase from Crithidia fasciculata. Purification, characterization, and similarity to Leishmanial and trypanosomal enzymes. J. Biol. Chem., 258, 125-130 (1983) [72] Lee, Y.M.; Ayala, F.J.; Misra, H.P.: Purification and properties of superoxide dismutase from Drosophila melanogaster. J. Biol. Chem., 256, 85068509 (1981) [73] Gregory, E.M.: Characterization of the O2 -induced manganese-containing superoxide dismutase from Bacteroides fragilis. Arch. Biochem. Biophys., 238, 83-89 (1985) [74] Duke, M.V.; Salin, M.L.: Purification and characterization of an iron-containing superoxide dismutase from a eucaryote, Ginkgo biloba. Arch. Biochem. Biophys., 243, 305-314 (1985) [75] Misra, H.P.; Keele, B.B.: The purification and properties of superoxide dismutase from a blue-green alga. Biochim. Biophys. Acta, 379, 418-425 (1975) [76] Sato, S.; Nakazawa, K.: Purification and properties of superoxide dismutase from Thermus thermophilus HB8. J. Biochem., 83, 1165-1171 (1978) [77] Calabrese, L.; Rotilio, G.; Mondovi, B.: Cobalt erythrocuprein: preparation and properties. Biochim. Biophys. Acta, 263, 827-829 (1972) [78] Lumsden, J.; Cammack, R.; Hall, D.O.: Purification and physicochemical properties of superoxide dismutase from two photosynthetic microorganisms. Biochim. Biophys. Acta, 438, 380-392 (1976) [79] Yamakura, F.: Purification, crystallization and properties of iron-containing superoxide dismutase from Pseudomonas ovalis. Biochim. Biophys. Acta, 422, 280-294 (1976) [80] Kirby, T.W.; Lancaster, J.R.; Fridovich, I.: Isolation and characterization of the iron-containing superoxide dismutase of Methanobacterium bryantii. Arch. Biochem. Biophys., 210, 140-148 (1981)

423

Superoxide dismutase

1.15.1.1

[81] Ramanaiah, M.; Venkaiah, B.: Characterization of superoxide dismutase from South Indian scorpion venom. Biochem. Int., 26, 113-123 (1992) [82] Tanaka, K.; Takio, S.; Yamamoto, I.; Satoh, T.: Purification of the cytosolic CuZn-superoxide dismutase (CuZn-SOD) of Marchantia paleacea var. diptera and its resemblance to CuZn-SOD from chloroplasts. Plant Cell Physiol., 37, 523-529 (1996) [83] Lamarre, C.; LeMay, J.D.; Deslauriers, N.; Bourbonnais, Y.: Candida albicans expresses an unusual cytoplasmic manganese-containing superoxide dismutase (SOD3 gene product) upon the entry and during the stationary phase. J. Biol. Chem., 276, 43784-43791 (2001) [84] Holdom, M.D.; Lechenne, B.; Hay, R.J.; Hamilton, A.J.; Monod, M.: Production and characterization of recombinant Aspergillus fumigatus Cu,Zn superoxide dismutase and its recognition by immune human sera. J. Clin. Microbiol., 38, 558-562 (2000) [85] Barnes, A.C.; Balebona, M.C.; Horne, M.T.; Ellis, A.E.: Superoxide dismutase and catalase in Photobacterium damselae subsp. piscicida and their roles in resistance to reactive oxygen species. Microbiology, 145, 483-494 (1999) [86] Fujii, J.; Myint, T.; Seo, H.G.; Kyanoki, Y.; Ikeda, Y.; Taniguchi, N.: Characterization of wild-type and amyotrophic lateral sclerosis-related mutant Cu,Zn-superoxide dismutases overproduced in baculovirus-infected insect cells. J. Neurochem., 64, 1456-1461 (1995) [87] Kim, S.M.; Eum, W.S.; Kang, J.H.: Expression, purification, and characterization of a familial amyotrophic lateral sclerosis-associated D90A Cu,Znsuperoxide dismutase mutant. Mol. Cells, 8, 478-482 (1998) [88] Ozturk-Urek, R.; Tarhan, L.: Purification and characterization of superoxide dismutase from chicken liver. Comp. Biochem. Physiol. B, 128, 205-212 (2001) [89] An, S.S.; Kim, Y.M.: Purification and characterization of a manganesecontaining superoxide dismutase from a carboxydobacterium, Pseudomonas carboxydohydrogena. Mol. Cells, 7, 730-737 (1997) [90] Kang, J.H.; Choi, B.J.; Kim, S.M.: Expression and characterization of recombinant human Cu,Zn-superoxide dismutase in Escherichia coli. J. Biochem. Mol. Biol., 30, 60-65 (1997) [91] Liu, W.; Zhu, R.H.; Li, G.P.; Wang, D.C.: cDNA cloning, high-level expression, purification, and characterization of an avian Cu,Zn superoxide dismutase from Peking duck. Protein Expr. Purif., 25, 379-388 (2002) [92] He, H.J.; Yuan, Q.S.; Yang, G.Z.; Wu, X.F.: High-level expression of human extracellular superoxide dismutase in Escherichia coli and insect cells. Protein Expr. Purif., 24, 13-17 (2002) [93] Ikeda, K.; Matsumi, S.; Magara, T.; Nakagawa, S.: Purification and characterization of canine manganese superoxide dismutase and its immunohistochemical localization in canine heart compared with that of copper-zinc superoxide dismutase. Int. J. Biochem. Cell Biol., 27, 1257-1265 (1995) [94] Holdom, M.D.; Hay, R.J.; Hamilton, A.J.: The Cu,Zn superoxide dismutases of Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, and Aspergillus terreus: purification and biochemical comparison with the Aspergil424

1.15.1.1

[95]

[96]

[97]

[98]

[99]

[100] [101] [102]

[103]

Superoxide dismutase

lus fumigatus Cu,Zn superoxide dismutase. Infect. Immun., 64, 3326-3332 (1996) Chen, H.Y.; Hu, R.G.; Wang, B.Z.; Chen, W.F.; Liu, W.Y.; Schroder, W.; Frank, P.; Ulbrich, N.: Structural studies of an eukaryotic cambialistic superoxide dismutase purified from the mature seeds of camphor tree. Arch. Biochem. Biophys., 404, 218-226 (2002) Palma, J.M.; Lopez-Huertas, E.; Corpas, F.J.; Sandalio, L.M.; Gomez, M.; Del Rio, L.A.: Peroxisomal manganese superoxide dismutase: purification and properties of the isoenzyme from pea leaves. Physiol. Plant., 104, 720726 (1998) Cannio, R.; Dngelo, A.; Rossi, M.; Bartolucci, S.: A superoxide dismutase from the archaeon Sulfolobus solfataricus is an extracellular enzyme and prevents the deactivation by superoxide of cell-bound proteins. Eur. J. Biochem., 267, 235-243 (2000) Banci, L.; Bertini, I.; Borsari, M.; Viezzoli, M.S.; Hallewell, R.A.: Mutation of the metal-bridging proton-donor His63 residue in human copper, zinc superoxide dismutase. Biochemical and biophysical analysis of the His63Cys mutant. Eur. J. Biochem., 232, 220-225 (1995) Dos Santos, W.G.; Pacheco, I.; Liu, M.Y.; Teixeira, M.; Xavier, A.V.; LeGall, J.: Purification and characterization of an iron superoxide dismutase and a catalase from the sulfate-reducing bacterium Desulfovibrio gigas. J. Bacteriol., 182, 796-804 (2000) Santos, R.; Bocquet, S.; Puppo, A.; Touati, D.: Characterization of an atypical superoxide dismutase from Sinorhizobium meliloti. J. Bacteriol., 181, 4509-4516 (1999) Lin, M.W.; Lin, M.T.; Lin, C.T.: Copper/zinc-superoxide dismutase from lemon cDNA and enzyme stability. J. Agric. Food Chem., 50, 7264-7270 (2002) Borders, C.L., Jr.; Bjerrum, M.J.; Schirmer, M.A.; Oliver, S.G.: Characterization of recombinant Saccharomyces cerevisiae manganese-containing superoxide dismutase and its H30A and K170R mutants expressed in Escherichia coli. Biochemistry, 37, 11323-11331 (1998) Carter, C.; Thornburg, R.W.: Tobacco nectarin I. Purification and characterization as a germin-like, manganese superoxide dismutase implicated in the defense of floral reproductive tissues. J. Biol. Chem., 275, 3672636733 (2000)

425

 



   
 1.15.1.2    rubredoxin:superoxide oxidoreductase    superoxide reductase    SOR desulfoferrodoxin [3] desulforedoxin neelaredoxin [2]
    250679-67-5

     Pyrococcus furiosus (hyperthermophilic anaerobe [1]) [1, 5, 9, 12] Treponema pallidum (microaerophilic obligate human pathogen [2]) [2, 12, 13] Desulfoarculus baarsii [3, 6, 10, 12, 13] Archaeoglobus fulgidus (strict anaerobic archaeon, the oxidized enzyme exhibits a characteristic blue color that prompted the name neelaredoxin, derived from the sanskrit word for blue [4]) [4, 12] Desulfovibrio vulgaris ( enzyme protects from exposure to superoxide but not hydrogen peroxide [8]) [7, 8, 11, 12, 14]

!    "
 # 

reduced rubredoxin + superoxide + 2 H+ = rubredoxin + H2 O2 ( very fast bimolecular reaction of iron center II with superoxide, followed by the formation of two successive intermediate species [6]; reduction of superoxide may proceed through Fe3+ -peroxo intermediates [10])

426

1.15.1.2

Superoxide reductase

   oxidation redox reaction reduction       reduced rubredoxin + superoxide + H+ ( rubredoxin is assumed to be the physiological electron carrier [1, 12] blue non-heme iron enzyme that functions in anaerobic microbes as a defense mechanism against reactive oxygen species by catalyzing the reduction of superoxide to H2 O2 [5]) (Reversibility: ? [1]) [1, 5, 12] $ oxidized rubredoxin + H2 O2 [1, 5, 12]      reduced acceptor + superoxide ( enzyme is able to both reduce and dismutate superoxide [4]) (Reversibility: ? [4]) [4] $ acceptor + H2 O2 + O2 [4]  reduced acceptor + superoxide + H+ ( enzyme can be fully reduced upon addition of NADH or NADPH under anaerobic conditions [4]) (Reversibility: ? [4]) [4] $ acceptor + H2 O2 [4]  reduced cytochrome c + superoxide + H+ ( enzyme shows only very weak superoxide dismutase activity [2, 3, 12]) (Reversibility: ? [1, 2, 3]) [1, 2, 3, 12] $ cytochrome c + H2 O2 [1, 12]  reduced rubredoxin + superoxide + H+ ( rubredoxin is assumed to be the physiological electron carrier [1]) (Reversibility: ? [1, 7]) [1, 7, 12] $ rubredoxin + H2 O2 [1, 7, 12]
"%  cytochrome c ( artificial electron carrier [1]) [1] rubredoxin ( assumed to be the physiological electron carrier [1]) [1] '(   Fe2+ /Fe3+ ( 0.5 iron atoms/mol subunit [1]; each subunit contains a single mononuclear non-heme iron center [5]; 0.67 iron atoms/subunit, iron atom exists as a mononuclear center in a mixture of high spin ferrous and ferric oxidation states [2]; center I is missing [13]; 1.97 iron atoms/subunit, enzyme contains two Fe-centers: center I contains a mononuclear ferric iron coordinated by four cysteines in distorted rubredoxin-type center, center II has a ferrous iron with square pyramidal coordination to four nitrogens from histidines as equatorial ligands and one sulfur from a cysteine as the axial ligand, the reduced form of center II can transfer 1 electron to superoxide anion very efficiently [3]; enzyme contains 1 iron atom/monomer [4]; in the oxidized state, the mononuclear ferric active site has a octahedral coordination with four equatorial histidyl ligands and axial cysteinate and monodentate glutamate ligands, in the reduced state 427

Superoxide reductase

1.15.1.2

the ferrous site has a square-pyramidal coordination geometry in frozen solution with four equatorial histidines and one axial cysteine [9]) [1, 2, 3, 4, 6, 9, 12, 13] Zn2+ ( approx. 0.25 atoms/subunit [2]) [2] " & *-% , Additional information ( 60.0 U/mg, 1 U is the amount of enzyme necessary to inhibit 50% of the reduction of cytochrome c by the xanthine/ xanthine oxidase system, superoxide dismutase activity of native enzyme [4]) [4] ) 

 *
, 25 ( enzyme functions efficiently in vitro at 25 C, which is 75 C below the organism's optimal growth temperature [1]) [1]

  #   ' 1  26000 ( gel filtration [2]) [2] 27000 ( gel filtration [3]) [3]   ? ( x * 14000, SDS-PAGE [1]; x * 14323, deduced from nucleotide sequence [1]) [1] dimer ( 2 * 14000, SDS-PAGE [2]; 1 * 13800 + 1 * 13670, ESIMS [2]; 2 * 14028, ES-MS [3]) [2, 3, 6] tetramer ( 4 * 14300 [5]) [5]

 2 %$ %' %

$"

(recombinant enzyme [1]) [1, 9] (recombinant enzyme [2]) [2] (recombinant enzyme, gel filtration, anion exchange [3,4]; wild-type and E47A and K48I mutant enzyme [6]; so far there is no suitable enzymatic assay for monitoring the purification from cell extracts [13]) [3, 4, 6, 13] (native and recombinant enzyme [4]) [4] (wild-type and E47A and K48A mutant [14]) [14]
 #

(sitting drop vapor diffusion method [5]) [5]
 

(expression in Escherichia coli [1, 2, 3, 4]) [1, 2, 3, 4, 5, 8, 9, 11, 12, 13]

428

1.15.1.2

Superoxide reductase



 

E47A ( mutation has almost no effect on the reaction with superoxide [6]; active site of the mutant can transiently stabilize an Fe3+ peroxo species [10]; E47 may interact with the iron atom of ferric center II, most likely by carboxylate ligation [11]) [6, 10, 11, 14] K48A ( lysyl side chain may participate in directing the superoxide toward the active site and in directing the protonation pathway of the ferric(hydro)peroxo intermediate toward release of hydrogen peroxide [14]) [14] K48I ( 20-fold lower second-order rate constant for the oxidation of the iron center by superoxide compared to wild-type enzyme, K48 may play a role in directing and stabilizing superoxide to the active site at center II [6]) [6]

"  [1] Jenney, F.E., Jr.; Verhagen, M.F.J.M.; Cui, X.; Adams, M.W.W.: Anaerobic microbes: oxygen detoxification without superoxide dismutase. Science, 286, 306-309 (1999) [2] Jovanovic, T.; Ascenso, C.; Hazlett, K.R.O.; Sikkink, R.; Krebs, C.; Litwiller, R.; Benson, L.M.; Moura, I.; Moura, J.J.G.; Radolf, J.D.; Huynh, B.H.; Naylor, S.; Rusnak, F.: Neelaredoxin, an iron-binding protein from the syphilis spirochete, Treponema pallidum, is a superoxide reductase. J. Biol. Chem., 275, 28439-28448 (2000) [3] Lombard, M.; Fontecave, M.; Touati, D.; Niviere, V.: Reaction of the desulfoferrodoxin from Desulfoarculus baarsii with superoxide anion. Evidence for a superoxide reductase activity. J. Biol. Chem., 275, 115-121 (2000) [4] Abreu, I.A.; Saraiva, L.M.; Carita, J.; Huber, H.; Stetter, K.O.; Cabelli, D.; Teixeira, M.: Oxygen detoxification in the strict anaerobic archaeon Archaeoglobus fulgidus: superoxide scavenging by Neelaredoxin. Mol. Microbiol., 38, 322-334 (2000) [5] Yeh, A.P.; Hu, Y.; Jenney, F.E., Jr.; Adams, M.W.W.; Rees, D.C.: Structures of the superoxide reductase from Pyrococcus furiosus in the oxidized and reduced states. Biochemistry, 39, 2499-2508 (2000) [6] Lombard, M.; Houee-Levin, C.; Touati, D.; Fontecave, M.; Niviere, V.: Superoxide reductase from Desulfoarculus baarsii: reaction mechanism and role of glutamate 47 and lysine 48 in catalysis. Biochemistry, 40, 5032-5040 (2001) [7] Coulter, E.D.; Kurtz, D.M., Jr.: A role for rubredoxin in oxidative stress protection in Desulfovibrio vulgaris: catalytic electron transfer to rubrerythrin and two-iron superoxide reductase. Arch. Biochem. Biophys., 394, 76-86. (2001) [8] Lumppio, H.L.; Shenvi, N.V.; Summers, A.O.; Voordouw, G.; Kurtz, D.M., Jr.: Rubrerythrin and rubredoxin oxidoreductase in Desulfovibrio vulgaris: a novel oxidative stress protection system. J. Bacteriol., 183, 101-108 (2001) [9] Clay, M.D.; Jenney, F.E., Jr.; Hagedoorn, P.L.; George, G.N.; Adams, M.W.W.; Johnson, M.K.: Spectroscopic studies of Pyrococcus furiosus superoxide re429

Superoxide reductase

[10]

[11]

[12] [13] [14]

430

1.15.1.2

ductase: implications for active-site structures and the catalytic mechanism. J. Am. Chem. Soc., 124, 788-805 (2002) Mathe, C.; Mattioli, T.A.; Horner, O.; Lombard, M.; Latour, J.M.; Fontecave, M.; Niviere, V.: Identification of iron(III) peroxo species in the active site of the superoxide reductase SOR from Desulfoarculus baarsii. J. Am. Chem. Soc., 124, 4966-4967 (2002) Coulter, E.D.; Emerson, J.P.; Kurtz, D.M., Jr.; Cabelli, D.E.: Superoxide reactivity of rubredoxin oxidoreductase (desulfoferrodoxin) from Desulfovibrio vulgaris: a pulse radiolysis study. J. Am. Chem. Soc., 122, 11555-11556 (2000) Rusnak, F.; Ascenso, C.; Moura, I.; Moura, J.J.G.: Superoxide reductase activities of neelaredoxin and desulfoferrodoxin metalloproteins. Methods Enzymol., 349, 243-258 (2002) Niviere, V.; Lombard, M.: Superoxide reductase from Desulfoarculus baarsii. Methods Enzymol., 349, 123-129 (2002) Emerson, J.P.; Coulter, E.D.; Cabelli, D.E.; Phillips, R.S.; Kurtz, D.M., Jr.: Kinetics and mechanism of superoxide reduction by two-iron superoxide reductase from Desulfovibrio vulgaris. Biochemistry, 41, 4348-4357 (2002)

' *22, 

4

   
 1.16.1.1    Hg:NADP+ oxidoreductase    mercury(II) reductase    Mer A MerA protein [22] mercurate(II) reductase mercuric ion reductase mercuric reductase mercury reductase reduced NADP:mercuric ion oxidoreductase reductase, mercurate(II)
    67880-93-7

     Pseudomonas aeruginosa (PAO9501 [1,9,17]; Tn501-encoded [11]; enzyme is encoded by the transposon Tn501 [19]) [1, 4, 8, 9, 11, 17, 18, 19, 20] Escherichia coli (containing the cloned mercury resistance genes from plasmid NR1 [2]; W3110 lacIq containing the plasmid pPSO1 [12]; Tn501 mercuric ion reductase [26]; PWS1 [29]) [2, 10, 12, 17, 18, 22, 26, 29] Thiobacillus ferrooxidans (TFI 29 [14]) [3, 7, 14, 21] Pseudomonas stutzeri [4] Pseudomonas cepacia [4] Bacillus sp. (strain RC607 [5,28,30]) [5, 10, 18, 28, 30] Penicillium sp. (MR-2 strain [6]) [6] Bacillus sphaericus [10, 18] Bacillus polymyxa [10, 18] Rhodococcus sp. [10, 18]

431

Mercury(II) reductase





















1.16.1.1

Bacillus licheniformis [10, 18] Bacillus megaterium [10] Oerskovia sp. [10, 18] Staphylococcus saprophyticus [10, 18] Staphylococcus aureus [10, 18] Mycobacterium sp. [10] Citrobacterium [10] Micrococcus roseus [10, 18] Micrococcus luteus [10] Arthrobacter sp. [10] Streptomyces espinosus (strain 5 [13]) [13] Streptomyces lividans (strain 8 [13]; strain 1326 [13]) [13] Streptomyces coelicolor (strain M130 [13]) [13] Streptococcus agalactiae [13] Mycobacterium scrofulaceum (enzyme is encoded by the plasmid pVT1 [15]) [15] Yersinia enterolytica (138A14 [16,24]) [16, 24] Acinetobacter lwoffi [18] Acinetobacter calcoaceticus [18] Xanthomonas sp. [18] Pseudomonas sp. [18] Aeromonas sp. [18] Pseudomonas mendocina [18] Pseudomonas alcaligenes [18] Pseudomonas fluorescens [18] Xanthomonas campestris [18] Flavobacterium rigense (strain PR2 [27]) [27] Erwinia sp. [18] Pseudomonas putida (KT2442:mer-73 [23]) [23] Azotobacter chroococcum (SS2 [25]) [25] Geobacillus stearothermophilus [18]

!    "
 # 

Hg + NADP+ + H+ = Hg2+ + NADPH + H+    redox reaction       Hg2+ + NADPH (, the enzyme is a key component of an organomercurial detoxification system [6]; , inducible enzyme [13]; , last step in bacterial mercury detoxification pathway [26]; , mercury resistance is due to the sequential action of two mercury-detoxificating enzymes, organomercurial lyase and mercuric re-

432

1.16.1.1

Mercury(II) reductase

ductase. Enzyme is induced by Hg2+ and organomercurials [27]) (Reversibility: ? [6, 13, 26, 27]) [6, 13, 26, 27] $ Hg + NADP+ + H+      2,4,6-trinitrobenzenesulfonate + NADPH (Reversibility: ? [17]) [17] $ ? + NADP+ [17]  Hg2+ + NADH (, very little activity with NADH [6]; , nearly identical activity with NADPH or NADH [15]) (Reversibility: ? [6, 15, 16, 27]) [6, 15, 16, 27] $ Hg + NADP+ + H+ [6]  Hg2+ + NADPH (, nearly identical activity with NADPH or NADH [15]) (Reversibility: ? [1-30]) [1-30] $ Hg + NADP+ + H+ [1, 2]  Hg2+ + azure A (Reversibility: ? [29]) [29] $ Hg + ?  Hg2+ + neutral red (Reversibility: ? [29]) [29] $ Hg + ?  merthiolate + NADPH (Reversibility: ? [24]) [24] $ ? + NADP+  Additional information (, Cys558 plays a more important role in forming the reducible complex with Hg(II), while both Cys558 and Cys559 seem to be involved in efficient scavenginig of Hg(II) [26]; , Tyr264 and Tyr605 are involved in substrate binding, Tyr264 is important for catalysis, possibly by destabilizing the binding of Hg(II) to the two ligating thiolates at the active site [30]) [26, 30] $ ? 2  Ag+ (, 0.1 mM [25]; , 0.1 mM AgNO3, complete inhibition [27]; , AgNO3 [2,14]) [2, 14, 25, 27] Ag2+ [24] AuCl3 [2] Bi3+ (, 0.1 mM, 40% inhibition [25]; , 0.1 mM Bi(NO3 )3 , 34% inhibition [27]) [25, 27] Cd2+ (, weak inhibition [24]) [2, 24, 25] CoCl2 (, 0.1 mM CoCl2 , 10% inhibition [27]; , CoCl2 [14]) [14, 25, 27] Cu2+ (, 0.1 mM, 44% inhibition [25]; , 0.1 mM, 40% inhibition [27]; , CuCl2 [2,14]) [2, 14, 24, 25, 27] CuCl [14] FeCl3 [14] HgCl2 (, activity is inhibited by an excess of HgCl2 [24,25]) [24, 25] KCN (, 0.1 mM, 20% inhibition [27]) [27] Mn2+ (, weak inhibition [24]) [24]

433

Mercury(II) reductase

1.16.1.1

NADPH (, substrate inhibition of the reaction with 2,4,6-trinitrobenzenesulfonate and NADPH [17]) [17] NEM (, 2 mM, 60% inhibition [25]; , 0.1 mM, 50% inhibition [27]) [25, 27] NaN3 (, 0.1 mM, 50% inhibition [27]) [25, 27] Ni2+ (, 0.1 mM, 62% inhibition [25]) [24, 25] Pb(NO3 )2 (, 0.1 mM, 78% inhibition [27]) [27] Pb2+ (, 0.1 mM, 60% inhibition [25]) [25] Zn2+ (, 0.1 mM, 60% inhibition [25]; , 0.1 mM, 40% inhibition [27]) [25, 27] Additional information (, no inhibition by 2,4,6-trinitrobenzenesulfonate [17]) [17]
"%  FAD (, contains FAD [8,9,14,24]; , flavoprotein [1,19]; , activity is dependent upon [6]) [1, 6, 8, 9, 14, 19, 24] NADH (, very little activity [6]; , slowly oxidized [16]; , some activation [11]; , strain 1326, NADPH stimulates more effectively than NADH [13]; , NADPH stimulates more effectively than NADH [13]; , nearly identical activity with NADPH or NADH [15]) [6, 11, 13, 15, 16, 27] NADPH (, in presence of 1 mM cysteine only one equivalent of NADPH per FAD is required for full activation [11]; , nearly identical activity with NADPH or NADH [15]; , optimal with a NADPH concentration near 0.05 mM [16]; , in the presence of an excess of NADPH, the final product of the reaction is probably an NADPH complex of two-electronreduced enzyme, but below pH 6 the final spectrum becomes less intense suggesting a partial formation of four-electron-reduced enzyme [20]) [1-30] &

  2-mercaptoethanol (, activates, optimal concentration is 3.3 mM [6]; , increases activity [16]; , required for activity with merthiolate [24]; , thiol compound required, optimal concentration is 0.5 mM [7]; , required [25]; , optimal concentration: 2 mM [25]) [6, 7, 16, 24, 25] EDTA (, required for maximal activity [7]; , no effect [11]) [7] NADP+ (, strong stimulation of the reaction with 2,4,6-trinitrobenzenesulfonate and NADPH [17]) [17] cysteine (, thiol compound required [7]) [7] dithiothreitol (, thiol compound required [7]) [7] thioglycolate (, thiol compound required [7]) [7] thiol (, exogenous thiols are required for catalytic reduction of Hg(II) to Hg2+ , due to prevention or reversal of formation of an abortive complex of Hg(II) with the thiol/thiolate pair of two-electron reduced enzyme [12]) [12] '(   Mg2+ (, required [25]) [25]

434

1.16.1.1

Mercury(II) reductase

) &  * +, 23 (Hg2+ , , mutant enzyme C558A, anaerobic conditions [26]) [26] 30 (Hg2+ , , mutant enzyme Y605F [22]; , mutant enzyme C558A, aerobic conditions [26]) [22, 26] 291 (Hg2+ , , mutant enzyme C559A, anaerobic conditions [26]) [26] 446 (Hg2+ , , wild-type enzyme, aerobic conditions [26]) [26] 458 (Hg2+ , , mutant enzyme C559A, aerobic conditions [26]) [26] 720 (Hg2+ ) [30] 746 (HgCl2 ) [14] 810 (Hg2+ , , wild-type enzyme, aerobic conditions [26]) [26] Additional information [11] " & *-% , 2.08 [24] 6 [1] 6.8 [27] 12.8 [14] 18.32 [25] Additional information [2] . / *', 0.0004 (NADPH, , in presence of 1 mM cysteine [11]) [11] 0.0019 (Hg2+ , , mutant enzyme Y605F [22]) [22] 0.0032 (Hg2+ , , in presence of 1 mM cysteine [11]) [11] 0.005 (Hg2+ , , wild-type enzyme, aerobic conditions [26]) [26] 0.0057 (Hg2+ , , wild-type enzyme, anaerobic conditions [26]) [26] 0.0088 (HgCl2 ) [6] 0.0089 (HgCl2 ) [14] 0.011 (HgCl2 ) [25] 0.012 (HgCl2 ) [1] 0.015 (Hg2+ ) [7] 0.021 (Hg2+ , , mutant enzyme C559A, anaerobic conditions [26]) [26] 0.025 (Hg2+ , , mutant enzyme C559A, aerobic conditions [26]) [26] 0.03 (Hg2+ ) [30] 0.045 (Hg2+ , , mutant enzyme C558A, aerobic conditions [26]) [26] 0.052 (Hg2+ , , mutant enzyme C558A, anaerobic conditions [26]) [26] 0.2 (HgCl2 , , 20 mM 2-mercaptoethanol [24]) [24] Additional information (, the Km -value for HgCl2 is dependent on the concentration of exogenous thiol compounds [24]) [2, 23, 24] . / *', 0.016 (Ag2+ ) [24] 0.0175 (Ni2+ ) [24] 0.018 (Cu2+ ) [24]

435

Mercury(II) reductase

1.16.1.1

0  7 (, soluble and immobilized mercuric reductase [29]) [29, 30] 7.3 [16] 7.4 [25] 7.5 [14] 7.5-8 [6] 0 

 6.5-9 (, pH 6.5: about 60% of maximal activity, pH 9.0: about 50% of maximal activity [14]) [14] )  *
, 34 [6] 37-43 [16] 45 [25]

  #   ' 1  58000 (, gel filtration [6]) [6] 110000 (, gel filtration [2]) [2] 123000 (, gel filtration [1]) [1] 130000 (, gel filtration [14]) [14] 142000 (, gel filtration [25]) [25] 200000 (, gel filtration [24]) [24] Additional information (, the enzyme shows extensive sequence homology and functional similarities in the active site of mercuric reductase and nicotinamide disulfide oxidoreductase [19]; , nucleotide sequence of the gene encoding mercuric reductase [21]) [19, 21]   ? (, x * 69000, SDS-PAGE [30]) [30] dimer (, 1 * 54000 + 1 * 69000, SDS-PAGE [25]; , 2 * 56000, SDS-PAGE [2]; , x * 54000 + x * 62000, SDS-PAGE [14]; , 1 * 56000 + 1 * 62000, SDS-PAGE [1]) [1, 2, 14, 25] monomer (, 1 * 58000, SDS-PAGE [6]) [6] trimer (, 3 * 70000, SDS-PAGE [24]) [24] Additional information (, along with ageing, as well as limited proteolytic digestion, the enzyme evolves to give a dimeric molecule of 105000 Da composed of two identical subunits of 52000 [24]) [24]

 2 %$ %' %

% mycelium [13]

436

1.16.1.1

Mercury(II) reductase

3#

soluble [7] $"

(purification of the major 14 C-labeled peptide from a tryptic digestion of labeled mercuric reductase [19]) [1, 19] [29] [14] [5, 30] [6] [24] [25]
 #

(hanging-drop vapor-diffusion method [5]) [5]
 

(cloned and expressed constitutively in Escherichia coli [3]) [3] (expression in Escherichia coli [30]) [30] 

 

C558A (, mutation results in a total disruption of the Hg(II) detoxification pathway in vivo, compared to wild-type enzyme the mutant shows a 20fold reduction in turnover number and a 10fold increase in Km [26]) [26] C559A (, mutation results in a total disruption of the Hg(II) detoxification pathway in vivo, compared to wild-type enzyme less than a 2fold reduction in turnover number and an increase in Km -value of 4-5fold [26]) [26] C628A (, HgX2 substrates with small ligands can rapidly access the redox-active cysteines in the absence of the C-terminal cysteines, but those with large ligands require the C-terminal cysteines for rapid access. The Cterminal cysteines play a critical role in removing the high-affinity ligands before Hg(II) reaches the redox-active cysteines [28]) [28] C629A (, HgX2 substrates with small ligands can rapidly access the redox-active cysteines in the absence of the C-terminal cysteines, but those with large ligands require the C-terminal cysteines for rapid access. The Cterminal cysteines play a critical role in removing the high-affinity ligands before Hg(II) reaches the redox-active cysteines [28]) [28] Y264F (, Km -value for Hg2+ is 5fold lower compared to the Km -value of the wild-type enzyme, turn-over number is reduced by 164fold [30]) [30] Y264F/Y605F (, Km -value for Hg2+ is 5fold lower than the Km -value of the wild-type enzyme, turnover-number is reduced by 1091fold [30]) [30] Y605F (, Km -value for Hg2+ is 1.3fold higher compared to the Km value of the wild-type enzyme, turnover-number is reduced by 6.3fold [30]) [30] Y605H (, 24fold decrease in turnover number and a 15fold decrease in Km -value [22]) [22]

437

Mercury(II) reductase

1.16.1.1



environmental protection (, application of the immobilized mercuric reductase for continuous treatment of Hg(II)-containing water in a fixed bed reactor [23]; , detoxification of mercury by immobilized mercuric reductase [29]) [23, 29]

4  )  80 (, stable at [7]; , 15 min, 15% loss of activity [16]) [7, 16] 100 (, 15 min, complete inactivation [16]) [16] 5    "

, immobilization of the enzyme appears to significantly enhance storage stability [29] , stable to repeated freeze-thaw cycles [7] , operational stability: after 1.5 h decline of activity up to 20% [23]    , 4 C, 50 mM potassium phosphate buffer, pH 7.2, 0.5 mM EDTA, 1% 2mercaptoethanol, half-life of soluble enzyme is 3 weeks, immobilized enzyme shows large decline at the beginning and almost no further decrease of activity after 3 weeks [23]

"  [1] Fox, B.; Walsh, C.T.: Mercuric reductase. Purification and characterization of a transposon-encoded flavoprotein containing an oxidation-reductionactive disulfide. J. Biol. Chem., 257, 2498-2503 (1982) [2] Rinderle, S.J.; Booth, J.E.; Williams, J.W.: Mercuric reductase from R-plasmid NR1: characterization and mechanistic study. Biochemistry, 22, 869876 (1983) [3] Kusano, T.; Ji, G.; Inoue, C.; Silver, S.: Constitutive synthesis of a transport function encoded by the Thiobacillus ferrooxidans merC gene cloned in Escherichia coli. J. Bacteriol., 172, 2688-2692 (1990) [4] Barkay, T.; Gillman, M.; Liebert, C.: Genes encoding mercuric reductases from selected gram-negative aquatic bacteria have a low degree of homology with merA of transposon Tn501. Appl. Environ. Microbiol., 56, 16951701 (1990) [5] Moore, M.J.; Distefano, M.D.; Walsh, C.T.; Schiering, N.; Pai, E.F.: Purification, crystallization, and preliminary X-ray diffraction studies of the flavoenzyme mercuric ion reductase from Bacillus sp. strain RC607. J. Biol. Chem., 264, 14386-14388 (1989) [6] Tezuka, T.; Someya, J.: Purification and some properties of mercuric reductase from the organomercury-resistant Penicillium sp. MR-2 strain. Agric. Biol. Chem., 54, 1551-1552 (1990) 438

1.16.1.1

Mercury(II) reductase

[7] Olson, G.J.; Porter, F.D.; Rubinstein, J.; Silver, S.: Mercuric reductase enzyme from a mercury-volatilizing strain of Thiobacillus ferrooxidans. J. Bacteriol., 151, 1230-1236 (1982) [8] Sahlman, L.; Lambeir, A.M.; Lindskog, S.; Dunford, H. B.: The reaction between NADPH and mercuric reductase from Pseudomonas aeruginosa. J. Biol. Chem., 259, 12403-12408 (1984) [9] Sahlman, L.; Lindskog, S.: A stopped-flow study of the reaction between mercuric reductase and NADPH. Biochem. Biophys. Res. Commun., 117, 231-237 (1983) [10] Bogdanova, E.S.; Mindlin, S.Z.: Two structural types of mercury reductases and possible ways of their evolution. FEBS Lett., 247, 333-336 (1989) [11] Sandstroem, A.; Lindskog, S.: Activation of mercuric reductase by the substrate NADPH. Eur. J. Biochem., 164, 243-249 (1987) [12] Miller, S.M.; Ballou, D.P.; Massey, V.; Williams, C. H.; Walsh, C.T.: Two-electron reduced mercuric reductase binds Hg(II) to the active site dithiol but does not catalyze Hg(II) reduction. J. Biol. Chem., 261, 8081-8084 (1986) [13] Nakahara, H.; Schottel, J.L.; Yamada, T.; Miyakawa, Y.; Asakawa, M.; Harville, J.; Silver, S.: Mercuric reductase enzymes from Streptomyces species and group B Streptococcus. J. Gen. Microbiol., 131, 1053-1059 (1985) [14] Booth, J.E.; Williams, J.W.: The isolation of a mercuric ion-reducing flavoprotein from Thiobacillus ferrooxidans. J. Gen. Microbiol., 130, 725-730 (1984) [15] Meissner, P.S.; Falkinham, J.O.: Plasmid-encoded mercuric reductase in Mycobacterium scrofulaceum. J. Bacteriol., 157, 669-672 (1984) [16] Blaghen, M.; Lett, M.C.; Vidon, D.J.M.: Mercuric reductase activity in a mercury-resistant strain of Yersinia enterolytica. FEMS Microbiol. Lett., 19, 93-96 (1983) [17] Carlberg, I.C.; Sahlman, L.; Mannervik, B.: The effect of 2,4,6-trinitrobenzenesulfonate on mercuric reductase, glutathione reductase and lipoamide dehydrogenase. FEBS Lett., 180, 102-106 (1985) [18] Bogdanova, E.S.; Mindlin, S.Z.; Kalyaeva, E.S.; Nikiforov, V.G.: The diversity of mercury reductases among mercury-resistant bacteria. FEBS Lett., 234, 280-282 (1988) [19] Fox, B.S.; Walsh, C.T.: Mercuric reductase: homology to glutathione reductase and lipoamide dehydrogenase. Iodoacetamide alkylation and sequence of the active site peptide. Biochemistry, 22, 4082-4088 (1983) [20] Sahlman, L.; Lambeir, A.M.; Lindskog, S.: Rapid-scan stopped-flow studies of the pH dependence of the reaction between mercuric reductase and NADPH. Eur. J. Biochem., 156, 479-488 (1986) [21] Inoue, C.; Sugawara, K.; Shiratori, T.; Kusano, T.; Kitagawa, Y.: Nucleotide sequence of the Thiobacillus ferrooxidans chromosomal gene encoding mercuric reductase. Gene, 84, 47-54 (1989) [22] Rennex, D.; Pickett, M.; Bradley, M.: In vivo and in vitro effects of mutagenesis of active site tyrosine residues of mercuric reductase. FEBS Lett., 355, 220-222 (1994)

439

Mercury(II) reductase

1.16.1.1

[23] Anspach, F.B.; Hueckel, M.; Brunke, M.; Schuette, H.; Deckwer, W.D.: Immobilization of mercuric reductase from a Pseudomonas putida strain on different activated carriers. Appl. Biochem. Biotechnol., 44, 135-150 (1994) [24] Blaghen, M.; Vidon, D.J.M.; El Kebbaj, M.S.: Purification and properties of mercuric reductase from Yersinia enterocolitica 138A14. Can. J. Microbiol., 39, 193-200 (1993) [25] Ghosh, S.; Sadhukhan, P.C.; Chaudhuri, J.; Ghosh, D.K.; Mandal, A.: Purification and properties of mercuric reductase from Azotobacter chroococcum. J. Appl. Microbiol., 86, 7-12 (1999) [26] Moore, M.J.; Miller, S.M.; Walsh, C.T.: C-Terminal cysteines of Tn501 mercuric ion reductase. Biochemistry, 31, 1677-1685 (1992) [27] Gachhui, R.; Chaudhuri, J.; Ray, S.; Pahan, K.; Mandal, A.: Studies on mercury-detoxicating enzymes from a broad-spectrum mercury-resistant strain of Flavobacterium rigense. Folia Microbiol., 42, 337-343 (1997) [28] Engst, S.; Miller, S.M.: Alternative routes for entry of HgX2 into the active site of mercuric ion reductase depend on the nature of the X ligands. Biochemistry, 38, 3519-3529 (1999) [29] Chang, J.S.; Hwang, Y.P.; Fong, Y.M.; Lin, P.J.: Detoxification of mercury by immobilized mercuric reductase. J. Chem. Technol. Biotechnol., 74, 965-973 (1999) [30] Rennex, D.; Cummings, R.T.; Pickett, M.; Walsh, C.T.; Bradley, M.: Role of tyrosine residues in Hg(II) detoxification by mercuric reductase from Bacillus sp. strain RC607. Biochemistry, 32, 7475-7478 (1993)

440

9"  " 

4

   
 1.16.1.2    transferrin[Fe(II)]2 :NAD+ oxidoreductase    diferric-transferrin reductase    NADH diferric transferrin reductase diferric transferrin reductase transferrin reductase
    105238-49-1

     Homo sapiens [3] Rattus norvegicus [2, 3] Plasmodium falciparum (malaria parasite, erythrocytes infected with Plasmodium falciparum [1]) [1] Mycobacterium paratuberculosis [4]

!    "
 # 

transferrin[Fe(II)]2 + NAD+ = transferrin[Fe(III)]2 + NADH + H+ ( amino acid composition [4])    oxidation redox reaction reduction       transferrin[Fe(III)]2 + NADH ( enzyme of the malaria parasite Plasmodium falciparum together with parasite-derived transferrin receptor in the erythrocyte membrane forms a transferrin receptor-mediated 441

Diferric-transferrin reductase

1.16.1.2

uptake mechanism of iron [1]; reduction of diferric transferrin at the cell surface may be an important function for diferric transferrin in stimulation of cell growth [2]; role in iron transport [2]) (Reversibility: ? [1, 2]) [1, 2] $ transferrin[Fe(II)]2 + NAD+      ferric ammonium citrate + NADH (Reversibility: ? [4]) [4] $ ferrous ammonium citrate + NAD+  ferritin[Fe(III)] + NADH (Reversibility: ? [1-4]) [1-4] $ ferritin[Fe(II)] + NAD+  transferrin[Fe(III)]2 + NADH (Reversibility: ? [1-4]) [1-4] $ transferrin[Fe(II)]2 + NAD+ [1-4] 2  adriamycin [2] all-trans retinoic acid [3] amiloride [2] apotransferrin [1, 2] atebrin [2] chloroquinone [2] Additional information ( sensitive to proteinase K treatment [4]; retinol and retinyl acetate have very little inhibitory effect [3]; sensitive to treatment with trypsin [1,2]) [1-4]
"%  NADH ( dependent on, highest activity at about 0.1 mM [4]; preferred electron donor [2]) [1-4] NADPH ( about 50% of the activity with NADH [2]) [2] &

  3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate ( detergent, activates [2]) [2] '(   K+ ( potassium phosphate buffer leads to a slightly higher activity than sodium phosphate buffer [2]) [2] Mg2+ ( activates and stabilizes [4]) [4] " & *-% , 0.0057 [2] . / *', 0.0061 (NADH) [2] 0.033 (diferric transferrin) [2] 0.213 (ferric ammonium citrate) [4] 0  5-10 [4] 8.5 [2]

442

1.16.1.2

Diferric-transferrin reductase

0 

 7-10 ( pH 7.0: about 40% of activity maximum, pH 10.0: about 35% of activity maximum [2]) [2] )  *
, 37 [4]

  #   ' 1  17000 ( immunoblotting analysis [4]) [4]

 2 %$ %' %

% HeLa cell [3] erythrocyte ( infected with Plasmodium falciparum, synthesis of the enzyme by the intraerythrocytic parasite [1]) [1] liver [2, 3] 3#

cytoplasmic membrane ( erythrocytes infected with Plasmodium falciparum, synthesis and incorporation of the enzyme by the intraerythrocytic parasite [1]) [1-3] extracellular [4] Additional information ( enzyme is found intracellular in Mycobacterium paratuberculosis infected bovine gut tissue [4]; low activity in the membranes of Golgi apparatus, endoplasmic reticulum and mitochondria, about 10% of the activity in the plasma membrane [2]) [2, 4] $"

[4]

"  [1] Fry, M.: Diferric transferrin reductase in Plasmodium falciparum-infected erythrocytes. Biochem. Biophys. Res. Commun., 158, 469-473 (1989) [2] Sun, I.L.; Navas, P.; Crane, F.L.; Morre, D.J.; Löw, H.: NADH diferric transferrin reductase in liver plasma membrane. J. Biol. Chem., 262, 15915-15921 (1987) [3] Sun, I.L.; Toole-Simms, W.; Crane, F.L.; Golub, E.S.; Diaz de Pagan, T.; Morre, D.J.; Löw, H.: Retinoic acid inhibition of transplasmalemma diferric transferrin reductase. Biochem. Biophys. Res. Commun., 146, 976-982 (1987) [4] Homuth, M.; Valentin-Weigand, P.; Rohde, M.; Gerlach, G.F.: Identification and characterization of a novel extracellular ferric reductase from Mycobacterium paratuberculosis. Infect. Immun., 66, 710-716 (1998)

443

? 

4!

   
 1.16.1.3    cob(II)alamin:NAD+ oxidoreductase    aquacobalamin reductase    B12a reductase EC 1.6.99.8 (formerly) NADH-linked aquacobalamin reductase NADH2 :cob(III)alamin oxidoreductase aquacobalamin reductase reductase, aquacobalamin reductase, vitamin B12a vitamin B12a reductase Additional information ( enzyme is probably part of the b-type cytochrome/cytochrome b5 reductase complex in rat mitochondria [7, 9]) [7, 9]
    37256-39-6

    







444

Clostridium tetanomorphum [2] Rattus norvegicus [1, 3, 6, 7, 9] Macaca sp. (monkey [1]) [1] Bos taurus [1] Sus scrofa [1] Gallus gallus [1] Rana catesbeiana (frog [1]) [1] Girella punctate (sea fish) [1] Cyprinus auratus (freshwater fish [1]) [1] Homo sapiens [4-6, 8]

1.16.1.3

Aquacobalamin reductase

!    "
 # 

2 cob(II)alamin + NAD+ = 2 aquacob(III)alamin + NADH + H+    oxidation redox reaction reduction       NADH + aquacob(III)alamin ( part of conversion of vitamin B12 to its principal coenzyme form, adenosyl-B12 [2,4,5]) (Reversibility: ? [2, 4-9]) [2, 4-9] $ NAD+ + cob(II)alamin [2, 4-9]  Additional information ( determination of aquacobalamin uptake as pro-coenzyme for cytosolic methionine synthase, EC 2.1.1.13, and mitochondrial methylmalonyl-CoA mutase, EC 5.4.99.2 into fibroblasts and glial cells [5]) [5] $ ?      NADH + aquacob(III)alamin ( specific for aquacobalamin, cyanocobalamin cannot be reduced [7, 9]; dithioerythritol cannot replace NADH as reductant [2]) (Reversibility: ? [2, 4-9]) [2, 4-9] $ NAD+ + cob(II)alamin [2, 4-9]  NADPH + hydroxycobalamin (Reversibility: ? [1]) [1] $ NADP+ + 5'-deoxyadenosylcobalamin [1]
"%  FAD ( flavoprotein, utilizes FAD better than FMN [2, 6]) [2, 6] FMN ( flavoprotein, utilizes FAD better than FMN [2]) [2] NADH ( dependent on [6,7]; specific for [2, 4, 8]) [2, 4, 6-9] NADPH ( about 12% of activity with NADH [6]) [6] Additional information ( dithiothreitol cannot replace NADH as electron donor [2]) [2] " & *-% , 0.00028 ( liver enzyme [6]) [6] 0.0224 ( mitochondrial, mutant cbl C [8]) [8] 0.025-0.126 ( mitochondrial, several mutants cbl C [6]) [6] 0.029 ( mitochondrial, mutant cbl A [8]) [8] 0.037 ( mitochondrial, wild-type [8]) [8] 0.0385 ( microsomal, mutant cbl A [8]) [8] 0.0435 ( microsomal, wild-type [8]) [8] 0.044 ( microsomal, mutant cbl C [8]) [8] 0.069 ( liver enzyme [4]) [4] 0.1 ( mitochondrial, mutant cbl D [6]) [6]

445

Aquacobalamin reductase

1.16.1.3

0.107 ( mitochondrial, wild-type [6]) [6] 0.11 ( mitochondrial [7,9]; partially purified enzyme [7]) [7, 9] 0.111 ( mitochondrial, mutant cbl E [6]) [6] 0.118-0.186 ( microsomal, several mutants cbl C [6]) [6] 0.1275 ( microsomal, liver [9]) [9] 0.257 ( microsomal, mutant cbl D [6]) [6] 0.448 ( microsomal, wild-type [6]) [6] 0.458 ( microsomal, mutant cbl E [6]) [6] 0.578 ( partially purified enzyme [6]) [6] 3.3 ( purified cytochrome b5 /cytochrome b5 reductase complex from microsomes, substrate aquacobalamin [9]) [9] Additional information ( overview, activity in several tissues [1,4]) [1, 4, 6] . / *', 0.01 (NADH, microsomal [9]) [9] 0.0144 (NADH, microsomal [7,9]; in presence of 0.1 mM aquacobalamin [7]) [7, 9] 0.015 (FAD) [2] 0.015 (FMN) [2] 0.016 (NADH) [6] 0.032 (aquacob(III)alamin, microsomal [9]) [9] 0.042 (aquacob(III)alamin, microsomal [7,9]) [7, 9] 0.057 (aquacob(III)alamin) [8] 0.08 (aquacob(III)alamin) [6] 0.08-0.099 (aquacob(III)alamin, mutant cbl C [8]) [8] Additional information ( cytochrome b5 /cytochrome b5 reductase complex from microsomes, substrate aquacobalamin [9]) [8, 9] 0  6.6 ( microsomal, liver [9]) [9] 6.8 ( assay at [5]; at 40 C [6]) [5, 6] 7 ( assay at [1,3]) [1, 3] 7.1 ( mitochondrial [7,9]) [7, 9] )  *
, 40 ( mitochondrial [7,9]; assay at [1,5,8]) [1, 5, 7-9] 40-42 [6] 50 ( assay at [3]; microsomal, liver [9]) [3, 9]

 2 %$ %' %

% adrenal gland [1] adrenal gland [4] bone marrow [1, 4] brain [1]

446

1.16.1.3

Aquacobalamin reductase

cerebellum [4] cerebrum [4] colon [4] duodenum [4] fibroblast ( skin [6,8]) [5, 6, 8] glial cell [5] heart [1] ileum [1, 4] jejunum [1, 4] kidney [1, 4] large intestine [1] leukocyte [6] liver [1, 3, 4, 6, 7, 9] lung [1, 4] pancreas [1] spleen [1, 4] stomach [1, 4] testis [1] 3#

microsome [3, 4, 6, 8, 9] mitochondrion ( membrane [7,9]; inside of outer membrane [3,4,6]) [3, 4, 6-9] $"

(partially [6,7]; both microsomal and mitochondrial [9]; enzyme is probably part of the b-type cytochrome/cytochrome b5 reductase complex in rat mitochondria, identical elution behaviour in gel chromatography [7,9]) [6, 7, 9] 

 

Additional information ( mitochondrial enzyme mutant cbl A, which is uneffected in the NADH-linked enzyme, and cbl C with lowered affinity for NADH and therefore decreased activity [8]; mutant cell lines WG1952, WG1936, and WG1998, and enzyme mutants cbl C, cbl D, and cbl E from human fibroblasts and leukocytes, cbl C mutants show normal or reduced activity, cbl D reduced activity, cbl E mutation affects the methione synthase EC 2.1.1.13 [6]) [6, 8]

4  )  50 ( 5 min, about 80% loss of activity [2]) [2] 60 ( 5 min, about 90% loss of activity [2]) [2] 70 ( 5 min, complete loss of activity [2]) [2]

447

Aquacobalamin reductase

1.16.1.3

5    "

, dithioerythritol stabilizes [2] , enzyme solutions rapidly lose activity, especially if frozen and thawed [2] , lyophilized preparations of cell-free extract stable to storage at ±20 C for several weeks [2] , proteolytically unstable during purification of microsomes [6]

"  [1] Watanabe, F.; Nakano, Y.; Tachikake, N.; Tamura, Y.; Yamanaka, H.; Kitaoka, S.: Occurrence and tissue distribution of both NADH- and NADPH-linked aquacobalamin reductases in some vertebrates. J. Nutr. Sci. Vitaminol., 36, 349-356 (1990) [2] Walker, G.A.; Murphy, S.; Huennekens, F.M.: Enzymatic conversion of vitamin B12a to adenosyl-B 12: evidence for the existence of two separate reducing systems. Arch. Biochem. Biophys., 134, 95-102 (1969) [3] Watanabe, F.; Nakano, Y.; Maruno, S.; Tachikake, N.; Tamura, Y.; Kitaoka, S.: NADH- and NADPH-linked aquacobalamin reductases occur in both mitochondrial and microsomal membranes of rat liver. Biochem. Biophys. Res. Commun., 165, 675-679 (1989) [4] Watanabe, F.; Nakano, Y.; Tachikake, N.; Kitaoka, S.; Tamura, Y.; Yamanaka, H.; Haga, S.; Imai, S.; Saido, H.: Occurrence and subcellular location of NADH- and NADPH-linked aquacobalamin reductases in human liver. Int. J. Biochem., 23, 531-533 (1991) [5] Pezacka, E.H.; Jacobsen, D.W.; Luce, K.; Green, R.: Glial cells as a model for the role of cobalamin in the nervous system: impaired synthesis of cobalamin coenzymes in cultured human astrocytes following short-term cobalamin-deprivation. Biochem. Biophys. Res. Commun., 184, 832-839 (1992) [6] Pezacka, E.H.: Identification and characterization of two enzymes involved in the intracellular metabolism of cobalamin. Cyanocobalamin b-ligand transferase and microsomal cob(III)alamin reductase. Biochim. Biophys. Acta, 1157, 167-177 (1993) [7] Saido, H.; Watanabe, F.; Tamura, Y.; Miyatake, K.; Ito, A.; Yubisui, T.; Nakano, Y.: Cytochrome b5 -like hemoprotein/cytochrome b5 reductase complex in rat liver mitochondria has NADH-linked aquacobalamin reductase activity. J. Nutr., 124, 1037-1040 (1994) [8] Watanabe, F.; Saido, H.; Yamaji, R.; Miyatake, K.; Isegawa, Y.; Ito, A.; Yubisui, T.; Rosenblatt, D.S.; Nakano, Y.: Mitochondrial NADH- or NADPH-linked aquacobalamin reductase activity is low in human skin fibroblasts with defects in synthesis of cobalamin coenzymes. J. Nutr., 126, 2947-2951 (1996) [9] Watanabe, F.; Nakano, Y.: Purification and characterization of aquacobalamin reductases from mammals. Methods Enzymol., 281, 295-305 (1997)

448


*22, 

4

   
 1.16.1.4    cob(I)alamin:NAD+ oxidoreductase    cob(II)alamin reductase    B12r reductase EC 1.6.99.9 (formerly) NADH2:cob(II)alamin oxidoreductase reductase, vitamin B12r vitamin B12r reductase
    37256-40-9

     Clostridium tetanomorphum [1] Salmonella enterica (serovar typhimurium LT2, strain TR6583 [2]) [2]

!    "
 # 

2 cob(I)alamin + NAD+ = 2 cob(II)alamin + NADH + H+ ( N-terminal amino acid sequence is identical with EC 1.6.8.1 NAD(P)H:flavin oxidoreductase, but the enzymic reactions are differing [2])    oxidation redox reaction reduction

449

Cob(II)alamin reductase

1.16.1.4

      NADH + cob(II)alamine ( part of conversion of vitamin B12 to its principal coenzyme form, adenosyl-B12 [1, 2]; dithioerythritol can replace NADH as reductant [1]) (Reversibility: ? [1, 2]) [1, 2] $ NAD+ + cob(I)alamin [1, 2]      NADH + cob(II)alamine ( dithioerythritol can replace NADH as reductant [1, 2]) (Reversibility: ? [1, 2]) [1, 2] $ NAD+ + cob(I)alamin [1, 2]
"%  FAD ( flavoprotein, stimulated equally well by FAD or FMN [1]) [1] FMN ( dependent on [2]; flavoprotein, stimulated equally well by FAD or FMN [1]) [1, 2] NADH ( cannot be replaced by NADPH, but by dithiothreitol [1]) [1, 2] " & *-% , 0.00021 [1] 0.00022 ( FAD repalced by FMN [1]) [1] 0.00032 ( with dithiothreitol as electron donor [1]) [1]

 2 %$ %' %

$"

[2]

4  )  4 ( under N2 -atmosphere, cell-free extract, several days [1]) [1] 50 ( 5 min, about 40% loss of activity [1]) [1] 60 ( 5 min, 75% loss of activity [1]) [1] 70 ( 5 min, about 90% loss of activity [1]) [1]

"  [1] Walker, G.A.; Murphy, S.; Huennekens, F.M.: Enzymatic conversion of vitamin B12a to adenosyl-B12 : evidence for the existence of two separate reducing systems. Arch. Biochem. Biophys., 134, 95-102 (1969) [2] Fonseca, M.V.; Escalante-Semerena, J.C.: Reduction of cob(III)alamin to cob(II)alamin in Salmonella enterica serovar typhimurium LT2. J. Bacteriol., 182, 4304-4309 (2000)

450

?  *9$0,

4

   
 1.16.1.5    cob(II)alamin:NADP+ oxidoreductase    aquacobalamin reductase (NADPH)    EC 1.6.99.11 (formerly) NADPH-linked aquacobalamin reductase NADPH:aquacob(III)alamin oxidoreductase aquacobalamin (reduced nicotinamide adenine dinucleotide phosphate) reductase reductase, aquacobalamin (reduced nicotinamide adenine dinucleotide phosphate) Additional information ( microsomal aquacobalamin reductase has microsomal NADPH-cytochrome c (P-450) reductase like activity, the enzymes are probably identical [10]; mitochondrial aquacobalamin reductase and microsomal NADPH-cytochrome c (P-450) reductase are distinct proteins [8]; immunohistochemically crossreactive and in elution behaviour as well as molecular weight identical with NADPH-diaphorase domain of pyruvate:NADP+ oxidoreductase, pyruvate:NADP+ oxidoreductase also has aquacob(III)alamin reductase activity with NADPH [9]; enzyme has a NADPH-diaphorase-like activity [7,9]) [7-10]
    110777-32-7

    





Rattus norvegicus [1, 2, 5, 8, 10] Macaca sp. (monkey [1]) [1] Bos taurus [1] Sus scrofa [1] Gallus gallus [1] Rana catesbeiana (frog [1]) [1] Girella punctate (sea fish [1]) [1]

451

Aquacobalamin reductase (NADPH)

1.16.1.5

Cyprinus uratus (freshwater fish [1]) [1] Euglena gracilis (strain SM-ZK [3,4]) [3, 4, 7, 9] Homo sapiens [6]

!    "
 # 

2 cob(II)alamin + NADP+ = 2 aquacob(III)alamin + NADPH + H+ (A flavoprotein. Acts on aquacob(III)alamin and hydroxycobalamin, but not on cyanocobalamin)    oxidation redox reaction reduction       NADPH + aquacob(III)alamin (Reversibility: ? [1-9]) [19] $ NADP+ + cob(II)alamin [1-9]      NADPH + 2,6-dichlorophenolindophenol ( mitochondrial enzyme [5,7,8,9]; aerobic conditions [7]) (Reversibility: ? [5, 7, 8, 9]) [5, 7, 8, 9] $ ?  NADPH + aquacob(III)alamin ( the microsomal enzyme is specific for aquacobalamin [5]; aerobic conditions [7]) (Reversibility: ? [1-10]) [1-10] $ NADP+ + cob(II)alamin [1-10]  NADPH + cytochrome c ( mitochondrial enzyme [5,7,8,9]; aerobic conditions [7]) (Reversibility: ? [5, 7, 8, 9]) [5, 7, 8, 9] $ ?  NADPH + ferricyanide ( mitochondrial enzyme [5,7,8,9]; aerobic conditions [7]) (Reversibility: ? [5, 7, 8, 9]) [5, 7, 8, 9] $ NADP+ + ferrocyanide  NADPH + hydroxycobalamin ( aqua- and hydroxocobalamin are interconvertible in solution depending on pH. Above pH 8.0, hydroxocobalamin tends to predominate over aquacobalamin at equilibrium [7]) (Reversibility: ? [1, 3, 4, 7]) [1, 3, 4, 7] $ NADP+ + 5'-deoxyadenosylcobalamin [1, 3, 4]  Additional information ( under anaerobic conditions enzyme reduces FAD, FMN, methyl viologen, and benzyl viologen as well [7,9]; mitochondrial enzyme, specific for aquacobalamin, but not cyanocobalmin [7]; no actvity of the microsomal enzyme with cyanocobalamin [5]; no activity with cyanocobalamin [4]) [4, 5, 7, 9] $ ?

452

1.16.1.5

Aquacobalamin reductase (NADPH)

2  5,5'-dithiobis(2-nitrobenzoate) ( complete inhibition at 1 mM, mitochondrial enzyme [7]) [4, 7] Al3+ ( mitochondrial enzyme [5,7]) [4, 5, 7] Ba2+ ( mitochondrial enzyme [5,8]) [5, 8] Ca2+ ( mitochondrial enzyme [5,8]) [5, 8] Co2+ ( mitochondrial enzyme [5,8]) [5, 8] Cu2+ ( complete inhibition at 1 mM, mitochondrial enzyme [5,8]) [5, 8] Fe2+ ( mitochondrial enzyme [5,8]) [5, 8] Hg2+ ( complete inhibition at 1 mM, mitochondrial enzyme [5,8]) [5, 8] Mg2+ ( mitochondrial enzyme [5,8]) [5, 8] Mn2+ ( mitochondrial enzyme [5,8]) [5, 8] N-ethylmaleimide ( complete inhibition at 1 mM, mitochondrial enzyme [5,7,8]) [4, 5, 7, 8] Zn2+ ( complete inhibition at 1 mM, mitochondrial enzyme [5,8]) [4, 5, 7, 8] mersalyl ( complete inhibition at 1 mM, mitochondrial enzyme [5,7,8]) [4, 5, 7, 8] Additional information ( no inhibition with Na+ , K+ , Ni2+ , Mg2+ , Mn2+ , Co2+, Ca2+ , EDTA [4,7]; nearly no inhibition of the mitochondrial enzyme by K+ , Na+ , Li+ [5,8]) [4, 5, 7, 8]
"%  FAD ( 0.9 mol FAD and 0.94 mol FMN per mol of mitochondrial enzyme [5,8]; flavoprotein, contains 1 molecule of FAD or FMN as prosthetic group, FAD or FMN not required as cofactors [4,7]) [4, 5, 7, 8] FMN ( 1 mol FAD and FMN per mol of mitochondrial enzyme [5,8]; flavoprotein, contains 1 molecule of FMN or FAD as prosthetic group, FAD or FMN not required as cofactors [4,7]) [4, 5, 7, 8] NADPH ( specific for NADPH [3-10]) [3-10] Additional information ( no activity with NADH [3,4,7,8]) [3, 4, 7, 8] &

  EDTA ( slightly activating at 1 mM, mitochondrial enzyme [5,8]) [5, 8] o-phenanthroline ( slightly activating at 1 mM, mitochondrial enzyme [5,8]) [5, 8] phenobarbital ( mitochondrial enzyme [8]) [8] " & *-% , 0.0045 ( 0.0046 ( 0.0064 ( 0.0069 ( 0.0083 (

mitochondrial, mutant cbl C [6]) [6] mitochondrial, wild-type [6]) [6] microsomal, mutant cbl A [6]) [6] microsomal, mutant cbl C [6]) [6] microsomal, wild-type [6]) [6]

453

Aquacobalamin reductase (NADPH)

1.16.1.5

0.0152 ( mitochondrial enzyme, crude extract [3]) [3] 0.023 ( microsomal crude extract [8]) [8] 0.024 ( mitochondrial crude extract [8]) [8] 0.0365 ( mitochondrial crude extract from phenobarbital-treated rats [8]) [8] 0.0807 ( microsomal crude extract from phenobarbital-treated rats [8]) [8] 2.24 ( purified enzyme from mitochondria [4]) [4] 2.9 ( substrate aquacobalamin, purified mitochondrial enzyme, aerobic conditions [7,9]) [7, 9] 4.9 ( purified mitochondrial enzyme, substrate potassium ferricyanide [8]) [8] 6.4 ( purified mitochondrial enzyme, substrate aquacobalamin [5,8]) [5, 8] 11.22 ( purified microsomal enzyme [5,10]) [5, 10] 14.2 ( purified mitochondrial enzyme, substrate 2,6-dichlorophenolindophenol, aerobic conditions [7,9]) [7, 9] 15.4 ( purified mitochondrial enzyme, substrate cytochrome c [8]) [8] 16.8 ( purified mitochondrial enzyme, substrate 2,6-dichlorophenolindophenol [8]) [8] 25 ( purified mitochondrial enzyme, substrate potassium ferricyanide, aerobic conditions [7,9]) [7, 9] 37 ( purified mitochondrial enzyme, substrate cytochrome c, aerobic conditions [7,9]) [7, 9] Additional information ( overview: activity in several tissues [1]; overiew of cosubstrate specificity [7,9]) [1, 7, 9] . / *', 0.0048 (NADPH, mitochondrial enzyme [5,8]) [5, 8] 0.014 (NADPH, microsomal enzyme [5]) [5] 0.043 (NADPH) [4, 7] 0.055 (hydroxycobalamin) [4, 7] 0.058 (aquacobalamin, microsomal enzyme [5]) [5] 0.208 (aquacobalamin, mitochondrial enzyme [5,8]) [5, 8] 0  7 ( assay at [1,3,7]) [1, 4, 3, 7] 7.5 ( microsomal enzyme [5]) [5] 8.2 ( mitochondrial enzyme [5]) [5] )  *
, 40 ( assay at [1,3,4,6]; mitochondrial enzyme [4,6,7]) [1, 3, 4, 6, 7] 45 ( microsomal enzyme [5]) [5] 50 ( assay at [2]; mitochondrial enzyme [5]) [2, 5]

454

1.16.1.5

Aquacobalamin reductase (NADPH)

  #   ' 1  65000 ( mitochondrial enzyme, gel filtration [5,8]) [5, 8] 66000 ( gel filtration, SDS-PAGE [4,7,9]) [4, 7, 9] 79000 ( microsomal enzyme, gel filtration [5,10]) [5, 10] Additional information ( MW 166000 Da of native mitochondrial enzyme as part of pyruvate:NADP+ oxidoreductase, immunoblotting [9]) [9]   monomer ( 1 * 65000, SDS-PAGE [7]; 1 * 65000, mitochondrial enzyme, SDS-PAGE [5,8]; 1 * 79000, microsomal enzyme, SDSPAGE [5,10]) [5, 7, 8, 10]

 2 %$ %' %

% brain [1] fibroblast ( skin [6]) [6] heart [1] ileum [1] jejunum [1] kidney [1] large intestine [1] liver [1, 2, 5, 8, 10] lung [1] pancreas [1] spleen [1] stomach [1] testis [1] 3#

microsome [1, 2, 5, 6, 8, 10] mitochondrion ( membrane [8]; soluble fraction [7]; inside of outer membrane [2]) [1-8] $"

(partially [1]; from microsomes [10]; mitochondrial, light yellow coloured [8]; from mitochondria and microsomes [5]) [1, 5, 8, 10] (partially [3]; mitochondrial enzyme [4,7]; pale yellow coloured [7]) [3, 4, 7, 9] 

 

Additional information ( mitochondrial enzyme mutants cbl C and cbl A, cbl C mutant is unchanged compared to wild-type, while cbl A mutant shows no NADPH-linked activity [6]) [6]

455

Aquacobalamin reductase (NADPH)

1.16.1.5

4  0  5 ( below, complete loss of activity [7]) [7] 6-8 ( 10 min, 55 C, stable [4,7]) [4, 7] )  50 ( 10 min, stable [3]; 10 min, pH 7.0, stable up to [4,7]) [3, 4, 7] 60 ( 10 min, pH 7.0, complete loss of activity [4,7]) [4, 7]    , -20 to 80 C, 10 mM Tris-acetate, pH 7.0, potassium chloride, 1 mM EDTA, 0.001 mM dithiothreitol, concentrated, no loss of activity in several months [7] , 0 C, 2 weeks [3]

"  [1] Watanabe, F.; Nakano, Y.; Tachikake, N.; Tamura, Y.; Yamanaka, H.; Kitaoka, S.: Occurrence and tissue distribution of both NADH- and NADPH-linked aquacobalamin reductases in some vertebrates. J. Nutr. Sci. Vitaminol., 36, 349-356 (1990) [2] Watanabe, F.; Nakano, Y.; Maruno, S.; Tachikake, N.; Tamura, Y.; Kitaoka, S.: NADH- and NADPH-linked aquacobalamin reductases occur in both mitochondrial and microsomal membranes of rat liver. Biochem. Biophys. Res. Commun., 165, 675-679 (1989) [3] Watanabe, F.; Oki, Y.; Nakano, Y.; Kitaoka, S.: Occurence and subcellular location of aquacobalamin reductase in Euglena graciclis. Agric. Biol. Chem., 51, 273-274 (1987) [4] Watanabe, F.; Oki, Y.; Nakano, Y.; Kitaoka, S.: Purification and characterization of aquacobalamin reductase (NADPH) from Euglena gracilis. J. Biol. Chem., 262, 11514-11518 (1987) [5] Watanabe, F.; Nakano, Y.: Purification and characterization of aquacobalamin reductases from mammals. Methods Enzymol., 281, 295-305 (1997) [6] Watanabe, F.; Saido, H.; Yamaji, R.; Miyatake, K.; Isegawa, Y.; Ito, A.; Yubisui, T.; Rosenblatt, D.S.; Nakano, Y.: Mitochondrial NADH- or NADPHlinked aquacobalamin reductase activity is low in human skin fibroblasts with defects in synthesis of cobalamin coenzymes. J. Nutr., 126, 2947-2951 (1996) [7] Watanabe, F.; Nakano, Y.: Purification and characterization of aquacobalamin reductase from Euglena gracilis. Methods Enzymol., 281, 289-295 (1997) [8] Saido, H.; Watanabe, F.; Tamura, Y.; Funae, Y.; Imaoka, S.; Nakano, Y.: Mitochondrial NADPH-linked aquacobalamin reductase is distinct from the

456

1.16.1.5

Aquacobalamin reductase (NADPH)

NADPH-linked enzyme from microsomal membranes in rat liver. J. Nutr., 123, 1868-1874 (1993) [9] Watanabe, F.; Yamaji, R.; Isegawa, Y.; Yamamoto, T.; Tamura, Y.; Nakano, Y.: Characterization of aquacobalamin reductase (NADPH) from Euglena gracilis. Arch. Biochem. Biophys., 305, 421-427 (1993) [10] Watanabe, F.; Nakano, Y.; Saido, H.; Tamura, Y.; Yamanaka, H.: NADPH-cytochrome c (P-450) reductase has the activity of NADPH-linked aquacobalamin reductase in rat liver microsomes. Biochim. Biophys. Acta, 1119, 175177 (1992)

457


   *    

,

44

   
 1.16.1.6    cob(I)alamin, cyanide:NADP+ oxidoreductase    cyanocobalamin reductase (cyanide-eliminating)    EC 1.6.99.12 (formerly) NADPH2 :cyanocob(III)alamin oxidoreductase (cyanide-eliminating) cyanobalamin reductase (NADPH, cyanide-eliminating) cyanocobalamin reductase cyanocobalamin reductase (NADPH, CN-eliminating)
    131145-00-1

     Euglena gracilis [1]

!    "
 # 

cob(I)alamin + cyanide + NADP+ = cyanocob(III)alamin + NADPH + H+ (A flavoprotein)    decyanation oxidation redox reaction reduction       NADPH + cyanocob(III)alamin (Reversibility: ? [1]) [1] $ NADP+ + cob(I)alamin + cyanide [1]

458

1.16.1.6

Cyanocobalamin reductase (cyanide-eliminating)

     NADPH + cyanocob(III)alamin (Reversibility: ? [1]) [1] $ NADP+ + cob(I)alamin + cyanide [1]
"%  FAD [1] FMN [1] NADPH [1] " & *-% , 3 [1] )  *
, 45 [1] ) 

 *
, 10-60 [1]

 2 %$ %' %

3#

mitochondrion [1]

4  0  5-8 [1] )  30-40 [1]

"  [1] Watanabe, F.; Nakano, Y.: Comparative biochemistry of intracellular vitamin B12 transport and the synthesis of coenzyme form. Vitamins (Japan), 64, 273-284 (1990)

459

C  

   
 1.16.1.7    Fe(II):NAD+ oxidoreductase    ferric-chelate reductase    EC 1.6.99.13 (formerly) Fe(III)-ethylenediaminetetraacetic complex reductase Fe3+ -chelate reductase NADH-linked FeEDTA reductase NADH-linked ferric chelate (turbo) reductase NADH:Fe3+ oxidoreductase [Fe(III)-EDTA] reductase ferric chelate reductase iron chelate reductase reductase, iron chelate
    122097-10-3

     Lycopersicon esculentum (Mill. [1]) [1] Beta vulgaris [2] Plantago lanceolata [3]

!    "
 # 

2 Fe(II) + NAD+ = 2 Fe(III) + NADH + H+    oxidation redox reaction reduction

460

48

1.16.1.7

Ferric-chelate reductase

      NADH + Fe3+ (, iron deficiency results in a 2fold increase in specific activity [3]) (Reversibility: ? [3]) [3] $ NAD+ + Fe2+      NADH + Fe(III)-ethylenediaminetetraacetic complex (, turbo ferric chelate reductase activity of Fe-deficient plants at low pH appears to be different from the constitutive ferric chelate reductase [2]) (Reversibility: ? [2]) [2] $ NAD+ + ?  NADH + ferric dicitrate (Reversibility: ? [1]) [1] $ NAD+ + ?  NADPH + Fe(III)-ethylenediaminetetraacetic complex (, activity with NADPH is 10-20% of the activity with NADH [2]) (Reversibility: ? [2]) [2] $ NADP+ + ? 2  cycloheximide [3] &

  Brij 58 (, stimulates [3]) [3] Triton X-100 (, stimulates [3]) [3] . / *', Additional information (, turbo ferric chelate reductase activity of Fe-deficient plants at low pH appears to be different from the constitutive ferric chelate reductase [2]) [1, 2] 0  6.5 [1] Additional information (, turbo ferric chelate reductase activity of Fe-deficient plants at low pH appears to be different from the constitutive ferric chelate reductase [2]) [2] 0 

 5.5-8 (, pH 5.5: about 35% of maximal activity, pH 8.0: about 50% of maximal activity [1]) [1]

 2 %$ %' %

% root [1, 2, 3] 3#

plasma membrane (, in vitro both donor and acceptor sites are located on the cytosolic face of the membrane [3]) [1, 2, 3]

461

Ferric-chelate reductase

1.16.1.7

"  [1] Holden, M.J.; Luster, D.G.; Chaney, R.L.; Buckhout, T.J, Robinson, C.: Fe3+ chelate reductase activity of plasma membranes isolated from tomato (Lycopersicon esculentum Mill.) Roots. Plant Physiol., 97, 537-544 (1991) [2] Susin, S.; Abadia, A.; Gonzalez-Reyes, J.A.; Lucena, J.J.; Abadia, J.: The pH requirement for in vivo activity of the iron-deficiency-induced ªturboª ferric chelate reductase: A comparison of the iron-deficiency-induced iron reductase activities of intact plants and isolated plasma membrane fractions in sugar beet. Plant Physiol., 110, 111-123 (1996) [3] Schmidt, W.; Bartels, M.: Orientation of NADH-linked ferric chelate (turbo) reductase in plasma membranes from roots of Plantago lanceolata. Protoplasma, 203, 186-193 (1998)

462

;'    < 

47

   
 1.16.1.8    [methionine synthase]-methylcob(I)alamin,S-adenosylhomocysteine:NADP+ oxidoreductase    [methionine synthase] reductase    EC 2.1.1.135 (formerly) MSR methionine synthase cob(II)alamin reductase (methylating) methionine synthase reductase reductase, methionine synthase
    207004-87-3

     Homo sapiens (patients with homocystinuria [1]) [1] Escherichia coli [2]

!    "
 # 

2 [methionine synthase]-methylcob(I)alamin + 2 S-adenosylhomocysteine + NADP+ = 2 [methionine synthase]-cob(II)alamin + NADPH + H+ + 2 S-adenosyl-l-methionine (, under anaerobic growth conditions, oxidized ferredoxin (flavodoxin):NADP+ oxidoreductase accepts a hydride from NADPH and transfers the electron to flavodoxin, generating primarily flavodoxin semiquinone. Under anaerobic conditions the decarboxylation of pyruvate is coupled to reduction of flavodoxin, forming the flavodoxin hydroquinone. These reduced forms of flavodoxin bind to inactive cob(II)alamin enzyme, causing a conformational change that is coupled with dissociation of His759 and protonation of the His759-Asp757-Ser810 triad. Although NADPH oxida-

463

[Methionine synthase] reductase

1.16.1.8

tion ultimately produces 2 equivalent of flavodoxin semiquinone, only one electron is transferred to methionine synthase during reductive methylation [2])    methyl group transfer       [methionine synthase]-cob(II)alamin + NADPH + S-adenosyl-l-methionine (, the enzyme is involved in reductive activation of methionine synthase: [1,2]; , patients of the cblE complementation group of disorders of folate/cobalamin metabolism who are defective in reductive activation of methionine synthase exhibit megablastic anemia, developmental delay, hyperhomocysteinemia, and hypomethioninemia [1]) [1, 2]      [methionine synthase]-cob(II)alamin + NADPH + S-adenosyl-l-methionine [1] $ [methionine synthase]methylcob(I)alamin + S-adenosylhomocysteine + NADP+ [1]
"%  NADH (, under anaerobic growth conditions, oxidized ferredoxin (flavodoxin):NADP+ oxidoreductase accepts a hydride from NADPH and transfers the electron to flavodoxin, generating primarily flavodoxin semiquinone. Under anaerobic conditions the decarboxylation of pyruvate is coupled to reduction of flavodoxin, forming the flavodoxin hydroquinone. These reduced forms of flavodoxin bind to inactive cob(II)alamin enzyme, leading to a conformational change that is coupled with dissociation of His759 and protonation of the His759-Asp757-Ser810 triad. Although NADPH oxidation ultimately produces 2 equivalent of flavodoxin semiquinone, only one electron is transferred to methionine synthase during reductive methylation) [2] &

  reduced flavodoxin (, under anaerobic growth conditions, oxidized ferredoxin (flavodoxin):NADP+ oxidoreductase accepts a hydride from NADPH and transfers the electron to flavodoxin, generating primarily flavodoxin semiquinone. Under anaerobic conditions the decarboxylation of pyruvate is coupled to reduction of flavodoxin, forming the flavodoxin hydroquinone. These reduced forms of flavodoxin bind to inactive cob(II)alamin enzyme, leading to a conformational change that is coupled with dissociation of His759 and protonation of the His759-Asp757-Ser810 triad. Although NADPH oxidation ultimately produces 2 equivalent of flavodoxin semiquinone, only one electron is transferred to methionine synthase during reductive methylation) [2]

464

1.16.1.8

[Methionine synthase] reductase

  #     ? (, x * 77000, calculation from nucleotide sequence) [1]

 2 %$ %' %


 

[1] 

medicine (cloning of the cDNA will permit the diagnostic characterization of cblE patients and investigation of the potential role of polymorphisms of this enzyme as a risk factor in hyperhomocysteinemia-linked vascular disease) [1]

"  [1] Leclerc, D.; Wilson, A.; Dumas, R.; Gafuik, C.; Song, D.; Watkins, D.; Heng, H.H.Q.; Rommens, J.M.; Scherer, S.W.; Rosenblatt, D.S.; Gravel, R.A.: Cloning and mapping of a cDNA for methionine synthase reductase, a flavoprotein defective in patients with homocystinuria. Proc. Natl. Acad. Sci. USA, 95, 3059-3064 (1998) [2] Jarrett, J.T.; Hoover, D.M.; Ludwig, M.L.; Matthews, R.G.: The mechanism of adenosylmethionine-dependent activation of methionine synthase: a rapid kinetic analysis of intermediates in reductive methylation of cob(II)alamin enzyme. Biochemistry, 37, 12649-12658 (1998)

465

C

   
 1.16.3.1    Fe(II):oxygen oxidoreductase    ferroxidase    Cp115 [33] Cp135 [33] Cp200 [33] FET3 gene product [34] Fet3 [38] apoferritin [24] blue copper oxidase [15] caeruloplasmin ceruloplasmin ferro-O2 -oxidoreductase [1] ferro:O2 oxidoreductase ferroxidase I ferroxidase II [8, 11] ferroxidase, iron II:oxygen oxidoreductase fet3p [34, 35] human ceruloplasmin form I [14] iron(II): oxygen oxidoreductase monophenol-o-monoxygenase [25] mushroom tyrosinase [25] non-ceruloplasmin ferroxidase [8] serum ferroxidase [1] xanthine oxidoreductase [26]
    9031-37-2

     no activity in Rana catesbeiana (bullfrog [12]) [12] Agaricus bisporus (mushroom [25]) [25] 466

4!

1.16.3.1









Ferroxidase

Anser sp. (goose [30]) [30] Bos taurus (bovine [16, 17, 30]; cow [35]) [16, 17, 26, 30, 35] Caretta caretta (turtle [32]) [32] Cuniculus sp. [12, 16, 17] Equus sp. (horse [12, 16, 17]) [12, 16, 17, 24] Gallus sp. (chicken [3, 21, 29, 30, 32, 36]) [3, 21, 29, 30, 32, 36] Homo sapiens (human [1-23, 26-28, 30, 32-39]; Cohn's fraction F-IV-1 of normal human pooled plasma [9-12]; human hepatoblastoma cell line HepG2 [33]) [1-23, 26-28, 30, 32-39] Ovis aries (sheep [29, 32]) [29, 32] Rana sp. (frog [12]) [12] Rattus norvegicus (rat [11, 12, 16, 21, 33]; Sprague-Dawley [11]) [11, 12, 16, 21, 33] Saccharomyces cerevisiae (yeast [34, 35, 38]) [34, 35, 38] Sus scrofa (pig, porcine [3, 4, 9, 12, 17, 30, 31]; hog [12]) [3, 4, 9, 12, 16, 17, 21, 30, 31]

!    "
 # 

4 Fe(II) + 4 H+ + O2 = 4 Fe(III) + 2 H2 O (A multi-copper protein: ceruloplasmin from animals, rusticyanin in Thiobacillus ferroxidans)    oxidation redox reaction reduction       Fe2+ + H+ + O2 ( multicopper oxidase essential for normal iron homeostasis [38]; iron acquisition pathway [34]) (Reversibility: ? [1-39]) [1-39] $ Fe3+ + H2 O      2-chloro-p-phenylenediamine + Fe2+ + O2 (Reversibility: ? [12]) [12] $ ?  2-methoxy-p-phenylenediamine + Fe2+ + O2 (Reversibility: ? [12]) [12] $ ?  2-methyl-p-phenylenediamine + Fe2+ + O2 (Reversibility: ? [12]) [12] $ ?  2-nitro-p-phenylenediamine + Fe2+ + O2 (Reversibility: ? [12]) [12] $ ?

467

Ferroxidase

1.16.3.1

 2-sulfonic acid-p-phenylenediamine + Fe2+ + O2 (Reversibility: ? [12]) [12] $ ?  3,4-dihydroxyphenethylamine + Fe2+ + O2 (Reversibility: ? [12]) [12] $ ?  4-methylcatechol + Fe2+ + O2 (Reversibility: ? [12]) [12] $ ?  5-hydroxyindol-3-ylacetic acid + Fe2+ + O2 (Reversibility: ? [12]) [12] $ ?  5-hydroxytryptamine + Fe2+ + O2 (Reversibility: ? [12]) [12] $ ?  5-hydroxytryptophan + Fe2+ + O2 (Reversibility: ? [12]) [12] $ ?  5-hydroxytryptophol + Fe2+ + O2 (Reversibility: ? [12]) [12] $ ?  Fe2+ + H+ + O2 (Reversibility: ? [1-39]) [1-39] $ Fe3+ + H2 O  l-epinephrine + Fe2+ + O2 (Reversibility: ? [12, 34]) [12, 34] $ ?  l-norepinephrine + Fe2+ + O2 (Reversibility: ? [12]) [12] $ ?  N,N'-dimethyl-p-phenylenediamine + Cu2+ + O2 (Reversibility: ? [1, 5]) [1, 5] $ N,N'-dimethyl-p-phenylenediamine radical + Cu+  N,N'-dimethyl-p-phenylenediamine + Fe2+ + O2 (Reversibility: ? [1, 5, 7, 12, 14, 15, 21, 28]) [1, 5, 7, 12, 14, 15, 21, 28] $ ?  N,N'-dimethyl-p-phenylenediamine + Fe3+ + O2 (Reversibility: ? [1]) [1] $ ?  N,N,N,N'-tetramethyl-p-phenylenediamine + Fe2+ (Reversibility: ? [12, 28]) [12, 28] $ ?  N,N-diethyl-p-phenylenediamine + Fe2+ + O2 (Reversibility: ? [12]) [12] $ ?  N,N-dimethyl-m-phenylenediamine + Fe2+ + O2 (Reversibility: ? [12]) [12] $ ?  N,N-dimethyl-p-phenylenediamine + Fe2+ + O2 (Reversibility: ? [6, 12]) [6, 12] $ ?  N-(p-methoxyphenyl)-p-phenylenediamine + Fe2+ + O2 (Reversibility: ? [12]) [12] 468

1.16.3.1

Ferroxidase

$ ?  N-acetyl-p-phenylenediamine + Fe2+ + O2 (Reversibility: ? [12]) [12] $ ?  N-ethyl-N-(2-hydroxyethyl)-p-phenylenediamine Fe2+ + O2 (Reversibility: ? [12]) [12] $ ?  N-ethyl-N-2(S-methylsulfonamido)-ethyl-p-phenylenediamine + Fe2+ + O2 (Reversibility: ? [12]) [12] $ ?  N-phenyl-p-phenylenediamine + Fe2+ + O2 (Reversibility: ? [12]) [12] $ ?  alimemazine + Fe2+ + O2 (Reversibility: ? [12]) [12] $ ?  ascorbate + Fe2+ + O2 (Reversibility: ? [4, 12, 28]) [4, 12, 28] $ ?  catechol + Fe2+ + O2 (Reversibility: ? [12, 34]) [12, 34] $ ?  catechol + O2 ( mushroom tyrosinase is able to catalyse the oxidation of Fe2+ to Fe3+ [25]) (Reversibility: ? [25]) [25] $ ?  chlorpromazine + Fe2+ + O2 (Reversibility: ? [12]) [12] $ ?  diethazine + Fe2+ + O2 (Reversibility: ? [12]) [12] $ ?  dihydroxyphenylethylamine + Fe2+ + O2 (Reversibility: ? [5]) [5] $ ?  durenediamine + Fe2+ + O2 (Reversibility: ? [12]) [12] $ ?  ferrous ammonium sulfate + O2 (Reversibility: ? [36]) [36] $ ?  fluphenazine + Fe2+ + O2 (Reversibility: ? [12]) [12] $ ?  m-phenylenediamine + Fe2+ + O2 (Reversibility: ? [12]) [12] $ ?  monophenol + O2 (Reversibility: ? [25]) [25] $ catechol + H2 O  o-aminophenol + Fe2+ + O2 (Reversibility: ? [12, 28]) [12, 28] $ ?  o-dianisidine + Fe2+ + O2 (Reversibility: ? [12, 28, 30]) [12, 28, 30] $ ?  o-phenylenediamine + Fe2+ + O2 (Reversibility: ? [12, 34]) [12, 34] $ ? 469

Ferroxidase

 $  $ 

$ 

$  $  $  $  $  $  $  $  $  $  $ 

470

1.16.3.1

p-aminophenol + Fe2+ + O2 (Reversibility: ? [12, 28]) [12, 28] ? p-anisidine + Fe2+ + O2 (Reversibility: ? [12, 28]) [12, 28] ? p-phenylenediamine + Cu2+ + O2 ( without iron p-phenylenediamine is directly oxidized by the enzyme-bound copper [1]; oxidation through this iron-ferroxidase-coupled system is faster than direct oxidation by the enzyme [1]) (Reversibility: ? [1]) [1] p-phenylenediamine radical + Cu+ p-phenylenediamine + Fe2+ + O2 ( no p-phenylenediamine oxidase activity by ferroxidase II [8]) (Reversibility: ? [1, 3-5, 12, 21, 28-30, 32, 34, 36]) [1, 3-5, 8, 12, 21, 28-30, 32, 34, 36] ? periciazine + Fe2+ + O2 (Reversibility: ? [12]) [12] ? perphenazine + Fe2+ + O2 (Reversibility: ? [12]) [12] ? prochlorperazine + Fe2+ + O2 (Reversibility: ? [12]) [12] ? promazine + Fe2+ + O2 (Reversibility: ? [12]) [12] ? prometazine + Fe2+ + O2 (Reversibility: ? [12]) [12] ? pyrogallol + Fe2+ + O2 (Reversibility: ? [12]) [12] ? quinone + Fe2+ + O2 (Reversibility: ? [12]) [12] ? thioridazine + Fe2+ + O2 (Reversibility: ? [12]) [12] ? trifluoperazine + Fe2+ + O2 (Reversibility: ? [12]) [12] ? triflupromazine + Fe2+ + O2 (Reversibility: ? [12]) [12] ? Additional information ( ferroxidase oxidizes in the presence of trace amounts of iron certain substances such as ascorbate, catechol and hydroquinone, which are not true substrates but can react with Fe3+ in a cyclic reaction [1]; ascorbate and aromatic amines are not directly oxidized by the enzyme, but through an iron-ferroxidase-coupled system in which iron is an electron mediator between the substance and the enzyme [5]; multifunctional protein, copper transport, molecule directly involved in iron mobilization to the plasma by means of its ferroxidase activity, regulator of circulating biogenic amine levels through its oxidase activity [12]; possesses superoxide dismutase activity [30]; only ceruloplasmin capable of complete reoxidation by oxygen [29]; caeruloplasmin inhibits lipid peroxidation and deoxyribose degradation stimulated by iron and copper

1.16.3.1

Ferroxidase

salts [20]; ferroxidase II does not catalyze the oxidation of benzylamine [8]; ascorbate oxidase EC 1.10.3.3 activity [4]; Fet3p is able to catalyze effectively the incorporation of iron onto apotransferrin [34]) [1, 4, 5, 8, 12, 20, 29, 30, 34] $ ? 2  6-aminohexanoic acid [12] Al3+ ( strong inhibitor [12]) [7, 12] Ba2+ ( weak inhibitor [12]) [12] Ca2+ ( weak inhibitor [12]) [12] Ce3+ ( strong inhibitor [12]) [7, 12] Cr3+ ( strong inhibitor [12]) [12] EDTA [34] Ga3+ ( strong inhibitor [12]) [7, 12] In3+ ( strong inhibitor [12]) [7, 12] K+ ( weak inhibitor [12]) [12] La3+ ( strong inhibitor [12]) [7, 12] Li+ ( weak inhibitor [12]) [12] N-3 ( anion behaves as an inhibitor of the oxidase activity versus Fe2+ [36]) [36] Na+ ( weak inhibitor [12]) [12] Rh3+ ( strong inhibitor [12]) [7, 12] Sc3+ ( strong inhibitor [12]) [7, 12] Sn2+ ( weak inhibitor [12]) [12] VO2+ ( strong inhibitor [12]) [7, 12] Y3+ ( strong inhibitor [12]) [7, 12] Zn2+ [24] ZrO2 + ( strong inhibitor [12]) [7, 12] azide [34] bathocuproinedisulfonic acid [34] catalase ( catalase in the assay system results in little inhibition of the ferroxidase activity of xanthine oxidoreductase [26]) [26] sodium azide ( irreversible inhibitor [28]; no inhibition of ferroxidase II [8]; amine oxidase activity is sensitive to inhibition by sodium azide [21]) [8, 21, 28] superoxide dismutase ( superoxide dismutase in the assay system results in little inhibition of the ferroxidase activity of xanthine oxidoreductase [26]) [26] '(   Cd2+ ( activating [12]) [7, 12] Co2+ ( activating [12]) [6, 12] Cr3+ ( 0.1 M, activity 102% [7]) [7] Cu2+ ( copper-binding protein [27]) [3, 5, 9-12, 14, 16-18, 21, 23, 27, 28, 30, 32, 33, 35-39]

471

Ferroxidase

1.16.3.1

Fe2+ ( potent activator in the oxidation of many aromatic amines and ascorbate catalyzed by ferroxidase [5]; substrate and activator for ferroxidase [7]) [5-7, 12] Mg2+ ( activating [12]) [6, 12] Mn2+ ( activating [12]) [6, 12] Ni2+ ( activating [12]) [12] Zn2+ ( activating [12]) [12] " & *-% , 0.28 [21] 0.29 ( p-phenylenediamine as substrate [30]) [30] 0.88 ( o-dianisidine as substrate [30]) [30] 1.15 ( o-dianisidine as substrate [30]) [30] 1.25 [31] 1.73 ( p-phenylenediamine as substrate [30]) [30] 3.13 [22] 3.29 ( o-dianisidine as substrate [30]) [30] 5.72 ( p-phenylenediamine as substrate [30]) [30] 30 ( ferroxidase II [8]) [8] . / *', 0.0006 (Fe2+ , pH 6.5, 30 C, 2 Km -values: 0.0006 and 0.050 mM [1,12,26]) [1, 12, 26] 0.0011 (O2 ) [35] 0.00126 (2-nitro-p-phenylenediamine) [12] 0.0013 (O2 ) [35] 0.0013 (perphenazine) [12] 0.0013 (promazine) [12] 0.0014 (alimemazine) [12] 0.0014 (thioridazine) [12] 0.00154 (p-aminophenol) [12] 0.002 (Fe2+ ) [34, 35] 0.002 (periciazine) [12] 0.0023 (diethazine) [12] 0.0023 (promethazine) [12] 0.00255 (l-epinephrine) [12] 0.00262 (2-sulfonic acid-p-phenylenediamine) [12] 0.0028 (trifluoperazine) [12] 0.00281 (l-norepinephrine) [12] 0.00285 (3,4-dihydroxyphenethylamine) [12] 0.00288 (o-aminophenol) [12] 0.00295 (o-phenylenediamine) [12] 0.00305 (N,N-dimethyl-m-phenylenediamine) [12] 0.0035 (chlorpromazine) [12] 0.0039 (Fe2+ ) [35] 0.0041 (Fe2+ ) [35] 0.0048 (Fe2+ ) [35] 0.005 (fluphenazine) [12] 472

1.16.3.1

Ferroxidase

0.0051 (5-hydroxytryptophol) [12] 0.0052 (ascorbate) [12] 0.00614 (p-anisidine) [12] 0.0074 (O2, pH 7.0, 0.2 M phosphate buffer [1]) [1] 0.00834 (5-hydroxyindol-3-ylacetic acid) [12] 0.009 (O2 ) [26] 0.01 (triflupromazine) [12] 0.0123 (N-acetyl-p-phenylenediamine) [12] 0.0163 (5-hydroxytryptophan) [12] 0.0182 (O2, pH 7.0, 0.2 M acetate buffer [1]) [1] 0.019 (p-phenylenediamine) [32] 0.021 (N-(p-methoxyphenyl)p-phenylenediamine) [12] 0.0276 (O2, pH 5.2, 0.0133 M phosphate buffer [1]) [1] 0.0286 (O2, pH 5.4, 0.2 M acetate buffer [1]) [1] 0.036 (m-phenylenediamine) [12] 0.046 (Fe2+ , xanthine oxidoreductase with ferroxidase activity [26]) [26] 0.048 (N-phenyl-p-phenylenediamine) [12] 0.05 (Fe2+ , pH 6.5, 30 C, 2 Km -values: 0.0006 and 0.050 mM [1,12]) [1, 12] 0.05 (O2, xanthine oxidoreductase with ferroxidase activity [26]) [26] 0.053 (p-phenylenediamine) [30] 0.0554 (O2, pH 6.3, 0.0133 M phosphate buffer [1]) [1] 0.0579 (pyrogallol) [12] 0.06 (N,N'-dimethyl-p-phenylenediamine) [21] 0.0603 (4-methylcatechol) [12] 0.0632 (O2, pH 6.5, 0.2 M acetate buffer [1]) [1] 0.0657 (quinone) [12] 0.085 (p-phenylenediamine) [32] 0.087 (N-ethyl-N-2(S-methylsulfonamido)-ethyl-p-phenylenediamine) [12] 0.11 (N,N'-dimethyl-p-phenylenediamine) [15, 21] 0.11 (N-ethyl-N-(2-hydroxyethyl)p-phenylenediamine) [12] 0.12 (Fe2+ ) [32] 0.12 (o-dianisidine) [30] 0.125 (Fe2+ ) [25] 0.13 (Fe2+ ) [32] 0.15 (Fe2+ ) [32] 0.15 (o-dianisidine) [30] 0.161 (2-methoxy-p-phenylenediamine) [12] 0.164 (N,N'-dimethyl-p-phenylenediamine) [12] 0.171 (durenediamine) [12] 0.18 (o-dianisidine) [12] 0.197 (N,N,N,N'-tetramethyl-p-phenylenediamine) [12] 0.203 (N,N-dimethyl-p-phenylenediamine) [12] 0.213 (2-methyl-p-phenylenediamine) [12] 473

Ferroxidase

1.16.3.1

0.22 (p-phenylenediamine) [32] 0.241 (2-chloro-p-phenylenediamine) [12] 0.282 (catechol) [12] 0.292 (p-phenylenediamine) [12] 0.36 (p-phenylenediamine) [30] 0.43 (o-dianisidine) [30] 0.556 (N,N-diethyl-p-phenylenediamine) [12] 0.64 (p-phenylenediamine) [30] 0.76 (o-dianisidine) [30] 0.78-2.5 (p-phenylenediamine) [3] 0.9 (p-phenylenediamine) [34] 0.9 (prochlorperazine) [12] 0.908 (5-hydroxytryptamine) [12] 1.1 (p-phenylenediamine) [3] 1.12 (p-phenylenediamine) [21, 30] 1.19 (p-phenylenediamine) [30] 4.15 (o-phenylenediamine) [34] 5.8 (l-epinephrine) [34] 0  5 ( o-dianisidine as substrate [30]) [30, 34] 5-5.2 ( schizophrenics [2]) [2] 5.5-5.6 ( normal persons [2]) [2] 5.7 ( phosphate buffer [1]) [1] 5.8 ( p-phenylenediamine as substrate [30]) [30] 6.5 ( most active in 0.2 M acetate buffer [1]) [1, 26] 6.8 [29] 7.4 ( xanthine oxidoreductase with ferroxidase activity [26]) [26] 0 

 2-9 [34] 5-7.5 [1]

  #   ' 1  61800 ( protein characterization [35]) [35] 72400 ( predicted from amino acid residues [35]) [35] 72870 ( electrospray mass spectrometry [35]) [35] 85000 ( holoceruloplasmin, SDS-PAGE [38]; SDS-PAGE [35]) [35, 38] 100000 ( 2 differentially glycosylated forms, SDS-PAGE [34]) [34] 100000-150000 ( glycerol gradient [34]) [34] 100000-200000 ( gel filtration [13]) [13] 113000 ( amino acid composition [30]) [30] 114900 ( amino acid composition [30]) [30] 115000 ( human ceruloplasmin antibody reaction [33]) [33] 474

1.16.3.1

Ferroxidase

120000 ( 2 differentially glycosylated forms, SDS-PAGE [34]) [34] 120000 ( recombinant ceruloplasmin [37]) [16, 37] 121000 ( amino acid composition [30]) [30] 121300 ( gel filtration [30]) [30] 123000 ( sedimentation-velocity [16]) [16] 124000 ( nonproteolyzed single-chain, sedimentation equilibrium [12]; gel filtration [21]) [12, 16, 21] 125000 ( amino acid composition [16]) [16] 129000 ( determination of peptide chain length [14]) [14] 130000 ( gel filtration, sedimentation equilibrium centrifugation [22]) [14, 16, 22, 30, 37] 131000 ( sedimentation equilibrium [14]) [14] 132000 ( amino acid sequence [15]; crystallographic investigation [14]) [12, 14, 15, 27, 28, 30, 32, 33, 35] 133000 [14, 21] 134000 ( nonproteolyzed single-chain, sedimentation equilibrium [12]; form I, meniscus depletion sedimentation equilibrium [14]) [12, 14] 135000 ( undegraded single-chain protein, gel filtration, analytical SDS-PAGE [15]; human ceruloplasmin antibody reaction [33]; apoprotein, SDS-PAGE [38]) [15, 33, 38] 137000 ( gel filtration [14]) [14] 140000 ( nonreducing SDS-PAGE [29]) [14, 29] 145000 [32] 150000 [12, 14] 155000 ( sedimentation equilibrium [14]) [14] 158000 ( sedimentation velocity experiments [3]) [3] 160000 ( light scattering [9]) [8, 9, 12, 14] 200000 ( human ceruloplasmin antibody reaction [33]) [33] 800000-2000000 ( ferroxidase II, gel filtration [8]) [8]   dimer ( 1 * 17000 + 1 * 59000 [12]; 1 * 24000 + 1 * 93000, cleaved by protease [12]) [12] monomer ( 1 * 130000, SDS-PAGE [21]; 1 * 140000, SDS-PAGE [29]; 1 * 121300, SDS-PAGE [30]) [12, 16, 17, 21, 29, 30, 34] tetramer ( 1 * 19000 + 1 * 25000 + 1 * 26000 + 1 * 67000, SDSPAGE [28]; 2 * 16000 + 2 * 35000, SDS-PAGE [12]; 2 * 16000 + 2 * 59000, SDS-PAGE [13]; 2 * 15900 + 2 * 58900, SDS-PAGE [13]; 1 * 50000 + 1 * 90000 + 1 * 50000, SDS-PAGE [16]) [12, 13, 16, 28] trimer ( 1 * 18650 + 1 * 50000 + 1 * 70000, limited proteolysis [15]; 1 * 19000 + 1 * 50000 + 1 * 67000, limited proteolysis [28]) [15, 28] $   "

glycoprotein ( only bi- and triantennary N-glycosidic glucans [18]) [13, 18, 27, 28, 39]

475

Ferroxidase

1.16.3.1

 2 %$ %' %

% ascites fluid [13] blood ( retroplacental blood [14]; venous blood [35]8) [2, 9, 12-15, 17, 23, 27, 28, 30, 31, 35, 38] erythrocyte [20] hepatocyte [28, 33] leukocyte [38] liver [12, 27, 33] lymphocyte ( nonadherent cells, T and B lymphocytes [27]) [27] macrophage [27] milk [26] monocyte [27] plasma [1, 9, 12, 13, 15, 16, 18, 22, 23, 28, 30, 32, 34, 35, 3739] serum [2-4, 9, 11, 12, 14-16, 21, 28, 31, 33, 38] urine (nephrotic urine) [13] 3#

membrane [34, 35] $"

[30] [16, 17] [32] [3, 21, 29, 32] (partially [5]; non-ceruloplasmin ferroxidase II [8,11]; recombinant ceruloplasmin, expressed in Pichia pastoris GS115 his4 [37]) [5, 8, 9, 11-13, 15, 19, 22, 28, 33, 36-38] [32] [12] (recombinant soluble Fet3p [35]) [34, 35] [12, 31]
 #

[24] [1, 5-7, 9, 12, 14, 18, 33, 36, 37, 39] [4]
 

(cDNA clones encoding human CP identified, CP gene mapped to human chromosome 3q21-25 by human-mouse somatic-cell-hybrid analysis [27]; fully active recombinant human ceruloplasmin produced in the yeast Pichia pastoris [37]; gene sequencing and site-directed mutagenesis [38]) [27, 37, 38] (FET3 gene cloned, strain M2 carrying plasmid pDY148 used as expression system, recombinant soluble Fet3p produced in yeast [35]) [35]

476

1.16.3.1

Ferroxidase



analysis ( enzyme can be used in analytical biochemistry, especially for the construction of enzyme sensors, enzymes immobilized in enzyme-containing membranes coating oxygen sensitive electrodes and serve for a specific amperometric determination of their substrates in biological materials [31]) [31] medicine ( clinical importance because of its role in iron and copper metabolism and transport [17]; clinical interest relates to its critical role in the diagnosis of Wilson's disease in serum from patients, marked reduction in serum of patients [2,12,13,20,33]; homogenous ceruloplasmin with ferroxidase activity for the treatment of aplastic anemics, aplastic anemics have low levels of this enzyme, clinical trials have shown effective in 56% of cases [22]; unusual antioxidant property of caeruloplasmin have important implications in vivo for conditions such as rheumatoid joint disease and Wilson's disease, where changes in copper homeostasis, caeruloplasmin and oxygen radicals are known to occur [20]; different forms of normal and pathological ceruloplasmins, increasing ceruloplasmin activity in the blood of schizophrenics [2]; mutations in human ceruloplasmin which result in a loss of activity, cause aceruloplasminemia, a neurodegenerative disease [39]) [2, 12, 17, 20, 22, 33, 39]

4  0  4.8-6.4 ( above pH 5.8 activity progressively decreases [2]) [2] )  65 ( above irreversible denaturation process of the protein active site [23]) [23] 5    "

, unstable [16] , unusually resistant to aging and proteolysis [32]    , -196 C, kept in liquid nitrogen exhibits only minor modifications [16] , -20 C, storage induced heterogeneity and decrease of the oxidase activity [16] , -20 C, 3 months [32] , 4 C, 0.5 M sodium phosphate buffer, pH 7.0, retains catalytic activity for at least 3 weeks [21] , -20 C, 0.5 M phosphate buffer, pH 6.9, concentration of approximately 2.5% without noticeable loss of blue colour during 1 month [15] , -20 C, A280:A610 ratio increases for several weeks on storage [6] , -20 C, frozen in liquid nitrogen 0.2% enzyme solution in 0.015 M phosphate buffer, pH 6.9, containing 0.1 M NaCl loses its blue colour completely after 2 weeks storage [15]

477

Ferroxidase

1.16.3.1

, -90 C, when frozen in dry ice and thawed, about 5% of the absorbance at 610 nm is lost, no change in the absorbance at 280 nm [14] , 4 C, 0.1 M Tris buffer, pH 8.0, decomposes into fragments when stored for 36-48 h [12] , 4 C, 0.2% enzyme solution in 0.015 M phosphate buffer, pH 6.9, containing 0.1 M NaCl, its A610/A280 ratio reduced by approximately 10% in 1 month [15] , 4 C, ferroxidase II, purified enzyme is stable for at least 2 weeks [8] , 4 C, purified enzyme sensitive to storage, no oxidase activity after 4 months [22]

"  [1] Osaki, S.: Kinetic studies of ferrous ion oxidation with crystalline human ferroxidase (ceruloplasmin). J. Biol. Chem., 241, 5053-5059 (1966) [2] Puzynski, S.; Kalinowski, A.: Investigations of some physio-biochemical properties of ceruloplasmin in schizophrenics and in normal subjects. Nature, 212, 399-400 (1966) [3] Starcher, B.; Hill, C.H.: Isolation and characterization of induced ceruloplasmin from chicken serum. Biochim. Biophys. Acta, 127, 400-406 (1966) [4] Mukasa, H.; Kaya, T.; Sato, T.: Comparative study on enzymatic activity of two forms of porcine caeruloplasmin. J. Biochem., 61, 485-490 (1967) [5] Osaki, S.; Walaas, O.: Kinetic studies of ferrous ion oxidation with crystalline human ferroxidase. II. Rate constants at various steps and formation of a possible enzyme-substrate complex. J. Biol. Chem., 242, 2653-2657 (1967) [6] Huber, C.T.; Frieden, E.: Substrate activation and the kinetics of ferroxidase. J. Biol. Chem., 245, 3973-3978 (1970) [7] Huber, C.T.; Frieden, E.: The inhibition of ferroxidase by trivalent and other metal ions. J. Biol. Chem., 245, 3979-3984 (1970) [8] Topham, R.W.; Frieden, E.: Identification and purification of a non-ceruloplasmin ferroxidase of human serum. J. Biol. Chem., 245, 6698-6705 (1970) [9] Nakagawa, O.: Purification and properties of crystalline human ceruloplasmin. Int. J. Pept. Protein Res., 4, 385-394 (1972) [10] Wever, R.; van Leeuwen, F.X.R.; van Gelder, B.F.: The reaction of nitric oxide with ceruloplasmin. Biochim. Biophys. Acta, 302, 236-239 (1973) [11] Topham, R.W.; Sung, C.S.; Morgan, F.G.; Prince, W.D.; Jones, S.H.: Functional significance of the copper and lipid components of human ferroxidase-II. Arch. Biochem. Biophys., 167, 129-137 (1975) [12] Frieden, E.; Hsieh, H.S.: Ceruloplasmin: the copper transport protein with essential oxidase activity. Adv. Enzymol. Relat. Areas Mol. Biol., 44, 187-236 (1976) [13] McCombs, M.L.; Bowman, B.H.: Biochemical studies on human ceruloplasmin. Biochim. Biophys. Acta, 434, 452-461 (1976) [14] Ryden, L.; Bjoerk, I.: Reinvestigation of some physicochemical and chemical properties of human ceruloplasmin (ferroxidase). Biochemistry, 15, 3411-3417 (1976) 478

1.16.3.1

Ferroxidase

[15] Noyer, M.; Dwulet, F.E.; Hao, Y.L.; Putnam, F.W.: Purification and characterization of undegraded human ceruloplasmin. Anal. Biochem., 102, 450-458 (1980) [16] Calabrese, L.; Malatesta, F.; Barra, D.: Purification and properties of bovine caeruloplasmin. Biochem. J., 199, 667-673 (1981) [17] Dooley, D.M.; Cote, C.E.; Coolbaugh, T.S.; Jenkins, P.L.: Characterization of bovine ceruloplasmin. FEBS Lett., 131, 363-365 (1981) [18] Endo, M.; Suzuki, K.; Schmid, K.; Fournet, B.; Karamanos, Y.; Montreuil, J.; Dorland, L.; van Halbeek, H.; Vliegenthart, J.F.G.: The structures and microheterogeneity of the carbohydrate chains of human plasma ceruloplasmin. A study employing 500-MHz 1H-NMR spectroscopy. J. Biol. Chem., 257, 8755-8760 (1982) [19] Tetaert, D.; Takahashi, N.; Putnam, F.W.: Purification of glycopeptides of human ceruloplasmin and immunoglobulin D by high-pressure liquid chromatography. Anal. Biochem., 123, 430-437 (1982) [20] Gutteridge, J.M.C.: Antioxidant properties of caeruloplasmin towards ironand copper-dependent oxygen radical formation. FEBS Lett., 157, 37-40 (1983) [21] Disilvestro, R.A.; Harris, E.D.: Purification and partial characterization of ceruloplasmin from chicken serum. Arch. Biochem. Biophys., 241, 438-446 (1985) [22] Oosthuizen, M.M.J.; Nei, L.; Myburgh, J.A.; Crookes, R.L.: Purification of undegraded ceruloplasmin from outdated human plasma. Anal. Biochem., 146, 1-6 (1985) [23] Sportelli, L.; Desideri, A.; Campaniello, A.: Isotopic effect on the kinetic of thermal denaturation of ceruloplasmin. Z. Naturforsch. C, 40, 551-554 (1985) [24] Bakker, G.R.; Boyer, R.F.: Iron incorporation into apoferritin. The role of apoferritin as a ferroxidase. J. Biol. Chem., 261, 13182-13185 (1986) [25] Boyer, R.F.; Mascotti, D.P.; Schori, B.E.: Ferroxidase activity of mushroom tyrosinase. Phytochemistry, 25, 1281-1283 (1986) [26] Topham, R.W.; Jackson, M.R.; Joslin, S.A.; Walker, M. C.: Studies of the ferroxidase activity of native and chemically modified xanthine oxidoreductase. Biochem. J., 235, 39-44 (1986) [27] Yang, F.; Naylor, S.L.; Lum, J.B.; Cutshaw, S.; McCombs, J.L.; Naberhaus, K.H.; McGill, J.R.; Adrian, G.S.; Moore, C.M.; Barnett, D.R.; Bowman, B.H.: Characterization, mapping, and expression of the human ceruloplasmin gene. Proc. Natl. Acad. Sci. USA, 83, 3257-3261 (1986) [28] Arnaud, P.; Gianazza, E.; Miribel, L.: Ceruloplasmin. Methods Enzymol., 163, 441-452 (1988) [29] Calabrese, L.; Carbonaro, M.; Musci, G.: Chicken ceruloplasmin. Evidence in support of a trinuclear cluster involving type 2 and 3 copper centers. J. Biol. Chem., 263, 6480-6483 (1988) [30] Hilewicz-Grabska, M.; Zgirski, A.; Krajewski, T.; Plonka, A.: Purification and partial characterization of goose ceruloplasmin. Arch. Biochem. Biophys., 260, 18-27 (1988)

479

Ferroxidase

1.16.3.1

[31] Kovar, J.: High performance liquid chromatography and biotechnological application of several oxidoreductases. Acta Biotechnol., 8, 103-110 (1988) [32] Musci, G.; Carbonaro, M.; Adriani, A.; Lania, A.; Galtieri, A.; Calabrese, L.: Unusual stability properties of a reptilian ceruloplasmin. Arch. Biochem. Biophys., 279, 8-13 (1990) [33] Sato, M.; Schilsky, M.L.; Stockert, R.J.; Morell, A. G.; Sternlieb, I.: Detection of multiple forms of human ceruloplasmin. A novel Mr 200,000 form. J. Biol. Chem., 265, 2533-2537 (1990) [34] De Silva, D.; Davis-Kaplan, S.; Fergestad, J.; Kaplan, J.: Purification and characterization of Fet3 protein, a yeast homolog of ceruloplasmin. J. Biol. Chem., 272, 14208-14213 (1997) [35] Hassett, R.F.; Yuan, D.S.; Kosman, D.J.: Spectral and kinetic properties of the Fet3 protein from Saccharomyces cerevisiae, a multinuclear copper ferroxidase enzyme. J. Biol. Chem., 273, 23274-23282 (1998) [36] Musci, G.; Bellenchi, G.C.; Calabrese, L.: The multifunctional oxidase activity of ceruloplasmin as revealed by anion binding studies. Eur. J. Biochem., 265, 589-597 (1999) [37] Bielli, P.; Bellenchi, G.C.; Calabrese, L.: Site-directed mutagenesis of human ceruloplasmin: production of a proteolytically stable protein and structureactivity relationships of type 1 sites. J. Biol. Chem., 276, 2678-2685 (2001) [38] Hellman, N.E.; Kono, S.; Mancini, G.M.; Hoogeboom, A.J.; de Jong, G.J.; Gitlin, J.D.: Mechanisms of copper incorporation into human ceruloplasmin. J. Biol. Chem., 277, 46632-46638 (2002) [39] Vachette, P.; Dainese, E.; Vasyliev, V.B.; Di Muro, P.; Beltramini, M.; Svergun, D.I.; De Filippis, V.; Salvato, B.: A key structural role for active site type 3 copper ions in human ceruloplasmin. J. Biol. Chem., 277, 4082340831 (2002)

480


9$  9  4   

8

   
 1.17.1.1    CDP-4-dehydro-3,6-dideoxy-d-glucose:NAD(P)+ 3-oxidoreductase    CDP-4-dehydro-6-deoxyglucose reductase    CDP-4-keto-6-deoxy-d-glucose-3-dehydrogenase system CDP-4-keto-deoxy-glucose reductase NAD(P)H:CDP-4-keto-6-deoxy-d-glucose oxidoreductase cytidine diphosphate 4-keto-6-deoxy-d-glucose-3-dehydrogenase reductase, cytidine diphospho-4-keto-6-deoxy-d-glucose Additional information (2 proteins E1 and E3 are involved but no partial reaction has been observed in the presence of either alone [1, 2]) [1, 2]
    37256-87-4

     Pasteurella pseudotuberculosis (25 VO [2,3]) [1-3] Salmonella typhimurium (LT-2 [2]) [2]

!    "
 # 

CDP-4-dehydro-3,6-dideoxy-d-glucose + NAD(P)+ + H2 O = CDP-4-dehydro6-deoxy-d-glucose + NAD(P)H + H+ ( mechanism [1-3]; role of the enzymes E1 and E3, as well as role of the cofactor. Multicomponent system consisting of substrate, NADH or NADPH, enzyme E1: recognition unit of the system by binding to the substrate through the amino group of the coenzyme, enzyme E3: acts as the dehydrogenase of the system, and cofactor: pyridoxamine 5'-phosphate [1, 3]; E1 binds the substrate pyridoxamine 5'-phosphate essential for binding, E3 possesses NADH oxidase activity, may be the reductase [3]; 2 proteins, E1 and E3, are involved but no partial reaction is observed in the presence of either alone [1, 2]) 481

CDP-4-Dehydro-6-deoxyglucose reductase

1.17.1.1

   oxidation redox reaction reduction       CDP-4-dehydro-6-deoxy-d-glucose + NAD(P)H ( biosynthesis of 3,6-dideoxysugars [1]) (Reversibility: ? [1]) [1] $ CDP-4-dehydro-3,6-dideoxy-d-glucose + NAD(P)+ + H2 O [1]      CDP-4-dehydro-6-deoxy-d-glucose + NAD(P)H ( the substrate undergoes substitution of a hydroxyl group by a hydrogen atom at carbon atom 3 from glucose, it is supposed that the reaction is triggered by an attack from a hydride ion from NAD(P)H to atom carbon 3 [1]) (Reversibility: ? [1, 2]) [1, 2] $ CDP-4-dehydro-3,6-dideoxy-d-glucose + NAD(P)+ + H2 O [1, 2] 2  5,5'-dithiobis(2-nitrobenzoate) ( inhibits the activity of the whole system [1]; 89% inhibition at 10 mM [2]) [1-3] N-ethylmaleimide ( inhibits the activity of the whole system [1]; 88% inhibition at 10 mM [2]; enzyme E1: dithiothreitol in excess protects against inhibition [3]) [1-3] iodoacetamide ( 85% inhibition at 100 mM [2]) [2] p-chloromercuribenzoate ( 91% inhibition at 1 mM [2]) [2] p-chlorophenylsulfonate ( 91% inhibition at 1 mM [2]) [2]
"%  NADH ( the second substrate of the reaction, the hydrogen donor [1]) [1] NADPH ( the second substrate of the reaction, the hydrogen donor [1]) [1, 2] pyridoxamine 5'-phosphate ( bound to enzyme E1 through an ionic interaction with a positive charge on the surface of the enzyme, the cofactor is needed for the binding of the substrate to the enzyme [1,3]) [1, 3] Additional information ( 3.8 SH groups per molecule of enzyme E1, in the presence and absence of SDS [1]; 0.37 SH groups per molecule of enzyme E3, in the absence of SDS and 1 SH group per molecule of enzyme E3 in the presence of SDS, the single SH group in enzyme E3 is essential for activity [1,3]; derivatives of vitamin B6 such as pyridoxine, pyridoxine 5'-phosphate, pyridoxal, pyridoxal 5'-phosphate, pyridoxamine and pyridoxamine 5'-phosphate have stimulatory effect on the reaction, indicating that the amino group and the phosphate group are necessary for the reaction [1]; 4.3 SH per mol of enzyme E1 in the presence of SDS, calculated using a molecular weight of 61000 [3]) [1, 3] '(   Additional information ( no divalent cation required [2]) [2]

482

1.17.1.1

CDP-4-Dehydro-6-deoxyglucose reductase

" & *-% , 0.11 ( E1 [1]) [1] 0.32 ( E3 [1]) [1] Additional information [2, 3] . / *', 0.15 (CDP-4-keto-6-deoxy-d-glucose) [2] 0  7.3 [2] 7.5 ( assay at [3]) [3] 0 

 5.5-8.5 ( pH 5.5: about 60% of activity maximum, pH 8.5: about 40% of activity maximum [2]) [2] )  *
, 25 ( assay at [3]) [3]

  #   ' 1  35000-45000 ( enzyme E3, thin-layer chromatography on Sephadex G-100 [1, 3]) [1, 3] 50000-70000 ( enzyme E1, thin-layer chromatography on Sephadex G-100 [3]) [3] 60000-70000 ( enzyme E1, thin-layer chromatography on Sephadex G-100 [1]) [1] Additional information ( cofactor: 200-400, gel filtration on Bio Gel P2 [1]) [1]   monomer ( enzyme E3: 1 * 40000, SDS-PAGE, enzyme E1: 1 * 61000, SDS-PAGE, 2 proteins E1 and E3 are involved but no partial reaction has been observed in the presence of either alone [1, 3]) [1, 3]

 2 %$ %' %

$"

(the first three steps are common to the purification of enzyme E1, enzyme E3 and cofactor, their separation can be accomplished after step 4, step 1: preparation of the crude extract, step 2: streptomycin sulfate precipitation, step 3: ammonium sulfate precipitation and dialysis, step 4: DEAE-cellulose chromatography, step 5: purification of enzyme E1, gel filtration on Sephadex G-100, step 6: second DEAE-cellulose chromatography, step 7: preparative polyacrylamide gel electrophoresis, step 5': purification of enzyme E3, gel filtration on Sephadex G-100, step 6': second DEAE-cellulose chromatogra-

483

CDP-4-Dehydro-6-deoxyglucose reductase

1.17.1.1

phy, step 7': third DEAE-cellulose chromatography [1, 3]; of cofactor, using streptomycin sulfate precipitation, ammonium sulfate precipitation and dialysis, DEAE-cellulose chromatography, ultrafiltration, Bio Gel P-2 filtration and ion exchange chromatography on Dowex-1 [1]; enzyme E1: using filtration on a Sephadex G-100 column and column chromatography on DEAE-cellulose, enzyme E3: using two filtrations on Sephadex G-100 columns [2]) [1-3] [2] 

 

Additional information ( a mutant which cannot synthesize CDPabequose is found to be missing E1 [2]) [2]

4     , frozen, for months without loss of activity, enzyme E1 [1] , lyophilized powder: enzyme E3 not stable [1, 3] , lyophilized powder: for months, enzyme E1 [1, 3]

"  [1] Gonzalez-Porque, P.: Cytidine diphosphate 4-keto-6-deoxy-d-glucose-3-dehydrogenase. Coenzymes and cofactors, Glutathione, Chem. Biochem. Med. Aspects Pt. A (Dolphin D, Poulson R, Avromonic O, eds.) John Wiley & Sons, New York, 1, 391-419 (1986) [2] Pape, H.; Strominger, J.L.: Enzymatic synthesis of cytidine diphosphate 3,6dideoxyhexoses. V. Partial purification of the two protein components required for introduction of the 3-deoxy group. J. Biol. Chem., 244, 3598-3604 (1969) [3] Gonzalez-Porque, P.; Strominger, J.L.: Enzymatic synthesis of cytidine diphosphate 3,6-dideoxyhexoses. VI. Purification to homogeneity and some properties of cytidine diphosphate-d-glucose oxidoreductase, enzyme E1 and enzyme E3. J. Biol. Chem., 247, 6748-6756 (1972)

484

 0  !       

8

   
 1.17.1.2    isopentenyl-diphosphate:NAD(P)+ oxidoreductase    4-hydroxy-3-methylbut-2-enyl diphosphate reductase
    512789-14-9

!    "
 # 

isopentenyl diphosphate + NAD(P)+ + H2 O = (E)-4-hydroxy-3-methylbut-2en-1-yl diphosphate + NAD(P)H + H+ (Forms part of an alternative, nonmevalonate pathway for terpenoid biosynthesis. The enzyme acts in the reverse direction producing a 5:1 mixture of isopentenyl diphosphate and dimethyallyl diphosphate)    oxidation redox reaction reduction

"  [1] Rohdich, F.; Hecht, S.; Gärtner, K.; Adam, P.; Krieger, C.; Amslinger, S.; Arigoni, D.; Bacher, A.; Eisenreich, W.: Studies on the nonmevalonate terpene biosynthetic pathway: Metabolic role of IspH (LytB) protein. Proc. Natl. Acad. Sci. USA, 99, 1158-1163 (2002) [2] Hintz, M.; Reichenberg, A.; Altincicek, B.; Bahr, U.; Gschwind, R.M.; Kollas, A.K.; Beck, E.; Wiesner, J.; Eberl, M.; Jomaa, H.: Identification of (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate as a major activator for human gd T cells in Escherichia coli. FEBS Lett., 509, 317-322 (2001) [3] Charon, L.; Pale-Grosdemange, C.; Rohmer, M.: On the reduction steps in the mevalonate independent 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis in the bacterium Zymomonas mobilis. Tetrahedron Lett., 40, 7231-7234 (1999)

485

3    

8!

   
 1.17.1.3    (2R,3S)-catechin:NADP+ 4-oxidoreductase    leucoanthocyanidin reductase    leucocyanidin reductase
    93389-48-1

!    "
 # 

(2R,3S)-catechin + NADP+ + H2 O = 2,3-trans-3,4-cis-leucocyanidin + NADPH + H+ (Catalyses the synthesis of catechin-4b-ol and the related flavan-3-ols afzelechin and gallocatechin, which are initiating monomers in the synthesis of plant polymeric proanthocyanidins or condensed tannins. While 2,3-trans-3,4-cis-leucocyanidin is the preferred flavan-3,4-diol substrate, 2,3trans-3,4-cis-leucodelphinidin and 2,3-trans-3, 4-cis-leucopelargonidin can also act as substrates, but more slowly. NADH can replace NADPH but is oxidized more slowly)

"  [1] Tanner, G.J.; Kristiansen, K.N.: Synthesis of 3,4-cis-[3 H]leucocyanidin and enzymatic reduction to catechin. Anal. Biochem., 209, 274-277 (1993) [2] Tanner, G.J.; Francki, K.T.; Abrahams, S.; Watson, J.M.; Larkin, P.J.; Ashton, A.R.: Proanthocyanidin biosynthesis in plants: Purification of legume leucoanthocyanidin reductase and molecular cloning of its cDNA. J. Biol. Chem., 278, 31647-31656 (2003)

486

$  

8!

   
 1.17.3.1    2-amino-4-hydroxypteridine:oxygen oxidoreductase (7-hydroxylating)    pteridine oxidase    oxidase, pteridine Additional information (different from EC 1.2.3.2)
    74082-65-8

     Ricinus communis (castor bean [1]) [1]

!    "
 # 

2-amino-4-hydroxypteridine + O2 = 2-amino-4,7-dihydroxypteridine + (?)    oxidation redox reaction reduction       2-amino-4-hydroxypteridine + O2 ( catabolism of purines via pteridines in plants [1]) (Reversibility: ? [1]) [1] $ 2-amino-4,7-dihydroxypteridine + ? [1]      2-amino-4-hydroxypteridine + O2 ( strictly dependent on oxygen [1]) (Reversibility: ? [1]) [1] $ 2-amino-4,7-dihydroxypteridine + ? [1]

487

Pteridine oxidase

1.17.3.1

 Additional information ( other electron acceptors such as ferricyanide, cytochrome c, 2,6-dichlorophenol indophenol and methylene blue are ineffective, enzyme does not act on hypoxanthine [1]) [1] $ ? 2  hypoxanthine [1] xanthine [1] . / *', 0.016 (hypoxanthine) [1] 0.016 (xanthine) [1] 0  6.8 [1] 7 ( assay at [1]) [1] 0 

 5.2-8.1 ( half-maximal activity at pH 5.2 and 8.1 [1]) [1] )  *
, 37 ( assay at [1]) [1]

  #   ' 1  310000 ( gel filtration [1]) [1]

 2 %$ %' %

% seed ( germinating, endosperm tissue [1]) [1] 3#

cytoplasm [1] $"

(partial, using ammonium sulfate precipitation followed by gradient elution from a Celite column [1]) [1]

"  [1] Hong, Y.N.: Detection of a pteridine oxidase in plants. Plant Sci. Lett., 18, 169-175 (1980)

488

   

8

   
 1.17.4.1    2'-deoxyribonucleoside-diphosphate:thioredoxin-disulfide 2'-oxidoreductase    ribonucleoside-diphosphate reductase    2'-deoxyribonucleoside-diphosphate:oxidized-thioredoxin 2'-oxidoreductase ADP reductase CDP reductase UDP reductase nucleoside diphosphate reductase reductase, ribonucleoside diphosphate ribonucleoside 5'-diphosphate reductase ribonucleoside diphosphate reductase ribonucleotide diphosphate reductase ribonucleotide reductase
    9047-64-7

     Mus musculus (Ehrlich ascites tumor cells [9, 12, 14, 24, 27, 28, 55, 72]; wild-type and reductase inhibitor resistant L1210 cell line [20]; mutant line of T-lymphoma S49 cells [57]) [1, 3, 9, 12, 14, 20, 24, 27, 28, 30, 38, 41, 45, 46, 54, 55, 57, 66, 72, 77, 79, 81, 83, 84] Rattus norvegicus (Novikoff hepatoma cells [12, 62]; Morris hepatoma cells [62]; non-proliferating cells inactivate 88000-90000 Da M1 subunit by degradation into 40000 Da fragments [19]) [2, 12, 19, 31, 46, 56, 62, 63] Escherichia coli (overproducing strain [7]) [3, 7, 12, 15, 18, 21, 22, 30-36, 39, 40, 47, 49, 51-53, 59, 70, 76, 77] Bacteriophage T4 (enzyme induced in Escherichia coli by infection with bacteriophage T4 [5, 6, 31]) [3, 5, 6, 12, 31, 48, 50, 74] Homo sapiens (Molt 4F cells [12]; HeLa cells [8, 59]) [8, 12, 31, 69] Bos taurus (calf [59]) [3, 10, 12, 29, 31, 59]

489

Ribonucleoside-diphosphate reductase

1.17.4.1

Mesocricetus auratus (baby hamster kidney cells, enzyme induced by Herpes simplex, virus type 1, HSV-1 [11]; Chinese hamster overy cells [13]) [11-13] Scenedesmus obliquus (green algae [12]) [12, 23, 26, 61] Saccharomyces cerevisiae [12, 64, 78] Oryctolagus cuniculus [12, 31, 43, 60, 65, 71] monkey (monkey kidney cells BSC-40 induced by vaccina virus strain WR, activity may be virally encoded [16]) [16] Herpes simplex (type 1, HSV-1 [17, 42, 44]; type 2, HSV-2 [4, 25]) [4, 17, 25, 30, 42, 44, 58] Bacteriophage T2 [31] Bacteriophage T5 (may use triphosphates as substrates [31]) [12, 31] Bacteriophage T6 [12, 31] Chlorella pyrenoidosa [61] Mus musculus (BALB/3T3 cells, ATCC CCL 163 [37]) [37] Corynebacterium nephridii [67] Vaccinia virus [68] Salmonella typhimurium (class I ribonucleotide reductase [70]) [70] Streptomyces aureofaciens [73] Corynebacterium ammoniagenes (class Ib reductase [75]) [75] Chlamydia trachomatis [80] Bacteroides fragilis (nrdA and nrdB genes encoding a CDP reductase are induced by oxidative stress [82]) [82]

!    "
 # 

2'-deoxyribonucleoside diphosphate + thioredoxin disulfide + H2 O = ribonucleoside diphosphate + thioredoxin ( proposed mechanism [12, 30, 32, 47]; postulated mechanism, radical cation intermediates [30]; NDP reduction requires cleavage of the 3'-C-H bond of the substrate, hypothesis of enzyme mechanism [49]; allosteric regulation of the catalytic activity of subunit R1 [81]; kinetics and mechanism of formation of the tyrosyl radical and micro-oxo-diiron cluster in the R2 subunit [83])    oxidation redox reaction reduction       ribonucleoside diphosphate + reduced thioredoxin ( enzyme catalyzes the first unique step in DNA synthesis [4, 12, 24]; possible role in HSV-2-induced transformation [4]; thioredoxin is the physiological reductant [26, 62]; critical and ratecontrolling step in pathway leading to DNA synthesis and cell replication [62]) (Reversibility: ? [4, 12, 24]) [4, 12, 24, 26, 62] 490

1.17.4.1

Ribonucleoside-diphosphate reductase

$ 2'-deoxyribonucleoside diphosphate + oxidized thioredoxin [4, 12, 24, 26, 62]      2'-methyladenosine 5'-diphosphate + reduced dithiothreitol (Reversibility: ? [67]) [67] $ adenine + 2'-deoxy-2'-methyladenosine + H2 O ( 5% reduction to 2'-deoxy-2'-methyladenosine, adenine is the major product besides other unidentified products [67]) [67]  2'-methyluridine 5'-diphosphate + reduced dithiothreitol (Reversibility: ? [67]) [67] $ uracil + H2 O ( uracil is the major product, unidentified minor product may be 2'-deoxy-2'-methyluridine [67]) [67]  2,6-diaminopurineriboside diphosphate + reduced thioredoxin (Reversibility: ? [12]) [12] $ 2'-deoxy-2,6-diaminopurineriboside diphosphate + H2 O [12]  2-aminopurineriboside diphosphate + reduced thioredoxin (Reversibility: ? [12]) [12] $ 2'-deoxy-2-aminopurineriboside diphosphate + H2 O [12]  ADP + reduced thioredoxin (Reversibility: ir [1]) [1] $ 2'-deoxy-ADP + oxidized thioredoxin [1]  CDP + reduced thioredoxin (Reversibility: ir [1]) [1] $ 2'-deoxy-CDP + oxidized thioredoxin [1]  GDP + reduced thioredoxin (Reversibility: ir [1]) [1] $ 2'-deoxy-GDP + oxidized thioredoxin [1]  UDP + reduced thioredoxin (Reversibility: ir [1]) [1] $ 2'-deoxy-UDP + oxidized thioredoxin [1]  benzimidazoleriboside diphosphate + reduced thioredoxin (Reversibility: ? [12]) [12] $ 2'-deoxybenzimidazolriboside diphosphate + H2 O [12]  purineriboside diphosphate + reduced thioredoxin (Reversibility: ? [12]) [12] $ 2'-deoxypurineriboside diphosphate + H2 O [12]  ribonucleoside diphosphate + reduced glutaredoxin (Reversibility: ir [70]) [70] $ 2'-deoxyribonucleoside diphosphate + oxidized glutaredoxin + H2 O [70]  ribonucleoside diphosphate + reduced glutaredoxin 1 (Reversibility: ir [70]) [70] $ 2'-deoxyribonucleoside diphosphate + oxidized glutaredoxin 1 + H2 O [70]  ribonucleoside diphosphate + reduced thioredoxin ( high concentration of dithiothreitol serves as in vitro hydrogen donor, thioredoxin B of Scenedesmus obliqus and yeast thioredoxin are most effective donors [26]; dithiothreitol at higher concentrations i.e. 100 mM can partially substitute for reduced T4 thioredoxin, the rate of CDP reduction is 10% of that obtained with the complete system [5]; maximal activ-

491

Ribonucleoside-diphosphate reductase

1.17.4.1

ity with E. coli thioredoxin [64]; dithiothreitol serves as in vitro electron donor, maximal activity with 50-75 mM [68]; dithiothreitol serves as in vitro electron donor, maximal activity with 40 mM [70]; NrdH-redoxin obtained from an overproducing strain, no activity with E. coli thioredoxin [75]; CDP is the only substrate that is reduced with a significant activity even in the absence of allosteric effectors [84]) (Reversibility: ir [1-81]) [1-81, 84] $ 2'-deoxyribonucleoside diphosphate + oxidized thioredoxin + H2 O ( in the presence of in vivo concentrations of effectors and all 4 substrates 43% dCDP, 14% dUDP, 31% dADP and 12% dGDP are formed [74]; CDP, ADP and GDP are reduced very poorly in the absence of allosteric effectors [81]) [1-81, 84]  tubercidin diphosphate + reduced thioredoxin ( 14% of GDP reduction rate [12]) (Reversibility: ? [12]) [12] $ 2'-deoxytubercidin diphosphate + oxidized thioredoxin [12] 2  1,10-phenanthroline ( 0.2 mM, 50% inhibition [26]) [12, 26] 1-formylisoquinoline thiosemicarbazone ( 0.0006 mM, 81% inhibition, 0.1 mM desferal reverses inhibition [9]) [9] 1-methyl-1-hydroxyurea ( 10 mM, 55% inhibition [3]) [3] 2',3'-dideoxy-ATP ( less potent inhibitor than dATP, 0.1 mM, 50% inhibition of CDP reduction [24]) [24] 2'-azido-2'-deoxynucleotides [30] 2'-chloro-2'-deoxycytidine 5'-diphosphate [30] 2'-deoxy-2'-azidocytidine diphosphate ( inactivation [59]; thymus enzyme, reversible inhibition [59]) [59] 2'-halo-2'-deoxynucleotides [30] 2'-methyladenosine 5'-diphosphate (, probably mechanism based inhibition, competitive inhibition vs. ADP and GDP [67]) [67] 2'-methyluridine 5'-diphosphate ( probably mechanism based inhibition, competitive inhibition vs. UDP and CDP [67]) [67] 2,3,4-trihydroxybenzamide [8] 2,3,4-trihydroxybenzohydroxamic acid ( 0.009 mM, 50% inhibition, reversible [3]; 0.012 mM, 50% inhibition, hydroxyurea-resistant cells [3]; 0.0035 mM, 50% inhibition [8]) [3, 8] 2,3-dihydro-1H-pyrazolo[2,3-a]imidazole ( mechanism of inhibition [14]; noncompetitive vs. CDP [17]) [9, 12, 14, 17] 2,3-dihydroxybenzohydroxamic acid ( 0.008 mM, 50% inhibition [8]) [8] 2,4-dichlorobenzohydroxamic acid ( 0.45 mM, 50% inhibition [8]) [8] 2,4-dihydroxybenzohydroxamic acid ( 0.3 mM, 50% inhibition [8]) [8] 2,5-dihydroxybenzohydroxamic acid ( 0.2 mM, 50% inhibition [8]) [8]

492

1.17.4.1

Ribonucleoside-diphosphate reductase

2,6-dihydroxybenzohydroxamic acid ( 0.1 mM, 50% inhibition [8]) [8] 2-aminobenzohydroxamic acid ( 0.12 mM, 50% inhibition [8]) [8] 2-azido-UDP ( rapid time dependent inactivation [30]) [30] 2-hydroxy-3-methylbenzohydroxamic acid ( 0.15 mM, 50% inhibition [8]) [8] 2-hydroxy-4-aminobenzohydroxamic acid ( 0.2 mM, 50% inhibition [8]) [8] 2-hydroxybenzohydroxamic acid ( 0.15 mM, 50% inhibition [8]) [8] 2-nitro-imidazole ( trivial name azomycin [12]) [12] 3,4,5-trihydroxybenzohydroxamic acid [8] 3,4,5-trihydroxybenzohydroxamic acid ( 0.01 mM, 50% inhibition [8]) [8, 30] 3,4,5-trihydroxybenzamide [8] 3,4,5-trihydroxyhydroxamic acid ( 0.012 mM, 50% inhibition, reversible [3]) [3] 3,4,5-trimethoxybenzohydroxamic acid ( 0.1 mM, 50% inhibition [8]) [8] 3,4-diaminobenzohydroxamic acid ( 0.04 mM, 50% inhibition [8]) [8] 3,4-dichlorobenzohydroxamic acid ( 0.3 mM, 50% inhibition [8]) [8] 3,4-dihydroxybenzamide [8] 3,4-dihydroxybenzohydroxamic acid ( 0.03 mM, 50% inhibition [8]) [8] 3,4-dihydroxybenzohydroxamic acid ( 0.033 mM, 50% inhibition, reversible [3]; 2.5 mM, 50% inhibition [3]; 0.3 mM, 50% inhibition [3]) [3] 3,4-dimethoxybenzohydroxamic acid ( 0.3 mM, 50% inhibition [8]) [8] 3,4-dimethylbenzohydroxamic acid ( 0.3 mM, 50% inhibition [8]) [8] 3,5-diamino-1H-1,2,4-triazole ( trivial name guanazole [9,12,17]; 2 mM, 41% inhibition, presence of 0.1 mM desferal pontentiates inhibition [9]; 2.3 mM, 50% inhibition of CDP reduction, 2.6 mM, 50% inhibition of ADP reduction [11]; 0.001 mM, 50% inhibition of CDP and UDP reduction, 0.05 mM, 50% inhibition of ADP reduction [31]; noncompetitive vs. CDP [17]) [9, 11, 12, 17, 31] 3,5-dihydroxybenzohydroxamic acid ( 0.4 mM, 50% inhibition [8]) [8] 3-aminobenzohydroxamic acid ( 0.35 mM, 50% inhibition [8]) [8] 3-hydroxybenzohydroxamic acid ( 0.35 mM, 50% inhibition [8]) [8] 3-methyl-1-hydroxyurea ( 10 mM, 57% inhibition [3]) [3] 3-methyl-4-nitrophenol [12] 4-amino-2-phenylimidazole-5-carboxamide [12] 4-aminobenzohydroxamic acid ( 0.15 mM, 50% inhibition [8]) [8] 4-dimethylaminobenzohydroxamic acid ( 0.5 mM, 50% inhibition [8]) [8] 493

Ribonucleoside-diphosphate reductase

1.17.4.1

4-hydroxybenzohydroxamic acid ( 0.30 mM, 50% inhibition [8]) [8] 4-methoxybenzohydroxamic acid ( 0.5 mM, 50% inhibition [8]) [8] 4-methyl-5-amino isoquinoline-1-carboxaldehyde thiosemicarbazone ( inhibits the non-heme iron subunit [14]) [12, 14] 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone ( 0.0003 mM, 93% inhibition, 0.1 mM desferal reverses inhibition [9]) [9] 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone ( inactivation, half-life: 3 min [17]) [9, 14, 17] 4-methylaminobenzohydroxamic acid ( 0.33 mM, 50% inhibition [8]) [8] 4-nitrobenzohydroxamic acid ( 0.5 mM, 50% inhibition [8]) [8] 5-(1-aziridinyl)-2,4-dinitrobenzamide [12] 8-hydroxyquinoline [12] 8-hydroxyquinoline 5-sulfonate ( no inhibition in the presence of excess iron [60]) [12, 60] ADP ( competitive inhibition of CDP reduction [25]) [25] ATP ( free ATP, 0.32 mM, 50% inhibition [25]; 4 mM, 50% inhibition of GDP reduction in the presence of dTTP [1]; CDP reduction is inhibited by free ATP [11]; 3 mM, 65% inhibition [58]) [1, 11, 25, 58] CDP ( competitive inhibition of ADP reduction [25]; competitive inhibition of UDP reduction [32]) [25, 32] Cibacron blue F3 GA [12] EDTA ( reversible stimulation of GDP reduction, irreversible inhibition of CDP reduction [1]; 1 mM, 72% inhibition [16]; 10 mM, 50% inhibition [26]) [1, 11, 12, 16, 26, 31, 59] FTLDADF ( last seven amino acid residues of carboxyl terminus of the R2 subunit of mouse enzyme and its N-a-acetyl derivative inhibit thymus enzyme [29]) [29] Fe2+ ( no effect of iron salts [63]; concentrations higher than 0.1-1 mM [12]; 0.2 mM, 50% inhibition [26]) [12, 26, 63] H2 O2 ( 0.01%, 81% inhibition [14]) [14] Mg2+ ( 1-5 mM, 10-20% inhibition of GDP reduction [1]; uncomplexed Mg2+ , 3.7 mM, 50% inhibition [25]; inhibition of CDP reduction [1]; uncomplexed Mg2+ [11]; 2 mM, 50% inhibition [26]) [1, 11, 25, 26] Mn2+ ( concentrations higher than 0.1-1 mM [12]; 0.2 mM, 50% inhibition [26]) [12, 26] N-ethylmaleimide ( 0.1 mM, 50% inhibition of intact enzyme, 0.05 mM, 50% inhibition of effector-binding subunit, 0.3 mM, 50% inhibition of non-heme iron subunit [24]) [24] N-hydroxy-a-aminoheptanoate ( 5 mM, 50% inhibition [26]) [26] N-hydroxy-a-aminohexanoate ( 15 mM, 50% inhibition [26]) [26] N-hydroxyguanidine ( 10 mM, 89% inhibition [3]) [3] N-hydroxyurethane ( 10 mM, 66% inhibition [3]) [3] N-methyl 3,4,5-trihydroxybenzamide [8] N-methylhydroxylamine ( 10 mM, 94% inhibition [3]) [3, 12] 494

1.17.4.1

Ribonucleoside-diphosphate reductase

NSFTLDADF ( inhibition of CDP reductase activity, peptide corresponds to the C-terminal region of the R2 subunit and competes with binding of R2 to the R1 subunit [72]) [72] UDP ( competitive inhibition of CDP reduction [32]; inhibition of CDP reduction [71]) [32, 33, 71] YAGAVVNDL ( peptide may prevent association of the two subunits by competing for the subunit binding site [42]) [42] YGAVVNDL [42] acetohydroxamic acid ( 1 mM, 50% inhibition [8]) [8, 12] aurintricarboxylate ( oligomeric form [12, 26]; 0.005 mM, 50% inhibition [26]) [12, 26] bathophenanthroline disulfonate [12] bathophenanthroline sulfonate ( 5 mM, almost complete inhibition of CDP and GDP reduction [1]; 1.5 mM, complete inactivation after 30 min, complete reactivation with FeCl3 [59]; no effect in the presence of excess iron [60]) [1, 59, 60] benzohydroxamic acid ( 0.4 mM, 50% inhibition [8]) [8] butylphenyl-dGTP ( 0.13 mM, 50% inhibition of ADP reduction [24]) [24] catechol derivatives [12] cisplatin ( more than 90% irreversible inhibition by inhibitor/enzyme ratios smaller than 2 under anaerobic conditions, 0.4 mM, 50% inhibition under aerobic conditions, inhibition of B1 subunit [22]) [22] dADP ( product inhibition [11]) [11] dATP ( 2.1 mM, 50% inhibition [11]; inhibition of CDP reduction [1, 2, 6, 11, 16, 31, 32, 33, 61, 63, 66]; inhibition of GDP reduction [1, 2, 6, 31]; inhibition of UDP reduction [2, 6, 32, 33]; inhibition of ADP reduction [2, 6, 31]; weak inhibition of ADP reduction [11]; 0.05 mM, 50% inhibition of CDP reduction in presence of optimum ATP concentration i.e. 6 mM, stimulation in absence of ATP [63]; 0.0033 mM, 50% inhibition of CDP reduction, 0.0036 mM, 50% inhibition of GDP reduction [1]; inhibition of CDP, UDP, GDP and ADP reduction [24, 66]; 0.005 mM, 50% inhibition of CDP and UDP reduction, 0.005 mM, 50% inhibition of GDP and ADP reduction [31]; inhibition of CDP and UDP reduction is reversed by ATP [33]; 0.1 mM, 96% inhibition of CDP reductase activity in dextran sulfate-treated cells, 85% inhibition of GDP reductase activity [54]; HSV type 2, 1 mM, 20% inhibition [58]; 3.5 mM, 92% inhibition of activity in extracts [61]; noncompetitive inhibition vs. ADP, GDP and CDP [66]; strong inhibition of the ATP activated enzyme, complete inhibition of GDP reduction, inhibition of ADP reduction [81]; inhibition of CDP reduction in the presence of ATP [82]) [1, 2, 6, 11, 16, 24, 26, 31, 32, 33, 54, 58, 61, 63, 66, 67, 81, 82] dCDP ( product inhibition [11]) [11] dCTP ( 1 mM, 50% inhibition of CDP reduction [1]; 1.2 mM, 50% inhibition of CDP reduction, 0.89 mM, 50% inhibition of ADP reduction [11]) [1, 11] 495

Ribonucleoside-diphosphate reductase

1.17.4.1

dGDP ( product inhibition [11]) [11] dGTP ( inhibition of CDP reduction [1, 2]; inhibition of UDP reduction [2, 31]; inhibition of GDP reduction [2, 66]; 0.08 mM, 50% inhibition of CDP reduction, 0.19 mM, 50% inhibition of GDP reduction [1]; inhibition of ATP- and dATP stimulated CDP reduction [6]; 1.2 mM, 50% inhibition of CDP reduction, 0.93 mM, 50% inhibition of ADP reduction [11]; 0.05 mM, 50% inhibition of GDP reduction [31]; 0.1 mM, 50% inhibition of CDP and UDP reduction [31]) [1, 2, 6, 11, 31, 66] dITP ( inhibition of CDP reduction [24]) [24] dTTP ( inhibition of UDP reduction [2, 31]; inhibition of CDP reduction [2, 11, 16, 61]; 0.2 mM, 50% inhibition of CDP reduction [1]; inhibition of ADP reduction [31]; HSV type 2, 1 mM, 20% inhibition [58]; inhibition of ADP- CDP- and UDP reduction [66]) [1, 2, 11, 16, 31, 58, 61, 66] dUDP ( product inhibition [11]) [11] dUTP ( inhibition of: CDP reduction, UDP reduction [2]) [2] desferrioxamine [12] dithiothreitol ( higher than 10 mM, activation below [71]) [71] ethyleneglycol-bis-(2-aminoethylether)-N,N,N',N'-tetraacetic acid ( trivial name EGTA [12]) [12] formohydroxamic acid ( 10 mM, 43% inhibition [3]) [3] g-l-glutaminyl-4-hydroxybenzene ( naturally occuring quinol from spores of Agaricus bisporus, 0.76 mM, 50% inhibition [45]) [45] glutaminyl-3,4-dihydroxybenzene ( 1.23 mM, 50% inhibition [45]) [45] glutathione ( analogs with aromatic substituents [12]) [12] hydroxylamine ( 10 mM, complete inhibition [3]) [3] hydroxyurea ( mechanism of inhibition [14]; thymus enzyme, reversible inhibition [59]; 1 mM, 98 and 81% inhibition of CDP and GDP reduction respectively [1]; 10 mM, complete inhibition, 0.3 mM, 50% inhibition [3]; 0.025 mM, 50% inhibition [3]; 0.01-0.03 mM, 50% inhibition, 0.2 mM, complete inhibition [50]; 0.5 mM, 50% inhibition [8]; 2 mM, 93% inhibition, presence of 0.1 mM desferal potentiates inhibition [9]; 1 mM, complete inactivation of thymus enzyme [12]; inhibits the non-heme iron subunit [14]; 1.5 mM, 50% inhibition [26]; 1 mM, approx. 90% inhibition [59]; approx. 0.5 mM, 50% inhibition [70]; 2 mM, approx. 80% inactivation [75]) [1, 3, 8, 9, 11, 12, 14, 16, 26, 30, 31, 50, 59, 63, 70, 71, 75, 80, 82] isoquinoline-1-carboxaldehyde thiosemicarbazone [12] meso-a,b-diphenylsuccinate [12] methyl 3,4,5-trihydroxybenzoate [8] monoclonal antibody raised against yeast tubulin ( CDP reductase activity is inhibited to a greater extent than ADP, UDP or GDP reductase activity, antibody recognizes a specific sequence in the C-terminal region on the R2 subunit [72]) [72] n-hexanohydroxamic acid [12] 496

1.17.4.1

Ribonucleoside-diphosphate reductase

nicotinohydroxamic acid ( 0.8 mM, 50% inhibition [8]) [8] nucleotide analogs ( overview [30]) [30] p-chloromercuribenzoate ( 0.35 mM, 50% inhibition of intact enzyme, 0.15 mM, 50% inhibition of effector-binding subunit, 1.5 mM, 50% inhibition of non-heme iron subunit [24]) [24] periodate-oxidized inosine ( inactivation, 1 mM, half-life: 6 min [17]) [9, 17] phenylacetohydroxamic acid ( 1 mM, 50% inhibition [8]) [8] picolinohydroxamic acid ( 0.5 mM, 50% inhibition [8]) [8] polyhydroxybenzohydroxamic acid [3] pyrazoloimidazol ( 2 mM, 79% inhibition, presence of 0.1 mM desferal pontentiates inhibition, inhibits the non-heme iron subunit [9]) [9, 14] pyridine-2-carboxaldehyde thiosemicarbazone [12] pyridoxal 5'-phosphate ( 1 mM, 65% inhibition, 3 mM, 90% inhibition [58]) [58] pyridoxal 5'-phosphate/NaBH4 [12] pyrogallol derivatives [12] sodium arsenite ( 0.025 mM, almost complete inhibition of CDP reduction, 86% inhibition of GDP reduction [1]) [1] synthetic peptides ( which specifically inhibit the activity of virusinduced enzyme [34]) [34] thenoyltrifluoroacetone ( 5 mM, almost complete inhibition of CDP and GDP reduction [1]) [1, 12] Additional information ( inhibition of reductase by hydroxyurea, guanazole and pyrazolo-imidazole is potentiated by iron-chelating agents e.g. EDTA, desferrioxamine mesylate and 8-hydroxyquinoline, inhibition by 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone and 1formylisoquinoline thiosemicarbazone is reversed by iron chelating agents [9]; each ribonucleoside diphosphate substrate is competitively inhibited by reduction of each other substrate [11]; overview: naturally occuring inhibitors e.g. proteins and nucleotides [12]; mechanism studied with inhibitors [17]; enzyme does not respond to feedback inhibition by dTTP or dATP [4]; L1210 cells with resistance to specific nucleotide reductase inhibitors [20]; overview [30]; mechanism-based inhibitors [32]; not inhibited by dATP and dTTP [12, 58]; not inibited by 8-hydroxyquinoline and o-phenanthroline [64]) [9, 11, 12, 17, 20, 30, 32, 58, 64]
"%  NADPH ( slight stimulation [1]; absolute requirement [5]) [1, 5] T4 thioredoxin ( enzyme induced in E. coli by infection with bacteriophage T4 [5,6]; absolute requirement [5]) [5] &

  2-mercaptoethanol ( 25 mM, maximal activation, 70% of activity with dithiothreitol [71]) [71] ADP [66] 497

Ribonucleoside-diphosphate reductase

1.17.4.1

ATP ( stimulation of CDP reduction [7, 12, 13, 31, 32, 43, 61]; stimulation of UDP reduction [7, 12, 31, 32]; stimulation of ADP reduction [13]; further stimulation of dTTP activated GDP reduction [2]; reduction of CDP is dependent on ATP or adenyl-5'-yl iminodiphosphate [1]; reduction of CDP and UDP requires 1-2 mM ATP [2]; most effective activator [68]; stimulation of CDP reduction [75]; required for CDP reduction [81]) [1, 2, 7, 12, 13, 31, 32, 43, 61, 63, 68, 75, 81, 82] E. coli thioredoxin reductase ( enzyme induced in E. coli by infection with bacteriophage T4 [5, 6]; absolute requirement [5, 6]) [5, 6] EDTA ( reversible stimulation of GDP reduction, irreversible inhibition of CDP reduction [1]) [1] GTP ( stimulation of CDP and ADP reduction [13]) [13] H2 O2 ( activation [21]) [21] P1,P5 -di(adenosine 5')tetraphosphate ( stimulation at low concentrations, inhibition above 0.3 mM [68]) [68] adenyl-5'-yl-imidodiphosphate ( maximal activation of CDP reduction at 4 mM [1]; can replace ATP as activator of CDP and UDP reduction [66]; stimulation at low concentration, inhibition above 0.3 mM [68]) [1, 66, 68] dATP ( stimulation of CDP and UDP reduction [6, 7]; 0.005 mM, stimulation of CDP reduction in absence of ATP, inhibition in presence of ATP [63]; stimulation of GDP reduction [2]; strong stimulation of CDP reduction [70]; stimulation of CDP reduction [75]; 6fold stimulation of GDP reduction [81]; stimulation of ADP reduction [81]; 0.2-0.4 mM, induces formation of dimers and tetramers of subunit R1, 1-2 mM, induces formation of hexamers of subunit R1 [84]) [2, 6, 7, 63, 70, 75, 81, 82] dCTP ( stimulation of UDP reduction [12]; stimulation of CDP and ADP reduction [13]; stimulation of CDP reduction [6]) [6, 12, 13] dGDP ( 40% less effective than dGTP [66]) [66] dGTP ( stimulation of GDP reduction [7]; stimulation of ADP reduction [7, 12, 13, 31]; stimulation of tubercidin diphosphate reduction [12]; stimulation of CDP reduction [13]; required for ADP reduction, maximal activity with 0.01 mM dGTP [2]; stimulation of ADP reduction [6]; activation of ADP reduction [66]; slight stimulation of CDP reduction [70]; required for ADP reduction [81]) [2, 6, 7, 12, 13, 31, 66, 70, 81] dITP ( activation of ADP reduction [24]) [24] dTTP ( stimulation of GDP reduction [1, 2, 6, 7, 12, 31, 67]; stimulation of ADP reduction [7]; absolutely required for GDP reduction, less than 10% activity in the absence of dTTP, maximal stimulation with 0.001-0.1 mM dTTP in the absence of ATP and 0.1-1 mM in the presence of ATP, inhibition above [2]; stimulation of CDP reduction [7, 12, 32]; stimulation of UDP reduction [7, 12, 32]; stimulation of 2-aminopurineriboside di498

1.17.4.1

Ribonucleoside-diphosphate reductase

phosphate reduction [12]; stimulation of 2,6-diaminopurine riboside reduction [12]; stimulation of purine riboside diphosphate reduction [12]; stimulation of benzimidazoleriboside diphosphate reduction [12]; required for ADP reduction [2]; slight stimulation of CDP reduction [70]; stimulation of CDP reduction [75]; required for GDP reduction [81]) [1, 2, 7, 12, 28, 31, 32, 70, 75, 81] dihydrolipoic acid ( slight stimulation [1]) [1] dithioerythritol ( 0.5 mM, maximal activation, 94% of activity with dithiothreitol [71]) [71] dithiols ( required for reduction of CDP in vitro [61]; required for in vitro reduction [43]) [43, 61] dithiothreitol ( required for optimal activity [13]; high concentrations serve as in vitro hydrogen donor [26]; slight activation [1]; optimal in vitro activity with 10 mM, inhibition above [71]) [1, 13, 26, 71] phosphate ( up to 50 mM, pH 6.7: necessary for activity, decrease of activity at higher values [26]; 50 mM, slight stimulation, inhibition at 200 mM [64]) [26, 64] thioredoxin reductase ( E. coli enzyme [5]) [5] Additional information ( enzyme of cells first treated with 2,6-dichlorophenolindophenol has a complete dependence on NADPH which can also be met by dithiothreitol or dihydrolipoic acid [1]; overview: stimulation of various enzymes [12]; stimulation with various substrates [12, 26]; stimulation by effector nucleotides [12, 24, 26]; overview: nucleoside 5'-diphosphates as effectors of mammalian ribonucleotide reductase [27]; ribonucleoside effectors are exclusively bound at effector binding sites on subunit B1 controlling substrate specificity and activity [7]; subunit R1 has 2 effector-binding sites per polypeptide chain: one activity site for dATP and ATP, with dATP-inhibiting and ATPstimulating catalytic activity and a second specificity site for dATP, ATP, dTTP and dGTP directing substrate specificity [81]) [1, 7, 12, 24, 26, 27, 81] '(   Ca2+ ( activtiy depends on Ca2+ [73]) [73] Mg2+ ( 5-10 mM, 2-3fold stimulation, not required for enzyme activity [5]; subunit B1 requires Mg2+ ions in the 10 mM concentration range for activity [12, 33]; absolutely required [43]; stimulates CDP but not ADP reduction [13]; thymus enzyme: about 50% activity in absence of added Mg2+ , optimal Mg2+ concentration varies with concentration of nucleotide effector [59]; 2fold stimulation [12]; 2fold activation [68]; activity depends on Mg2+ [70]) [5, 12, 13, 33, 43, 59, 68, 70, 80] Mn2+ ( EPR-silent Mn bound to the polypeptide chain, approx. 0.5 mol manganese ions/mol of R2F polypeptide [75]) [75] iron ( 2.3 atoms of nonheme iron per molecule [5]; subunit B2 contains iron, nonheme-like porphyrin complexes [12]; B2 subunit contains 2 dinuclear Fe3+ centers [21,30,32]; iron center

499

Ribonucleoside-diphosphate reductase

1.17.4.1

is composed of 2 high spin iron atoms antiferromagnetically coupled through a micro-oxo bridge [30]; 2 separate iron centers in subunit B2, 1 center on each b subunit, distance between iron centers: 25 A, distance between FeFe atoms: 3.3 A [35]; X-ray absorption fine structure, EXAFS, of ironcontaining subunit, Fe-Fe distance in subunit B2 is in the 3.26-3.48 A range [40]; iron center stabilizes tyrosyl radical, distance between the iron center and the tyrosyl radical is estimated to be 6-9.0 A [47]; B2 subunit contains 2 nonidentical high spin Fe3+ ions in an antiferromagnetically coupled binuclear complex that resembles both methydroxohemerythrin and oxyhemerythrin [51]; oxo- or carboxylate-bridge between the antiferromagnetically coupled pair of high spin Fe3+ , possibly with a binding oxogroup [53]; 120000 Da L2 subunit of regenerating liver contains iron [56]; nonheme iron is an essential component of the enzyme [59]; no stimulation by iron ions [61]; Fe2+ stimulates [43]; 2 iron atoms and a tyrosyl radical per 88000 Da subunit [30]; iron binds directly to the enzyme structure and not via sulfur [47]; Raman spectroscopy of B2 subunit shows Fe-O vibration of an oxygen-coordinated ligand [52]; 1.8 mol of iron per mol of R2F subunit, dinuclear iron center [70]; proposed in vitro mechanism for the assembly of the diferric tyrosyl radical cofactor of subunit R2 [76]) [5, 12, 21, 30, 32, 34, 35, 40, 43, 47, 51-53, 56, 59, 70, 76] Additional information ( salt-dependence of calf thymus enzyme: optimal activity in 40 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, pH 7.6, in the presence of 80-120 mM KCl, precipitation in lower salt concentration, inhibition in higher salt concentration [59]; Mg2+ is not required for activity in vitro [12,58]; no stimulation by Mg2+ or Fe2+ /Fe3+ [61]; model of enzyme regulation by nucleoside 5'-triphosphates [66]; the essential metallo-cofactor is a microoxo-micro-carboxylato-diiron cluster adjacent to a stable tyrosyl radical [83]) [12, 58, 59, 61, 66, 83] ) &  * +, 2.82 (CDP, CDP reduction in the absence of allosteric effectors [84]) [84] 9.6 (CDP, CDP reduction in the presence of 1 mM ATP [84]) [84] 15.6 (CDP, CDP reduction in the presence of 0.2 mM ATP [84]) [84] 17.4 (CDP, CDP reduction in the presence of 4 mM ATP [84]) [84] 200 (CDP, activity of subunit B1 assayed in the presence of an excess of subunit B2 [32]) [32] 775 (CDP, activity of subunit B2 assayed in the presence of an excess of subunit B1 [32]) [32] " & *-% , 0.0000045 ( partially purified enzyme, CDP reduction [71]) [71] 0.00028 ( partially purified enzyme [65]) [65] 0.0072 [64] 0.00837 [60] 0.0131 ( purified subunit L2 assayed in the presenc of L1 [56]) [56] 500

1.17.4.1

Ribonucleoside-diphosphate reductase

0.015 ( purified subunit L1 assayed in the presenc of L2, specific for CDP [56]) [56] 0.0237 [59] 0.024 ( subunit Y1, expressed in Saccharomyces cerevisiae [78]) [78] 0.034 [75] 0.035 ( recombinant R1 subunit, CDP reduction in the presence of ATP [80]) [80] 0.048 ( recombinant D1-248 R1 subunit, CDP reduction in the presence of ATP [80]) [80] 0.062 ( M1 subunit from a mutant cell line of S49 T-lymphoma cells [57]) [57] 0.1 [73] 0.122 ( recombinant enzyme [68]) [68] 0.18 ( recombinant R2 subunit, CDP reduction in the presence of ATP [80]) [80] 0.28 ( CDP reduction of subunit R1E in the presence of subunit R2F [70]) [70] 0.3 ( His-tagged subunit Y2-K387N, expressed in Saccharomyces cerevisiae [78]) [78] 0.59 ( purified subunit B1 [7]) [7] 0.83 ( CDP reduction of subunit R2F in the presence of subunit R1E [70]) [70] 1.09 [5] 2.86 ( purified subunit B2 [7]) [7] 4.21 ( recombinant subunit B2 [18]) [18] 4.3 ( Herpes simplex virus type 2 enzyme, CDP reduction [25]) [25] 6.3 ( recombinant B2 subunit [40]) [40] Additional information ( extracts from hydroxyurea resistant cell line HU-7 exhibit a 13fold higher ADP reductase activity and a 5fold higher CDP reductase activity when compared to wild-type [20]; optimal activity in 40 mM 4-(2-hydroxyethyl)-1-piperazineethansulfonic acid, pH 7.6, 80-120 mM KCl [59]; strong increase of specific activity during growth of Novikoff hepatoma cells [62]) [20, 59, 62] . / *', 0.00013 (glutaredoxin) [70] 0.00049 (CDP) [25] 0.00065 (CDP) [11] 0.0012 (GDP) [11] 0.0014 (CDP, in the presence of 5'-adenylimidodiphosphate [71]) [71] 0.0015 (dTTP, positive effector of CDP reduction [33]) [33] 0.0023 (CDP, in the presence of ATP [71]) [71] 0.003 (dATP, positive effector of CDP reduction [70]) [70] 0.003 (thioredoxin) [64] 0.006 (glutaredoxin 1) [70]

501

Ribonucleoside-diphosphate reductase

1.17.4.1

0.0078 (ADP) [25] 0.01 (CDP) [63] 0.012 (ADP) [11] 0.016 (CDP) [70] 0.021 (CDP, in the presence of 1.5 mM ATP [13]) [13] 0.033 (CDP, in the presence of 3.6 mM dCTP [13]) [13] 0.04 (GDP, in the presence of 0.4 mM dTTP as effector [6]) [6] 0.043 (CDP, in the presence of 0.002 mM dATP as effector [6]) [6] 0.048 (ADP, in the presence of 0.007 mM dGTP as effector [6]) [6] 0.05 (CDP) [1] 0.056 (CDP, in the presence of 0.5 mM ATP as effector [6]) [6] 0.057 (CDP, in the presence of 0.02 mM dTTP as effector [6]) [6] 0.07 (ATP, positive effector of CDP reduction [33]) [33] 0.08 (UDP) [11] 0.09 (CDP, subunit L1 [56]) [56] 0.095 (ADP, optimum dGTP concentration [13]) [13] 0.1 (UDP, in the presence of 0.3 mM ATP as effector [6]) [6] 0.2 (ADP, in the presence of 0.2 mM GTP [13]) [13] 0.22 (UDP) [33] 0.24 (GDP) [1] 0.3 (ADP) [6] 0.31 (CDP) [6] 0.37 (GDP) [6] 1.2 (UDP) [6] Additional information ( effect of different nucleoside triphosphates on Km [2, 5, 6]) [2, 5, 6] . / *', 0.0002 (dATP, in the presence of 0.15 mM dGTP [6]) [6] 0.0004 (dATP, in the presence of 0.55 mM dTTP [6]) [6] 0.0004 (dGTP, with 0.001 mM dATP as positive effector [6]) [6] 0.00042 (CDP, competitive inhibition of ADP reduction [25]) [25] 0.0005 (dATP, in the presence of 0.005 mM dGTP [6]) [6] 0.0005 (dGTP, with 0.02 mM dATP as positive effector [6]) [6] 0.00058 (CDP, vs. GDP [11]) [11] 0.0006 (GDP, vs. ADP [11]) [11] 0.0006 (dATP, in the presence of 0.05 mM dTTP [6]) [6] 0.0007 (CDP, vs. ADP [11]) [11] 0.0009 (dGTP, with 0.17 mM ATP as positive effector [6]) [6] 0.0012 (GDP, vs. CDP [11]) [11] 0.0018 (GDP, vs. UDP [11]) [11] 0.0018 (dGTP, with 1.7 mM ATP as positive effector [6]) [6] 0.0019 (CDP, vs. UDP [11]) [11] 0.0037 (2'-methyladenosine 5'-diphosphate, vs. ADP [67]) [67] 0.0055 (ADP, vs. GDP [11]) [11] 0.0058 (UDP, vs. GDP [11]) [11] 0.007 (GDP, competitive vs. CDP [33]) [33]

502

1.17.4.1

Ribonucleoside-diphosphate reductase

0.0079 (2'-methyladenosine 5'-diphosphate, vs. GDP [67]) [67] 0.011 (ADP, competitive inhibition of CDP reduction [25]) [25] 0.014 (ADP, vs. CDP [11]) [11] 0.02 (N-a-acetyl-FTLDADF, N-a-acetyl heptapeptide, identical to the last seven amino acids of R2 subunit C-terminus from mouse enzyme [29]) [29] 0.023 (2'-deoxy-2'-azidocytidine diphosphate) [59] 0.029 (UDP) [71] 0.036 (ADP, vs. UDP [11]) [11] 0.042 (UDP, vs. CDP [11]) [11] 0.043 (UDP, vs. ADP [11]) [11] 0.084 (2'-methyluridine 5'-diphosphate, vs. UDP [67]) [67] 0.115 (2'-methyluridine 5'-diphosphate, vs. CDP [67]) [67] 0.5 (UDP, competitive vs. CDP [33]) [33] 0.69 (hydroxyurea) [71] 1.1 (2,3-dihydro-1H-pyrazolo[2,3-a]imidazole, derived from slope [17]) [17] 1.5 (2,3-dihydro-1H-pyrazolo[2,3-a]imidazole, derived from intercept [17]) [17] 14 (guanozole, derived from intercept [17]) [17] 27 (guanozole, derived from slope [17]) [17] 0  6.5-7 [26] 7-8 [5] 8 [70] 8-8.8 [68] 8.1 [11] 0 

 6.8-8.8 ( 60% activity at pH 7.2 [68]) [68] )  *
, 25-30 [26] ) 

 *
, 15-42 ( 15 C: approx. 27% activity, 42 C: approx. 10% activity [26]) [26] 25-37 ( reaction rate is about twice as fast at 37 C as the rate at 25 C [59]) [59]

503

Ribonucleoside-diphosphate reductase

1.17.4.1

  #   ' 1  125900 (5 mM Ca2+ , gel filtration [73]) [73] 160000 (gel filtration [75]) [75]   ? ( x * 84000 + x * 58000, 84000 Da subunit is predominantly monomeric under experimental conditions, 58000 Da subunit may be oligomeric, SDS-PAGE [10]; 2 * 90000 + x * ?, Novikoff hepatoma cells [12]; 2 * 90000 + x * 75000, most likely 1 75000 Da subunit, SDS-PAGE [12,26]; x * 45000 + 1 * 75000 + 1 * 45000, holoenzyme may have an a4 bb' structure, SDS-PAGE [56]) [10, 12, 26, 56] dimer ( ab, 1 * 100000 + 1 * 100000, Molt F4 lymphoblast cells [12]; 1 * 136000 + 1 * 38000, molecular weight for subunit 1 deduced from sequence: 124017 Da, difference may be due to phosphorylation, SDS-PAGE [44]; 2 * 60200, dimer appears to dissociate in the absence of Ca2+ into monomers, SDS-PAGE [73]) [12, 44, 73] hexamer ( 4 * 45000 + 1 * 45000 + 1 * 75000, regenerating liver [12,56]) [12, 56] tetramer ( a2 b2 , 2 * 85000 + 2 * 35000, enzyme induced in E. coli after infection with bacteriophage T4, SDS-PAGE [5,12]; a2 b2 , 160000 Da subunit B1 and 78000 Da subunit B2, each consisting of 2 identical or similar proteins [7]; aa'b2 , 2 * 82000 + 2 * 78000, each subunit composed of 2 polypeptide chains, subunit B1, 82000 Da, sedimentation equilibrium centrifugation, subunit B2, low speed sedimentation equilibrium centrifugation [12,34]; 2 * 84000 + 2 * 55000, SDS-PAGE [12]; aa',b2 , 2 * 80000 + 2 * 39000, SDS-PAGE [12]; a2 b2 , 2 * 84000 + 2 * 43500, SDSPAGE [48]; a2 b2 , 2 * 70000 + 2 * 36000, SDS-PAGE [70]) [5, 7, 12, 34, 48, 70] trimer ( ab2 , 1 * 81200 + 2 * 37900, deduced from nucleotide sequence [75]) [75] Additional information ( enzyme in Ehrlich ascites tumor cells consits of two nonidentical subunits: an effector-binding subunit, EB, and a non-heme iron containing subunit, NHI, since their relative levels are not coordinately regulated the stoichiometry of the whole enzyme varies with the cell cycle [28]; composition of the enzyme is not constant, but is altered in presence of effectors [55]; 88000-90000 Da M1 subunits are degraded into 40000 Da fragments in proliferately quiescent liver cells, intact subunits are only accumulated when the cells replicate DNA [19]; the active form of B2 subunit contains a tyrosyl radical essential for activity [21,47]; catalytic subunit U2 contains a tyrosyl radical essential for activity [23]; tyrosyl radical is stabilized by an iron center [47]; large subunit R1 contains binding sites for substrates and allosteric effectors, smaller subunit R2 contains non-heme iron and a tyrosyl free-radical [72,79]; proposed in vitro mechanism for the assembly of the diferric tyrosyl radical cofactor of subunit R2 [76]; subunits Y1 and Y2 constitute the active

504

1.17.4.1

Ribonucleoside-diphosphate reductase

enzyme, large subunit Y3 has no activity, subunit Y4 may function as a chaperone [78]; nucleotide binding to the specificity site drives formation of an active R1,2R2,2 dimer, ATP or dATP binding to the adenine-specific site results in formation of an inactive tetramer and ATP binding to the hexamerization site drives formation of an active R1,6R2,6 hexamer which is probably the major active form in mammalian cells [84]) [19, 21, 23, 28, 47, 55, 72, 76, 78, 84]

 2 %$ %' %

% 3T6-Swiss albino [46] BHK-21 [11] BSC-40 [16] CHO [13] Ehrlich tumor carcinoma cell [9, 12, 14, 24, 27, 28, 55] HeLa cell [8, 69] L-1210 ( L1210 cells resistant to specific ribonucleotide reductase inhibitors [20,45]) [16, 20, 24, 27, 28, 31, 45, 46, 54, 55, 57, 68, 69] L-cell (cell line) [16, 20, 24, 27, 28, 31, 45, 46, 54, 55, 57, 68, 69] MDBK [46] Novikoff ascites tumor cell [12, 31] S49 ( mutant line of S49 mouse T-lymphoma cells [57]) [57] bone marrow [12, 43, 60] fibroblast [3] kidney ( baby hamster kidney cells [11]) [11] liver ( regenerating liver [12, 56, 63]) [12, 19, 56, 63] lymphoblast ( Molt 4F cells [12]) [12] thymus ( calf [10,12]) [10, 12, 29, 31, 59] 3#

cytoplasm ( M1 subunit is exclusively localized in cytoplasm [46]; enzyme from Novikoff hepatoma tumor cells is associated with a smooth membrane component of the cytoplasm [62]) [46, 62] intracellular ( almost entirely [54]) [54] mitochondrion ( enzyme may be specifically associated with mitochondria [69]) [69] $"

(Novikoff ascites tumor cells, partial [1]; M1 subunit from a mutant cell line of S49 T-lymphoma cells [57]; chimeric R2 genes in which C-terminal sequences in Escherichia coli subunit R2 are replaced by C-terminal sequences from the mouse subunit R2 [77]; Y370F and Y370w mutant enzymes [79]) [1, 57, 77, 79] (regenerating liver, dATP-Sepharose affinity chromatography [56]) [56] 505

Ribonucleoside-diphosphate reductase

1.17.4.1

(overproducing strain [7]; recombinant B2 subunit, Sephadex G-25, DEAE Bio gel, Sephadex QAE-50 [18]; chimeric R2 genes in which C-terminal sequences in Escherichia coli subunit R2 are replaced by C-terminal sequences from the mouse subunit R2) [7, 18, 40, 77] (enzyme induced in Escherichia coli after infection with bacteriophage T4, streptomycin, ammonium sulfate, dATP-Sepharose [5]) [5] (streptomycin, ammonium sulfate, DEAE-cellulose, hydroxylapatite, dATP-Sepharose [59]) [10, 59] [26] (partial [43]; ammonium sulfate, Sephadex G-200, dATP-Sepharose [60]; ammonium sulfate, Sephadex G-200, partially purified [65]; gel filtration, ATP-agarose, partial purification [71]) [43, 60, 65, 71] (HSV type 2 [25]; streptomycin sulfate, ammonium sulfate, partial purification [58]) [25, 58] (ammonium sulfate, Blue Sepharose, affinity resin [68]) [68] (R1E subunit, ammonium sulfate, DEAE-Sepharose, dATP-Sepharose [70]; R2F subunit, ammonium sulfate, DEAE-Sepharose, Mono Q, Superdex [70]) [70] (streptomycin, DEAE-Sepharose, Phenyl-Sepharose, Heparin-Sepharose, Sephacryl S-200 [73]) [73] (low-salt precipitation, DEAE-cellulose, Supredex 200, MonoQ [75]) [75] (recombinant wild-type R1 and R2 subunits and F127Y, Y129F and F127Y/Y129F R2 mutant subunits [80]) [80]
 #

(B2 subunit, hanging drops of 0.01 ml containing 25 mg/ml of enzyme with 1 ml of 1.5 M ammonium sulfate in the well and 750 mM ammonium sulfate starting concentration in the drop, pH 6.0, crystals appear after 1 week at room temperatur [15]; two-dimensional crystals of B1 dimer enzyme-effector complex, 18 A resolution [39]; B2 subunit, hanging drop method, crystallization in 20% polyethylene glycol 4000, 200 mM NaCl, 0.3% dioxane, 50 mM ethyl mercuric thiosalicylate, 50 mM MES buffer, pH 6.0, orthorhombic crystals, crystal structure at 2.2 A resolution [35, 36]) [15, 35, 36, 39]
 

(M2 subunit, expression in mouse BALB/3T3 cells [37]; nearly full length cDNA of M1 and M2 subunits, expression of M2 subunit in mouse 3T6 cells and monkey COS-7 cells [41]; expression of Y370F and Y370W mutant enzymes in Escherichia coli [79]; expression of wild-type and Y177F mutant subunit R2 in Escherichia coli [83]) [37, 41, 79, 83] (overexpression of B2 subunit [18]; expression of Y122F, Y356F and Y122F/Y356F mutant enzymes in Escherichia coli [76]; expression of chimeric R2 genes in which C-terminal sequences in Escherichia coli subunit R2 are replaced by C-terminal sequences from the mouse subunit R2 [77]) [18, 40, 76, 77]

506

1.17.4.1

Ribonucleoside-diphosphate reductase

(expression of subunits Y1, Y2, Y3 and Y4 in Escherichia coli, expression of Y1, His-tagged Y2 and His tagged Y2-K387N mutant enzyme subunit in Saccharomyces cerevisiae [78]) [78] (Herpes simplex virus type I and II [30]) [30] (expression of M1 mutant cDNA containing a G to A transition at codon 57 in chinese hamster ovary cells [37]) [37] (expression in Escherichia coli [68]) [68] (expression R1E and R2F subunits in Escherichia coli [75]) [75] (expression of wild-type and amino terminal deleted D1-248 R1 subunit and wild-type, F127Y, Y129F and F127Y/Y129F mutant R2 subunit in Escherichia coli [80]) [80] (expression of nrdA and nrdB genes encoding a CDP reductase in Escherichia coli [82]) [82] 

 

C225A ( 4-6% of wild-type activity [32]) [32] C225S ( C225 appears to be one of the participants in the direct reduction of substrate [30]; major product formed by interaction with CDP is cytosine [32]) [30, 32] C439A ( 4-6% of wild-type activity [32]) [32] C462S ( in the presence of dithiothreitol the major product formed by interaction with CDP is cytosine [32]) [32] C754A ( active with dithiothreitol as reductant, 3% of wild type activity with thioredoxin [32]) [32] C759A ( active with dithiothreitol as reductant, 3% of wild type activity with thioredoxin [32]) [32] C759S ( C759 may play a role in the relay of electrons between thioredoxin and subunit B1 [30]) [30] D57N ( mutation in R1 subunit, in contrast to wild-type dATP stimulates CDP reduction, GDP reduction is inhibited by dGTP, ADP reduction is inhibited by dTTP similar to wild-type, this suggests that the mutant enzyme binds both ATP and dATP to the activity site but does not distinguish between them when it comes to catalysis [81]) [81] F127Y ( similar CDP reductase activity as wild-type [80]) [80] F127Y/Y129F ( 10-15% of wild-type CDP reductase activity [80]) [80] K387N ( affords higher activity due to increased tyrosyl radical content [78]) [78] Y122F ( mutant enzyme cannot generate a Y122 tyrosyl radical necessary for catalysis, 0.5% of wild-type activity [76]) [76] Y122F/Y356F ( 0.5% of wild-type activity [76]) [76] Y129F ( no CDP reductase activity [80]) [80] Y177F ( tyrosyl residue involved in radical formation [83]) [83] Y356F ( similar properties as wild-type [76]) [76] Y370F ( mutation in R2 subunit, no activity [79]) [79]

507

Ribonucleoside-diphosphate reductase

1.17.4.1

Y370W ( mutation in R2 subunit, point mutation does not affect the ability to form a normal diferric iron/tyrosyl radical center, 1.7% of wild-type activity probably due to slow radical transfer [79]) [79]

4  )  50 ( half-life: 2.5 min [58]) [58] 5    "

, effector-binding subunit of mammalia is more sensitive to proteolysis by chymotrypsin, to heating at 55 C and to sulfhydryl reagents e.g. p-chloromercuribenzoate and N-ethylmaleimide, than the nonheme iron subunit, the latter is more sensitive to trypsin treatment [28] , dithiothreitol, 1 mM, stabilizes [5] , protein concentrations above 5 mg/ml stabilizes [5] , partially purified enzyme is very unstable in solution, half-life at 0 C: less than 24 h [64] , glycerol and ATP required for stabilization [60] , quite susceptible to denaturation [68]    , -80 C, 0.25 M sucrose, 100 mM Tris-HCl, pH 7.6, 10 mM MgCl2 , 2 mM dithiothreitol, 1 month, no loss of activty [56] , -70 C, 100 mM potassium phosphate, pH 7.0, 1 mM dithiothreitol, enzyme concentration 4.8 mg/ml, 6 months, no loss of activity [5] , -70 , 50 mM Tris-Cl, pH 7.6, 100 mM KCl, several months, no loss of activity [59] , quick-freezing in liquid nitrogen and subsequent storage at -20 C, several weeks, no loss of activity [64] , -15 C or -70 C, 10 mM histidine-HCl, pH 7.0, 2 mM dithiotreitol, 40% glycerol, 2 mM ATP, 6 months, no loss of activity [60] , -15 C, 10 mM histidine-HCl, pH 7.0, 2 mM dithiotreitol, 2 mM ATP, 6 months, 80% loss of activity [60] , -70 C, 10 mM histidine-HCl, pH 7.0, 2 mM dithiothreitol, 2 mM ATP, 6 months 60% loss of activity [60] , -70 C, up to 8 months, no loss of activity [58] , 4 C, at least 24 h, no loss of activity [58] , -80 C, several months, no loss of activity [68]

"  [1] Kucera, R.; Paulus, H.: Studies on ribonucleoside-diphosphate reductase in permeable animal cells. II. Catalytic and regulatory properties of the enzyme in mouse L cells. Arch. Biochem. Biophys., 214, 114-123 (1982)

508

1.17.4.1

Ribonucleoside-diphosphate reductase

[2] Moore, E.C.; Hurlbert, R.B.: Regulation of mammalian deoxyribonucleotide biosynthesis by nucleotides as activators and inhibitors. J. Biol. Chem., 241, 4802-4809 (1966) [3] Kjoller Larsen, I.; Sjöberg, B.M.; Thelander, L.: Characterization of the active site of ribonucleotide reductase of Escherichia coli, bacteriophage T4 and mammalian cells by inhibition studies with hydroxyurea analogues. Eur. J. Biochem., 125, 75-81 (1982) [4] Huszar, D.; Bacchetti, S.: Is ribonucleotide reductase the transforming function of herpes simplex virus 2?. Nature, 302, 77-79 (1983) [5] Berglund, O.: Ribonucleoside diphosphate reductase induced by bacteriophage Tr. I. Purification and characterization. J. Biol. Chem., 247, 72707275 (1972) [6] Berglund, O.: Ribonucleoside diphosphate reductase induced by bacteriophage T4. II. Allosteric regulation of substrate sepecificity and catalytic activity. J. Biol. Chem., 247, 7276-7281 (1972) [7] Thelander, L.; Sjöberg, B.M.; Eriksson, S.: Ribonucleoside diphosphate reductase (Escherichia coli). Methods Enzymol., 51, 227-237 (1978) [8] Elford, H.L.; Van't Riet, B.; Wampler, G.L.; Lin, A.L.; Elford, R.M.: Regulation of ribonucleotide reductase in mammalian cells by chemotherapeutic agents. Adv. Enzyme Regul., 19, 151-168 (1981) [9] Cory, J.G.; Sato, A.; Lasater, L.: Specific inhibition of the subunits of ribonucleotide reductase as a new approach to combination chemotherapy. Adv. Enzyme Regul., 19, 139-150 (1981) [10] Mattaliano, R.J.; Sloan, A.M.; Plumer, E.R.; Klippenstein, G.L.: Purification of the two complementary subunits of ribonucleotide reductase from calf thymus. Biochem. Biophys. Res. Commun., 102, 667-674 (1981) [11] Averett, D.R.; Lubbers, C.; Elion, G.B.; Spector, T.: Ribonucleotide reductase induced by herpes simplex type 1 virus. Characterization of a distinct enzyme. J. Biol. Chem., 258, 9831-9838 (1983) [12] Lammers, M.; Follmann, H.: The ribonucleotide reductases - a unique group of metalloenzymes essential for cell proliferation. Struct. Bonding, 54, 27-91 (1983) [13] Hards, R.G.; Wright, J.A.: Ribonucleotide reductase activity in intact mammalian cells: stimulation of enzyme activity by MgCl2 , dithiothreitol, and several nucleotides. Arch. Biochem. Biophys., 231, 9-16 (1984) [14] Sato, A.; Bacon, P.E.; Cory, J.G.: Studies on the differential mechanisms of inhibition of ribonucleotide reductase by specific inhibitors of the nonheme iron subunit. Adv. Enzyme Regul., 22, 231-241 (1984) [15] Joelson, T.; Uhlin, U.; Eklund, H.; Sjöberg, B.M.; Hahne, S.; Karlsson, M.: Crystallization and preliminary crystallographic data of ribonucleotide reductase protein B2 from Escherichia coli. J. Biol. Chem., 259, 9076-9077 (1984) [16] Slabaugh, M.B.; Johnson, T.L.; Mathews, C.K.: Vaccinia virus induces ribonucleotide reductase in primate cells. J. Virol., 52, 507-514 (1984) [17] Spector, T.; Jones, T.E.: Herpes simplex type 1 ribonucleotide reductase. Mechanism studies with inhibitors. J. Biol. Chem., 260, 8694-8697 (1985)

509

Ribonucleoside-diphosphate reductase

1.17.4.1

[18] Salowe, S.P.; Stubbe, J.: Cloning, overproduction, and purification of the B2 subunit of ribonucleoside-diphosphate reductase. J. Bacteriol., 165, 363-366 (1986) [19] Whitefield, J.F.; Sikorska, M.; Youdale, T.; Brewer, L.; Richards, R.; Walker, P.R.: Ribonucleotide reductase - new twists in an old tale. Adv. Enzyme Regul., 28, 113-123 (1989) [20] carter, G.L.; Cory, J.G.: Selective resistance of L1210 cell lines to inhibitors directed at the subunits of ribonucleotide reductase. Adv. Enzyme Regul., 29, 123-139 (1989) [21] Sahlin, M.; Sjöberg, B.M.; Backes, G.; Loehr, T.; Sanders-Loehr, J.: Activation of the iron-containing B2 protein of ribonucleotide reductase by hydrogen peroxide. Biochem. Biophys. Res. Commun., 167, 813-818 (1990) [22] Smith, S.L.; Douglas, K.T.: Stereoselective, strong inhibition of ribonucleotide reductase from E. coli by cisplatin. Biochem. Biophys. Res. Commun., 162, 715-723 (1989) [23] Harder, J.; Follmann, H.: Characterization of the free radical in a plant ribonucleotide reductase. FEBS Lett., 222, 171-174 (1987) [24] Cory, J.G.; Sato, A.; Brown, N.C.: Protein properties of the subunits of ribonucleotide reductase and the specificity of the allosteric site(s). Adv. Enzyme Regul., 25, 3-19 (1986) [25] Averett, D.R.; Furman, P.A.; Spector, T.: Ribonucleotide reductase of herpes simplex virus type 2 resembles that of herpes simplex virus type 1. J. Virol., 52, 981-983 (1984) [26] Hofmann, R.; Feller, W.; Pries, M.; Follmann, H.: Deoxyribonucleotide biosynthesis in green algae. Purification and characterization of ribonucleotide-diphosphate reductase from Scenedesmus obliquus. Biochim. Biophys. Acta, 832, 98-112 (1985) [27] Cory, J.G.; Rey, D.A.; Carter, G.L.; Bacon, P.E.: Nucleoside 5'-diphosphates as effectors of mammalian ribonucleotide reductase. J. Biol. Chem., 260, 12001-12007 (1985) [28] Sato, A.; Cory, J.G.: Differential sensitivities of the subunits of mammalian ribonucleotide reductase to proteases, sulfhydryl reagents, and heat. Arch. Biochem. Biophys., 244, 572-579 (1986) [29] Yang, F.D.; Spanevello, R.A.; Celiker, I.; Hirschmann, R.; Rubin, H.; Cooperman, B.S.: The carboxyl terminus heptapeptide of the R2 subunit of mammalian ribonucleotide reductase inhibits enzyme activity and can be used to purify the R1 subunit. FEBS Lett., 272, 61-64 (1990) [30] Stubbe, J.: Ribonucleotide reductases. Adv. Enzymol. Relat. Areas Mol. Biol., 63, 349-419 (1990) [31] Holmgren, A.: Regulation of ribonucleotide reductase. Curr. Top. Cell. Regul., 19, 47-76 (1981) [32] Stubbe, J.: Ribonucleotide reductases: amazing and confusing. J. Biol. Chem., 265, 5329-5332 (1990) [33] Larsson, A.; Reichard, P.: Enzymatic synthesis of deoxyribonucleotides. IX. Allosteric effects in the reduction of pyrimidine ribonucleotides by the ribonucleoside diphosphate reductase system of Escherichia coli. J. Biol. Chem., 241, 2533-2539 (1966) 510

1.17.4.1

Ribonucleoside-diphosphate reductase

[34] Thelander, L.: Physicochemical characterization of ribonucleoside diphosphate reductase from Escherichia coli. J. Biol. Chem., 248, 4591-4601 (1973) [35] Nordlund, P.; Sjöberg, B.M.; Eklund, H.: Three-dimensional structure of the free radical protein of ribonucleotide reductase. Nature, 345, 593-598 (1990) [36] Nordlund, P.; Uhlin, U.; Westergren, C.; Joelsen, T.; Sjöberg, B.M.; Eklund, H.: New crystal forms of the small subunit of ribonucleotide reductase from Escherichia coli. FEBS Lett., 258, 251-254 (1989) [37] Thelander, M.; Thelander, L.: Molecular cloning and expression of the functional gene encoding the M2 subunit of mouse ribonucleotide reductase: a new dominant marker gene. EMBO J., 8, 2475-2479 (1989) [38] Caras, I.W.; Martin, D.W.: Molecular cloning of the cDNA for a mutant mouse ribonucleotide reductase M1 that produces a dominant mutator phenotype in mammalian cells. Mol. Cell. Biol., 8, 2698-2704 (1988) [39] Ribi, H.O.; Reichard, P.; Kornberg, R.D.: Two-dimensional crystals of enzyme-effector complexes: ribonucleotide reductase at 18-A resolution. Biochemistry, 26, 7974-7979 (1987) [40] Bunker, G.; Petersson, L.; Sjöberg, B.M.; Sahlin, M.; Chance, M.; Chance, B.; Ehrenberg, A.: Extended X-ray absorption fine structure studies on the iron-containing subunit of ribonucleotide reductase from Escherichia coli. Biochemistry, 26, 4708-4716 (1987) [41] Thelander, L.; Berg, P.: Isolation and characterization of expressible cDNA clones encoding the M1 and M2 subunits of mouse ribonucleotide reductase. Mol. Cell. Biol., 6, 3433-3442 (1986) [42] Vitols, E.; Bauer, V.A.; Stanbrough, E.C.: Ribonucleotide reductase from Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun., 41, 71-77 (1970) [43] Hopper, S.: Ribonucleotide reductase of rabbit bone marrow. I. Purification, properties, and separation into two protein fractions. J. Biol. Chem., 247, 3336-3340 (1972) [44] Nikas, I.; McLauchlan, J.; Davison, A.J.; Taylor, W. R.; Clements, J.B.: Structural features of ribonucleotide reductase. Proteins Struct. Funct. Genet., 1, 376-384 (1986) [45] Fitzgerald, G.B.; Rosowsky, A.; Wick, M.M.: Inhibition of ribonucleotide reductase by naturally occurring quinols from spores of Agaricus bisporus. Biochem. Biophys. Res. Commun., 120, 1008-1014 (1984) [46] Engström, Y.; Rozell, B.; Hansson, H.A.; Stemme, S.; Thelander, L.: Localization of ribonucleotide reductase in mammalian cells. EMBO J., 3, 863-867 (1984) [47] Reichard, P.; Ehrenberg, A.: Ribonucleotide reductase - a radical enzyme. Science, 221, 514-519 (1983) [48] Cook, K.S.; Greenberg, G.R.: Properties of Bacteriophage T4 ribonucleoside diphosphate reductase subunits coded by nrdA and nrdB mutants. J. Biol. Chem., 258, 6064-6072 (1983) [49] Stubbe, J.; Ator, M.; Krenitsky, T.: Mechanism of ribonucleoside diphosphate reductase from Escherichia coli. Evidence for 3-C-H bond cleavage. J. Biol. Chem., 258, 1625-1630 (1983)

511

Ribonucleoside-diphosphate reductase

1.17.4.1

[50] Berglund, O.; Sjoberg, B.M.: Effect of hydroxyurea on T4 ribonucleotide reductase. J. Biol. Chem., 254, 253-254 (1979) [51] Atkin, C.L.; Thelander, L.; Reichard, P.; Lang, G.: Iron and free radical in ribonucleotide reductase. Exchange of iron and Mossbauer spectroscopy of the protein B2 subunit of the Escherichia coli enzyme. J. Biol. Chem., 248, 7464-7472 (1973) [52] Sjöberg, B.M.; Gräslund, A.; Loehr, J.S.; Loehr, T. M.: Ribonucleotide reductase: a structural study of the dimeric iron site. Biochem. Biophys. Res. Commun., 94, 793-799 (1980) [53] Petersson, L.; Gräslund, A.; Ehrenberg, A.; Sjöberg, B.M.; Reichard, P.: The iron center in ribonucleotide reductase from Escherichia coli. J. Biol. Chem., 255, 6706-6712 (1980) [54] Kucera, R.; Paulus, H.: Studied on ribonucleoside-diphosphate reductase in permeable animal cells. I. Reversible permeabilization of mouse L cells with dextran sulfate. Arch. Biochem. Biophys., 214, 102-113 (1982) [55] Cory, J.G.; Fleischer, A.E.: The molecular weight of Ehrlich tumor cell ribonucleotide reductase and its subunits: effector-induced changes. Arch. Biochem. Biophys., 217, 546-551 (1982) [56] Youdale, T.; MacManus, J.P.; Whitefield, J.F.: Rat liver ribonucleotide reductase: separation, purification, and properties of two nonidentical subunits. Can. J. Biochem., 60, 463-470 (1982) [57] Gudas, L.; Eriksson, S.; Ullman, B.; Martin, D.: Purification of a mutant ribonucleotide reductase from cultured mouse T-lymphoma cells. Adv. Enzyme Regul., 19, 129-137 (1981) [58] Huszar, S.; Bacchetti, S.: Partial purification and characterization of the ribonucleotide reductase induced by herpes simplex virus infection of mammalian cells. J. Virol., 37, 580-588 (1981) [59] Engström, Y.; Eriksson, S.; Thelander, L.; Akerman, M.: Ribonucleotide reductase from calf thymus. Purification and properties. Biochemistry, 18, 2941-2948 (1979) [60] Hopper, S.: Ribonucleotide reductase of rabbit bone marrow. Methods Enzymol., 51, 237-246 (1978) [61] Feller, W.; Follmann, H.: Ribonucleotide reductase activity in green algae. Biochem. Biophys. Res. Commun., 70, 752-758 (1976) [62] Elford, H.L.: Functional regulation of mammalian ribonucleotide reductase. Adv. Enzyme Regul., 10, 19-38 (1972) [63] Larsson, A.: Ribonucleotide reductase from regenerating rat liver. II. Substrate phosphorylation level and effect of deoxyadenosine triphosphate. Biochim. Biophys. Acta, 324, 447-451 (1973) [64] Vitols, E.; Bauer, V.A.; Stanbrough, E.C.: Ribonucleotide reductase from Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun., 41, 71-77 (1970) [65] Hopper, S.: Ribonucleotide reductase of rabbit bone marrow. I. Purification, properties, and separation into two protein fractions. J. Biol. Chem., 247, 3336-3340 (1972)

512

1.17.4.1

Ribonucleoside-diphosphate reductase

[66] Cory, J.G.; Rey, D.A.; Carter, G.L.; Bacon, P.E.: Nucleoside 5'-diphosphates as effectors of mammalian ribonucleotide reductase. J. Biol. Chem., 260, 12001-12007 (1985) [67] Ong, S.P.; McFarlan, S.C.; Hogenkamp, H.P.C.: 2'-C-Methyladenosine and 2'-C-methyluridine 5'-diphosphates are mechanism-based inhibitors of ribonucleoside diphosphate reductase from Corynebacterium nephridii. Biochemistry, 32, 11397-11404 (1993) [68] Slabaugh, M.B.; Davis, R.E.; Roseman, N.A.; Mathews, C.K.: Vaccinia virus ribonucleotide reductase expression and isolation of the recombinant large subunit. J. Biol. Chem., 268, 17803-17810 (1993) [69] Young, P.; Leeds, J.M.; Slabaugh, M.B.; Mathews, C.K.: Ribonucleotide reductase: evidence for specific association with HeLa cell mitochondria. Biochem. Biophys. Res. Commun., 203, 46-52 (1994) [70] Jordan, A.; Pontis, E.; Atta, M.; Krook, M.; Gibert, I.; Barbe, J.; Reichard, P.: A second class I ribonucleotide reductase in Enterobacteriaceae: characterization of the Salmonella typhimurium enzyme. Proc. Natl. Acad. Sci. USA, 91, 12892-12896 (1994) [71] Sinhababu, A.K.; Boehlert, C.C.; Gan, L.S.; Yanni, S.B.; Thakker, D.R.: Highperformance liquid chromatographic purification, optimization of the assay, and properties of ribonucleoside diphosphate reductase from rabbit bone marrow. Arch. Biochem. Biophys., 317, 285-291 (1995) [72] Cory, J.G.; Cory, A.H.; Downes, D.L.: Differential substrate properties of mammalian ribonucleotide reductase. Purine and pyrimidine metabolism in man, VIII ed. (Sahota A. and Taylor M. ed.), 631-635 (1995) [73] Racay, P.; Kollarova, M.: Purification and partial characterization of Ca2+ dependent ribonucleotide reductase from Streptomyces aureofaciens. Biochem. Mol. Biol. Int., 38, 493-500 (1996) [74] Hendricks, S.P.; Mathews, C.K.: Regulation of T4 phage aerobic ribonucleotide reductase. Simultaneous assay of the four activities. J. Biol. Chem., 272, 2861-2865 (1997) [75] Fieschi, F.; Torrents, E.; Toulokhonova, L.; Jordan, A.; Hellman, U.; Barbe, J.; Gibert, I.; Karlsson, M.; Sjoberg, B.M.: The manganese-containing ribonucleotide reductase of Corynebacterium ammoniagenes is a class Ib enzyme. J. Biol. Chem., 273, 4329-4337 (1998) [76] Tong, W.; Burdi, D.; Riggs-Gelasco, P.; Chen, S.; Edmondson, D.; Huynh, V.; Stubbe, J.; Han, S.; Arvai, A.; Tainer, J.: Characterization of Y122F R2 of Escherichia coli ribonucleotide reductase by time-resolved physical biochemical methods and X-ray crystallography. Biochemistry, 37, 5840-5848 (1998) [77] Hamann, C.S.; Lentainge, S.; Li, L.S.; Salem, J.S.; Yang, F.D.; Cooperman, B.S.: Chimeric small subunit inhibitors of mammalian ribonucleotide reductase: a dual function for the R2 C-terminus?. Protein Eng., 11, 219-224 (1998) [78] Nguyen, H.H.T.; Ge, J.; Perlstein, D.L.; Stubbe, J.: Purification of ribonucleotide reductase subunits Y1, Y2, Y3, and Y4 from yeast: Y4 plays a key role in diiron cluster assembly. Proc. Natl. Acad. Sci. USA, 96, 12339-12344 (1999) 513

Ribonucleoside-diphosphate reductase

1.17.4.1

[79] Rova, U.; Adrait, A.; Potsch, S.; Graslund, A.; Thelander, L.: Evidence by mutagenesis that Tyr370 of the mouse ribonucleotide reductase R2 protein is the connecting link in the intersubunit radical transfer pathway. J. Biol. Chem., 274, 23746-23751 (1999) [80] Roshick, C.; Iliffe-Lee, E.R.; McClarty, G.: Cloning and characterization of ribonucleotide reductase from Chlamydia trachomatis. J. Biol. Chem., 275, 38111-38119 (2000) [81] Reichard, P.; Eliasson, R.; Ingemarson, R.; Thelander, L.: Cross-talk between the allosteric effector-binding sites in mouse ribonucleotide reductase. J. Biol. Chem., 275, 33021-33026 (2000) [82] Smalley, D.; Rocha, E.R.; Smith, C.J.: Aerobic-type ribonucleotide reductase in the anaerobe Bacteroides fragilis. J. Bacteriol., 184, 895-903 (2002) [83] Yun, D.; Krebs, C.; Gupta, G.P.; Iwig, D.F.; Huynh, B.H.; Bollinger, J.M., Jr.: Facile electron transfer during formation of cluster x and kinetic competence of x for tyrosyl radical production in protein R2 of ribonucleotide reductase from mouse. Biochemistry, 41, 981-990 (2002) [84] Kashlan, O.B.; Scott, C.P.; Lear, J.D.; Cooperman, B.S.: A comprehensive model for the allosteric regulation of mammalian ribonucleotide reductase. Functional consequences of ATP- and dATP-induced oligomerization of the large subunit. Biochemistry, 41, 462-474 (2002)

514

   

8

   
 1.17.4.2    2'-deoxyribonucleoside-triphosphate:thioredoxin-disulfide 2'-oxidoreductase    ribonucleoside-triphosphate reductase    2'-deoxyribonucleoside-triphosphate:oxidized-thioredoxin 2'-oxidoreductase RNR [14, 18, 20] RTPR [10, 14-17] ribonucleoside triphosphate reductase ribonucleotide reductase
    9068-66-0

    













Anabaena sp. (strain 7119 [7]) [7, 12] Archaeoglobus fulgidus [11, 19] Astasia sp. (euglenoid flagellate [6]) [6] Brevundimonas diminuta [13] Brevundimonas vesicularis [13] Burkholderia cepacia [13] Chloroflexus aurantiacus (J-10-fl (ATCC 29366) [11]) [11] Clostridium sp. [6] Comamonas acidovorans [13] Corynebacterium ammoniagenes [20] Corynebacterium sp. [6] Deinococcus radiourans (strain R1, ATCC 13939 [11]) [11, 13] Escherichia coli [5, 7, 11-14] Euglena gracilis (strain Z [5]) [5] Euglena sp. (euglenoid flagellate [6]) [6] Halobacterium cutirubrum [12] Haloferax volcanii [12] Homo sapiens (human [12]) [12]

515

Ribonucleoside-triphosphate reductase

1.17.4.2

Hydrogenophaga flava [13] Lactobacillus leichmannii (strain ATCC 7630 [4]; strain ATCC 4797 [6]; mutant of strain ATCC 4797, produced during many years of transfers on litmus milk [1]; Lactobacillus leishmanii [13]) [1-8, 10-17, 19] Methanobacterium thermoautotrophicum [19] Methanococcus jannaschii [11] Mycobacterium tuberculosis [11-13] Paracoccus denitrificans [13] Pseudomonas aeruginosa [13] Pseudomonas putida [13] Pseudomonas sp. [6, 13] Pseudomonas stutzeri [13] Pyrococcus furiosus [11, 19] Ralstonia pickettii [13] Rhizobium sp. [6] Saccharomyces cerevisiae [18] Stenotrophomonas maltophila [13] Streptomyces aureofaciens (industrial strain BMK [9]) [9] Sulfolobus alcidocaldarius [19] Sulfolobus shibatae [19] Sulfolobus solfataricus [19] Thermoplasma acidophila (DSM 1728 [12]; strain MSB8 (DSM3109) [11]; Thermoplasma acidophilum [19]) [11, 12, 19] Thermotoga maritima (strain MSB8 (DSM 3109) [11]) [11, 19] Thermus aquaticus (strain YT-1 [3]; strain X1 [12]) [3, 12] Thermus sp. (X-1 [3]) [3] Xanthomonas campestris [13]

!    "
 # 

2'-deoxyribonucleoside triphosphate + thioredoxin disulfide + H2 O = ribonucleoside triphosphate + thioredoxin (requires a cobamide coenzyme and ATP)    redox reaction       2'-deoxyribonucleoside triphosphate + oxidized thioredoxin + H2 O (Reversibility: r [4]) [4] $ ribonucleoside triphosphate + reduced thioredoxin  ribonucleoside triphosphate + reduced thioredoxin + H2 O ( physiological hydrogen donor is unknown [2]; catalyzes the rate-determining step in DNA biosynthesis [14]; key enzyme in the pathway of DNA biosynthesis in all organisms [9]; hydrogen do-

516

1.17.4.2

Ribonucleoside-triphosphate reductase

nor is very likely thioredoxin or glutaredoxin [19]) (Reversibility: r [1-20]) [1-20] $ 2'-deoxyribonucleoside triphosphate + oxidized thioredoxin + H2 O      2'-deoxyribonucleoside triphosphate + oxidized thioredoxin + H2 O (Reversibility: r [4]) [4] $ ribonucleoside triphosphate + reduced thioredoxin  ADP + reduced thioredoxin ( most active diphosphate substrate [2]) (Reversibility: ? [2, 3, 5]) [2, 3, 5] $ dADP + oxidized thioredoxin + H2 O  ATP + reduced thioredoxin (Reversibility: ? [2, 3, 5, 7, 9, 20]) [2, 3, 5, 7, 9, 20] $ dATP + oxidized thioredoxin + H2 O  CDP + reduced thioredoxin ( poorly reduced [2]) (Reversibility: ? [2, 3, 5, 11, 19]) [2, 3, 5, 11, 19] $ dCDP + oxidized thioredoxin + H2 O  CMP + reduced thioredoxin ( poorly reduced [2]) (Reversibility: ? [2, 3]) [2, 3] $ dCMP + oxidized thioredoxin + H2 O  CTP + reduced thioredoxin (Reversibility: ? [1-3, 5, 7, 9, 11, 20]) [1-3, 5, 7, 9, 11, 20] $ dCTP + oxidized thioredoxin + H2 O  GDP + reduced thioredoxin ( poorly reduced [2]) (Reversibility: ? [2, 3]) [2, 3] $ dGDP + oxidized thioredoxin + H2 O  GTP + reduced thioredoxin (Reversibility: ? [2, 3, 5, 7, 20]) [2, 3, 5, 7, 20] $ dGTP + oxidized thioredoxin + H2 O  IDP + reduced thioredoxin (Reversibility: ? [3]) [3] $ dIDP + oxidized thioredoxin  ITP + reduced thioredoxin (Reversibility: ? [3]) [3] $ dITP + oxidized thioredoxin  UDP + reduced thioredoxin ( poorly reduced [2]) (Reversibility: ? [2, 3]) [2, 3] $ dUDP + oxidized thioredoxin + H2 O  UMP + reduced thioredoxin (Reversibility: ? [3]) [3] $ dUMP + oxidized thioredoxin + H2 O  UTP + reduced thioredoxin (Reversibility: ? [2, 3, 5, 7, 8, 20]) [2, 3, 5, 7, 8, 20] $ dUTP + oxidized thioredoxin + H2 O  ribonucleoside triphosphate + 2-hydroxyethyl disulfide (Reversibility: ? [7]) [7] $ 2'-deoxyribonucleoside triphosphate + 2-mercaptoethanol + H2 O  ribonucleoside triphosphate + GSSG (Reversibility: ? [7]) [7] $ 2'-deoxyribonucleoside triphosphate + GSH + H2 O

517

Ribonucleoside-triphosphate reductase

1.17.4.2

 ribonucleoside triphosphate + NADP+ (Reversibility: ? [7]) [7] $ 2'-deoxyribonucleoside triphosphate + NADPH + H2 O  ribonucleoside triphosphate + R(SH)2 (Reversibility: ? [6]) [6] $ deoxyribonucleoside triphosphate + RS2 + H2 O  ribonucleoside triphosphate + dihydrolipoate (Reversibility: ? [6, 7]) [6, 7] $ 2'-deoxyribonucleoside triphosphate + lipoate + H2 O  ribonucleoside triphosphate + dithioerythritol (Reversibility: ? [6, 7, 9]) [6, 7, 9] $ 2'-deoxyribonucleoside triphosphate + ? + H2 O  ribonucleoside triphosphate + dithiothreitol (Reversibility: ? [6, 7, 19]) [6, 7, 19] $ 2'-deoxyribonucleoside triphosphate + ? + H2 O  ribonucleoside triphosphate + reduced thioredoxin (Reversibility: r [1-20]) [1-20] $ 2'-deoxyribonucleoside triphosphate + oxidized thioredoxin + H2 O  Additional information ( GMP and AMP are no substrates [3]) [3] $ ? 2  2'-deoxyadenosylcobalamin [6] 3-isoadenosylcobalamin [6] K+ [7] l-adenosylcobalamin [6] Mg2+ ( strongly inhibitory in absence of ATP [2]) [2] Na+ [7] [3-(adenosin-5'-O-yl)propyl]cobalamin (C3 ) [16] [4-(adenosin-5'-O-yl)butyl]cobalamin (C4 ) [16] [5-(adenosin-5'-O-yl)pentyl]cobalamin (C5 ) [16] [6-(adenosin-5'-O-yl)hexyl]cobalamin (C6 ) [16] [7-(adenosin-5'-O-yl)heptyl]cobalamin (C7 ) [16] [w-(adenosin-5'-O-yl)alkyl]cobalamin [16] adeninylpentylcobalamin [16] cob(II)alamin [16] cyanocobalamin [16] dGTP ( inhibits CDP reduction [11]) [11] dTTP ( inhibits ATP reduction [2]; inhibits CTP, ATP and GTP reduction, reduction of UTP is unaffected by deoxyribonucleotides [5]; inhibits CDP reduction [11]) [2, 5, 11] isopropylideneadenosylcobalamin [6] nebularylcobalamin [6] tubercidylcobalamin [6]
"%  5,6-dimethylbenzimidazolycobamide [2] Coa-(aden-9-yl)-Cob-adenosylcobamide [6] 518

1.17.4.2

Ribonucleoside-triphosphate reductase

Coa-(benzimidazolyl)-Cob-adenosylcobamide [6] NADPH ( can utilize NADPH as hydrogen donor for ribonucleotide reduction [5]) [5] coenzyme B12 ( adenosylcobalamin [3, 6-8, 10, 11, 13-17, 19]) [2-4, 6-17, 19] dihydrolipoate [2] &

  2',5'-dideoxyadenosylcobalamin anilide [1] 2-mercaptoethanol [1] 5'-deoxy-5'-adenosylcobalamin [3] 5'-deoxyadenosylcobalamin [1, 5, 12, 16, 17] 5'-deoxyadenosylcobalamin anilide [1] 5'-deoxyuridylcobalamin [1] ATP ( stimulates CTP reduction [5]; stimulates CDP reduction [11]) [5, 11] NADPH ( stimulates reductase activity in crude extracts [2]) [2] cobinamide [1] cyanocobalamin [1] cyanocobalamin anilide [1] cyanocobalamin dibasic acid [1] cyanocobalamin ethylamide [1] cyanocobalamin monobasic acid [1] dATP ( specific activator for CTP reduction [5-7, 9]) [5-7, 9, 11] dATP ( stimulation of CTP reduction [4]) [2, 4] dCTP ( specific activator for UTP reduction [6]) [5, 6] dCTP ( stimulation of UTP reduction [4]) [4] dGTP ( specific activator for ATP reduction [2,6,7,9]) [2, 5-7, 9] dGTP ( stimulation of ATP reduction [4]) [4] dTTP ( specific activator for ITP reduction [6]; specific activator for GTP reduction [7]) [5-7] dTTP ( stimulation of GTP reduction [4]) [4] ethyl cobalamin [1] hydroxocobalamin [1] hydroxocobalamin methylamide [1] methyl cobalamin [1] '(   Ca2+ ( markedly stimulates activity [9]; absolutely dependent on [7]) [7, 9, 11] Cs+ ( activating effect [6]) [6] K+ ( activating effect [6]) [6] Li+ ( activating effect [6]) [6] Mg2+ ( markedly stimulates activity [9]; can stimulate the reaction [5]; stimulates enzyme activity [3]; stabilized by [7]) [3, 5, 7, 9, 11] 519

Ribonucleoside-triphosphate reductase

1.17.4.2

Mn2+ ( stimulates enzyme activity [3]) [3] NH+4 ( activating effect [6]) [6] Na+ ( activating effect [6]) [6] Rb+ ( activating effect [6]) [6] ) &  * +, 0.22 (CTP) [17] 0.87 (ATP) [17] 0.95 (UTP) [17] 1.1 (CTP) [17] 1.4 (CTP) [17] 1.8 (CTP) [17] " & *-% , 0.0018 [12] 0.167 [5] 0.3 [19] 0.51 [19] 1.1 [19] 2.82 [6] 16 [4] 77.25 [7] 576 [2] . / *', 0.0003 (adenosylcobalamin, presence of 1.0 mM CTP and 1 mM dATP [6]) [6] 0.00045 (adenosylcobalamin, presence of 0.1 mM CTP and 1 mM dATP [6]) [6] 0.001 (adenosylcobalamin) [19] 0.0055 (adenosylcobalamin, presence of 2 mM CTP without activator [6]) [6] 0.01 (CTP, immobilized enzyme [9]) [9] 0.01 (UTP, immobilized enzyme [9]) [9] 0.015 (ATP, immobilized enzyme [9]) [9] 0.017 (dGTP) [14] 0.02 (CTP, presence of 0.05 mM dATP [7]) [7] 0.06 (adenosylcobalamin) [14] 0.064 (NDP) [19] 0.07 (ATP, in presence of effector dGTP [9]) [9] 0.07 (CDP) [19] 0.08 (ADP, in presence of effector dGTP [9]) [9] 0.085 (CTP, presence of effector dATP [9]) [9] 0.09 (CTP, in presence of dATP [5]) [5] 0.09 (GTP, immobilized enzyme [9]) [9] 0.1 (adenosylcobalamin) [19] 0.11 (CDP, presence of effector dATP [9]) [9] 0.13 (CTP, presence of 1 mM dATP [6]) [6]

520

1.17.4.2

Ribonucleoside-triphosphate reductase

0.15 (CTP, in presence of dTTP [5]) [5] 0.22 (ATP, presence of 1 mM dGTP [6]) [6] 0.25 (ATP, in presence of dTTP [5]) [5] 0.29 (GTP, at 37 C [3]) [3] 0.32 (CTP, at 37 C [3]) [3] 0.44 (ATP) [5] 0.99 (GTP, at 70 C [3]) [3] 1 (ATP, at 70 C [3]) [3] 1.4 (CTP, at 70 C [3]) [3] 1.7 (CTP) [2] 2.3 (ATP) [2] 9.5 (CTP, without ATP [2]) [2] 20 (dithiothreitol) [19] . / *', 0.0013 0.0077 0.0128 0.0143 0.0189 0.0208 0.0246 0.0426 0.0558

(adeninylpentylcobalamin) [16] ([5-(adenosin-5'-O-yl)pentyl]cobalamin (C5 )) [16] ([7-(adenosin-5'-O-yl)heptyl]cobalamin (C7 )) [16] (cob(II)alamin, + adenosine [16]) [16] ([4-(adenosin-5'-O-yl)butyl]cobalamin (C4 )) [16] (cob(II)alamin) [16] ([6-(adenosin-5'-O-yl)hexyl]cobalamin (C6 )) [16] (cyanocobalamin) [16] ([3-(adenosin-5'-O-yl)propyl]cobalamin (C3 )) [16]

0  7.8-8.4 [3] 8 [7] 8-8.5 [9] 0 

 6.4-9.6 [9] )  *
, 30 [9] 50 [3] 55 [12] 70 [3] 80 [19] 80-90 [11] ) 

 *
, 20-60 [3] 20-80 [3]

521

Ribonucleoside-triphosphate reductase

1.17.4.2

  #   ' 1  69300 ( enzyme in 6 M guanidine-HCl, 0.1 M dithiothreitol, equilibrium ultracentrifugation [4]) [4] 72000 ( gel filtration [7]) [7] 72500 ( native enzyme in 0.1 M glycine buffer, pH 9.1, equilibrium ultracentrifugation [4]) [4] 72600 ( maleated enzyme in 0.05 M phosphate buffer, equilibrium ultracentrifugation [4]) [4] 74500 ( enzyme in 6 M guanidine-HCl, 0.1 M dithiothreitol, heated for 20 min at 60 C, equilibrium ultracentrifugation [4]) [4] 75300 ( native enzyme in 0.05 M phosphate buffer, pH 7.0, sedimentation equilibrium method [4]) [4] 76000 ( gel filtration [4]) [4, 6, 7] 78400 ( SDS-PAGE [4]) [4] 80000 ( gel filtration [3]) [3] 81800 ( MALDI-TOF mass spectrometry [17]) [17] 81850 ( predicted molecular weight based on the predicted amino acid sequence from the sequenced gene [17]) [17] 82000 [10] 90000 ( gel filtraton [11]; gel filtration [19]) [11, 19] 100000 ( size exclusion chromatography, SDS-PAGE [12]) [12] 107000 ( deduced amino acid sequence [11]) [11] 110000 ( sedimentation equilibrium analysis [2]) [2] 140000-150000 [5] 200000 ( deduced protein sequence [19]) [19] 240000 [7] 240000-278000 [5] 440000 ( gel filtration [5]) [5]   dimer [7] monomer ( 1 * 78400, SDS-PAGE [4]; 1 * 50000, SDS-PAGE [7]) [3, 4, 6, 7, 9, 10, 19] tetramer ( 4 * 100000, SDS-PAGE [5]) [5]

 2 %$ %' %

% mycelium [9] $"

[7] [11] [5] [5] 522

1.17.4.2







Ribonucleoside-triphosphate reductase

(from overexpressing Escherichia coli cells [16]) [2-6, 16, 17] [19] [18] [19] [19] [12, 19] [11, 19] (partially [3]) [3]


 #

[17]
 

(cloned by PCR from peptide sequence information [11]) [11] (cloning, sequencing and expression of the protein [10]; overexpressed in Escherichia coli [16,17]) [10, 16, 17] (sequence analysis, RNR gene discovered [19]) [19] (chromosomal DNA containg RNR gene PCR amplified, gene cloned, completely sequenced and expressed in Escherichia coli [12]) [12] (cloned by PCR from peptide sequence information, nrdj gene sequenced completely and expressed in Escherichia coli BL21(DE3) [11]) [11]

4    , activity is not air-sensitive [19] 5    "

, fairly stable under conditions of storage, losing only about 23% of its activity in 15 months [6] , immobilization stabilizes the enzyme substantially, compared with free enzyme, which has an inactivation half-life of less than 1 day, on immobilization the inactivation half-life is about 15 days [9] , extreme stability [19]    , -100 C, may be stored as frozen paste for at least 3 months without significant loss of activity [6] , -20 C, crude extracts relatively unstable, most of the activity disappears within a few weeks [2] , -65 C, no detectable loss of enzyme has been observed during several years of storage of packed bacteria [4] , 0-4 C, acetone and hydroxylapatite fractions are quite stable, at least 70-80% of their activities remain after 3 months [2] , 0-4 C, crude extracts relatively unstable, most of the activity disappears within a few weeks [2] , 20 C, 0.03 M dimethylglutarate buffer, pH 7.2, remains fully active on storage for 24 h [3] 523

Ribonucleoside-triphosphate reductase

1.17.4.2

"  [1] Bakley, R.L.: Cobamides and ribonucleotide reduction. I. Cobamide stimulation of ribonucleotide reduction in extracts of Lactobacillus leichmannii. J. Biol. Chem., 240, 2173-2180 (1965) [2] Goulian, M.; Beck, W.S.: Purification and properties of cobamide-dependent ribonucleotide reductase from Lactobacillus leichmannii. J. Biol. Chem., 241, 4233-4242 (1966) [3] Sando, G.N.; Hogenkamp, H.P.C.: Ribonucleotide reductase from Thermus X-1, a thermophilic organism. Biochemistry, 12, 3316-3322 (1973) [4] Chen, A.K.; Bhan, A.; Hopper, S.; Abrams, R.; Franzen, J.S.: Substrate and effector binding to ribonucleoside triphosphate reductase of Lactobacillus leichmannii. Biochemistry, 13, 654-661 (1974) [5] Hamilton, F.D.: Ribonucleotide reductase from Euglena gracilis. A 5-deoxyadenoslycobalamin-dependent enzyme. J. Biol. Chem., 249, 4428-4434 (1974) [6] Blakley, R.L.: Ribonucleoside triphosphate reductase from Lactobacillus leichmannii. Methods Enzymol., 51, 246-259 (1978) [7] Gleason, F.K.; Frick, T.D.: Adenosylcobalamin-dependent ribonucleotide reductase from the blue-green alga, Anabaena sp. Purification and partial characterization. J. Biol. Chem., 255, 7728-7733 (1980) [8] Ashley, G.W.; Harris, G.; Stubbe, J.: The mechanism of Lactobacillus leichmannii ribonucleotide reductase. Evidence for 3 carbon-hydrogen bond cleavage and a unique role for coenzyme B12 . J. Biol. Chem., 261, 39583964 (1986) [9] Halicky, P.; Kollarova, M.; Kois, P.; Zelinka, J.: Immobilization of ribonucleotide reductase from Streptomyces aureofaciens. Collect. Czech. Chem. Commun., 54, 2528-2541 (1989) [10] Booker, S.; Licht, S.; Broderick, J.; Stubbe, J.: Coenzyme B12 -dependent ribonucleotide reductase: evidence for the participation of five cysteine residues in ribonucleotide reduction. Biochemistry, 33, 12676-12685 (1994) [11] Jordan, A.; Torrents, E.; Jeanthon, C.; Eliasson, R.; Hellman, U.; Wernstedt, C.; Barbe, J.; Gibert, I.; Reichard, P.: B12 -dependent ribonucleotide reductases from deeply rooted eubacteria are structurally related to the aerobic enzyme from Escherichia coli. Proc. Natl. Acad. Sci. USA, 94, 13487-13492 (1997) [12] Tauer, A.; Benner, S.A.: The B12 -dependent ribonucleotide reductase from the archaebacterium Thermoplasma acidophila: an evolutionary solution to the ribonucleotide reductase conundrum. Proc. Natl. Acad. Sci. USA, 94, 53-58 (1997) [13] Jordan, A.; Torrents, E.; Sala, I.; Hellman, U.; Gibert, I.; Reichard, P.: Ribonucleotide reduction in Pseudomonas species: simultaneous presence of active enzymes from different classes. J. Bacteriol., 181, 3974-3980 (1999) [14] Licht, S.S.; Booker, S.; Stubbe, J.: Studies on the catalysis of carbon-cobalt bond homolysis by ribonucleoside triphosphate reductase: evidence for

524

1.17.4.2

[15]

[16]

[17]

[18] [19] [20]

Ribonucleoside-triphosphate reductase

concerted carbon-cobalt bond homolysis and thiyl radical formation. Biochemistry, 38, 1221-1233 (1999) Licht, S.S.; Lawrence, C.C.; Stubbe, J.: Thermodynamic and kinetic studies on carbon-cobalt bond homolysis by ribonucleoside triphosphate reductase: The importance of entropy in catalysis. Biochemistry, 38, 1234-1242 (1999) Suto, R.K.; Poppe, L.; Retey, J.; Finke, R.G.: Ribonucleoside triphosphate reductase from Lactobacillus leichmannii: Kinetic evaluation of a series of adenosylcobalamin competitive inhibitors, [w-(adenosin-5'-O-yl)alkyl]cobalamins, which mimic the post Co-C homolysis intermediate. Bioorg. Chem., 27, 451-462 (1999) Suto, R.K.; Whalen, M.A.; Finke, R.G.: Adenosylcobalamin-dependent ribonucleoside triphosphate reductase from Lactobacillus leichmannii. Rapid, improved purification involving dGTP-based affinity chromatography plus biophysical characterization studies demonstrating enhanced, crystallographic level purity. Prep. Biochem. Biotechnol., 29, 273-309 (1999) Chabes, A.; Domkin, V.; Larsson, G.; Liu, A.; Graslund, A.; Wijmenga, S.; Thelander, L.: Yeast ribonucleotide reductase has a heterodimeric iron-radical-containing subunit. Proc. Natl. Acad. Sci. USA, 97, 2474-2479 (2000) Fontecave, M.: Ribonucleotide reductase from Pyrococcus furiosus. Methods Enzymol., 334, 215-227 (2001) Hoegbom, M.; Huque, Y.; Sjoeberg, B.M.; Nordlund, P.: Crystal structure of the di-iron/radical protein of ribonucleotide reductase from Corynebacterium ammoniagenes. Biochemistry, 41, 1381-1389 (2002)

525

 0  !         

8!

   
 1.17.4.3    (E)-4-hydroxy-3-methylbut-2-en-1-yl-diphosphate:protein-disulfide oxidoreductase (hydrating)    4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase
    398144-56-4

!    "
 # 

(E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate + H2 O + protein-disulfide = 2-C-methyl-d-erythritol 2,4-cyclodiphosphate + protein-dithiol (Forms, in the reverse direction, part of an alternative, nonmevalonate pathway for terpenoid biosynthesis)

"  [1] Hecht, S.; Eisenreich, W.; Adam, P.; Amslinger, S.; Kis, K.; Bacher, A.; Arigoni, D.: Studies on the nonmevalonate pathway to terpenes: the role of the GcpE (IspG) protein. Proc. Natl. Acad. Sci. USA, 98, 14837-14842 (2001)

526


     *  

,

8::

   
 1.17.99.1    4-cresol:acceptor oxidoreductase (methyl-hydroxylating)    4-cresol dehydrogenase (hydroxylating)    PCMH p-cresol methylhydroxylase p-cresol methylhydroxylase A (, 2 enzyme forms with various MW and Km are formed during growth on 3,5-xylenol - hydroxylase A - and 4cresol - hydroxylase B [17]) [17] p-cresol methylhydroxylase B (, 2 enzyme forms with various MW and Km are formed during growth on 3,5-xylenol - formation of hydroxylase A - and on 4-cresol - formation of hydroxylase B [17]) [17] p-cresol-(acceptor) oxidoreductase (hydroxylating)
    66772-07-4

     Pseudomonas putida (2 enzyme forms with various MW and Km are formed during growth on 3,5-xylenol - hydroxylase A - and 4-cresol - hydroxylase B [17]; strain NCIB 9866 [5, 13]; strain NCIB 9869 [5, 6, 13, 16, 17]) [1-17, 19, 20, 21, 22, 23] denitrifying bacterium (PC-07 [18]) [18] Pseudomonas alcaligenes (strain NCIB 9867 [5]) [5] Pseudomonas testosteroni (strain NCIB 8955 [5]) [5]

!    "
 # 

4-cresol + acceptor + H2 O = 4-hydroxybenzaldehyde + reduced acceptor (, mechanism [16])

527

4-Cresol dehydrogenase (hydroxylating)

1.17.99.1

   oxidation redox reaction reduction       4-cresol + acceptor + H2 O (, degradation of the toxic p-cresol [19]) [19] $ 4-hydroxybenzaldehyde + reduced acceptor  4-cresol + azurin + H2 O (, azurin is the physiological acceptor [6,8]) (Reversibility: ? [6, 8]) [6, 8] $ 4-hydroxybenzaldehyde + reduced azurin  4-cresol + nitrate + H2 O (, nitrate is the natural terminal electron acceptor [18]) [18] $ 4-hydroxybenzaldehyde + ?  Additional information (, hydroxylase A is plasmid encoded and is produced constitutively, as long as the plasmid is maintained by growth on 3,5-xylenol. Hydroxylase B is induced by growth on 4-cresol [1]) [1] $ ?      (RS)-1-(4-hydroxyphenyl)ethanol + acceptor + H2 O (Reversibility: ? [11]) [11] $ ?  1,4-hydroxyquinone + acceptor + H2 O (Reversibility: ? [11]) [11] $ ?  2,4-xylenol + acceptor + H2 O (i.e. 2,4-dimethylphenol) (Reversibility: ? [1, 11, 17, 18]) [1, 11, 17, 18] $ 3-methyl-4-hydroxybenzaldehyde + reduced acceptor  2-bromo-4-methylphenol + acceptor + H2 O (Reversibility: ? [11]) [11] $ 3-bromo-4-hydroxybenzaldehyde + reduced acceptor  2-methoxy-4-methylphenol + acceptor + H2 O (Reversibility: ? [11]) [11] $ 3-methoxy-4-hydroxybenzaldehyde + reduced acceptor  3,4-dimethylphenol + acceptor + H2 O (Reversibility: ? [1, 11, 17, 18]) [1, 11, 17, 18] $ 2-methyl-4-hydroxybenzaldehyde + reduced acceptor  3-fluoro-4-methylphenol + acceptor + H2 O (Reversibility: ? [11]) [11] $ 2-fluoro-4-hydroxybenzaldehyde + reduced acceptor  4-cresol + acceptor + H2 O (, acceptor: phenazine methosulfate [1, 11]; , acceptor: nitrate [18]; , acceptor: 2,6-dichlorophenol-indophenol [1, 18]; , no activity with 2,6-dichlorophenol-indophenol as acceptor [11]; , acceptor: azurin [6, 8]; , no activity with O2, K3 Fe(CN)6 or methylene blue [11]; , 4-hydroxybenzyl alcohol is an enzyme-free intermediate [2]; , the enzyme catalyzes both 4-cre528

1.17.99.1

$  $  $  $  $  $  $  $ 

$

4-Cresol dehydrogenase (hydroxylating)

sol hydroxylation and further oxidation of the product, 4-hydroxybenzyl alcohol to 4-hydroxybenzaldehyde [1-3, 11, 19, 22]) (Reversibility: ? [1-23]) [1-23] 4-hydroxybenzaldehyde + reduced acceptor [1-23] 4-ethylphenol + acceptor + H2 O (, stereochemistry [7]) (Reversibility: ? [1, 7, 11, 16, 17, 18]) [1, 7, 11, 16, 17, 18] 1-(4'-hydroxyphenyl)ethanol + reduced acceptor (, 65-70% of the (S)-(-)-isomer [7]) [7] 4-hydroxybenzyl alcohol + acceptor + H2 O (Reversibility: ? [1, 2, 11, 17, 18, 19]) [1, 2, 11, 17, 18, 19] 4-hydroxybenzaldehyde + reduced acceptor 4-isopropylphenol + acceptor + H2 O (Reversibility: ? [11]) [11] ? 4-methyl-1-naphthol + acceptor + H2 O (Reversibility: ? [11]) [11] ? 4-methyl-3-nitrophenol + acceptor + H2 O (Reversibility: ? [11]) [11] 2-nitro-4-hydroxybenzaldehyde + reduced acceptor 4-methylcatechol + acceptor + H2 O (Reversibility: ? [11, 18]) [11, 18] 3,4-dihydroxybenzaldehyde + reduced acceptor 4-n-propylphenol + acceptor + H2 O (Reversibility: ? [11]) [11] ? Additional information (, no activity with 4-methyl-2-nitrophenol, 2,4,5-trimethylphenol, 2-chloro-4,5-dimethylphenol, 2,4,6-trimethylphenol, 2,6-dibromo-4-methylphenol, l-tyrosine, 4-hydroxybenzaldehyde 3-cresol [11]; , no activity with 2-cresol [11, 17]; , no activity with 2,3-xylenol and 2,5-xylenol [17]; , enzyme requires an alkyl-substituted ring with a hydroxyl group in the para position [18]) [11, 17, 18] ?


"%  8a-O-tyrosyl-FAD (, the covalently bound flavin is 8a-O-tyrosylFAD [3, 4]) [3, 4] FAD (, wild type enzyme and recombinant enzyme contains covalently bound FAD [13]; , a flavocytochrome c [1, 14, 18,19]; , enzyme has 2 subunits, one is a cytochrome c and the other a flavoprotein [1]; , enzyme contains covalently bound flavin and a cytochrome c [3]; , electron-transfer properties of the flavin and heme components [9]; , FAD is covalently attached to Tyr384 of the a subunit. Covalent flavinylation occurs by a self-catalytic mechanism. The cytochrome subunit is necessary for covalent FAD attachment to the flavoprotein subunit [21]; , FAD is covalently attached to Tyr384. The mutant Y384F of the flavoprotein subunit displays stoichiometric noncovalent FAD binding [22]) [1, 3, 9, 13, 14, 18, 19, 21, 22]

529

4-Cresol dehydrogenase (hydroxylating)

1.17.99.1

cytochrome c (, a flavocytochrome c [1, 14, 18,19]; , enzyme has 2 subunits, one is a cytochrome c and the other a flavoprotein [1]; , enzyme contains covalently bound flavin and a type c cytochrome [3, 11]; , electron-transfer properties of the flavin and heme components [9]; , redox potential of cytochrome c [5]; , the cytochrome subunit is necessary for covalent FAD attachment to the flavoprotein subunit [21]) [1, 3, 5, 9, 11, 14, 18, 19, 21] heme (, wild type enzyme and recombinant enzyme contains covalently bound heme [13]) [13] '(   copper (, contains one copper atom per molecule [6]) [6] ) &  * +, Additional information (, steady-state and stopped-flow kinetic measurements of the primary deuterium isotope effect [16]) [16, 21] " & *-% , 0.432 [18] 6.78 (, hydroxylase B [17]) [17] 7.52 [1] 9.95 (, hydroxylase A [17]) [17] 18.03 [12] Additional information [17, 18] . / *', 0.0036 (4-cresol, , hydroxylase B [17]) [17] 0.0073 (4-cresol) [1] 0.015 (4-cresol, , hydroxylase B, 4-hydroxybenzyl alcohol [17]) [17] 0.016 (4-cresol, , hydroxylase A [17]) [17] 0.0173 (2,4-xylenol) [1] 0.02 (2,4-dimethyl phenol) [18] 0.0264 (phenazine methosulfate, , reaction with 4-n-propylphenol [11]) [11] 0.027 (4-hydroxybenzyl alcohol, , hydroxylase A [17]) [17] 0.0476 (4-hydroxybenzyl alcohol) [1] 0.072 (3,4-xylenol) [1] 0.11 (4-cresol) [18] 0.17 (4-hydroxybenzyl alcohol) [18] 0.26 (4-methylcatechol) [18] 0.27 (3,4-dimethyl phenol) [18] 0.36 (4-ethylphenol) [1] 0.366 (phenazine methosulfate, , reaction with 2-bromo-4-methylphenol [11]) [11] 1.94 (phenazine methosulfate, , reaction with 4-ethylphenol [11]) [11] 2.17 (4-ethylphenol) [18] 3.26 (phenazine methosulfate, , reaction with 4-hydroxybenzyl alcohol [11]) [11] 530

1.17.99.1

4-Cresol dehydrogenase (hydroxylating)

5.23 (phenazine methosulfate, , reaction with 2,4-dimethylphenol [11]) [11] 6.76 (phenazine methosulfate, , reaction with 4-cresol [11]) [11] Additional information (, effect of pH-value and ionic strength on KM -value [11]) [11, 21] 0  8.3 [11]

  #   ' 1  14000 (, equilibrium sedimentation [6]) [6] 99000 (, hydroxylase B, equilibrium sedimentation [17]) [17] 100000 (, hydroxylase B, gel filtration [17]) [17] 108000 (, hydroxylase A, gel filtration [17]) [17] 114000 (, hydroxylase A, equilibrium sedimentation [17]) [17] 115000 (, sedimentation equilibrium ultracentrifugation [1]) [1]   dimer (, 2 * 56000, one subunit is a cytochrome c and the other a flavoprotein, SDS-PAGE after incubation with SDS and mercaptoethanol [1]; , 2 * 58000, hydroxylase A, SDS-PAGE after incubation with mercaptoethanol [17]; , 2 * 56000, hydroxylase B, SDS-PAGE after incubation with mercaptoethanol [17]) [1, 17] tetramer (, a2 b2 , 2 * 8500 + 2 * 49000, 95000 Da cytochrome subunit and 49000 flavoprotein subunit, amino acid analysis [14]; , a2 b2 , 2 * 8780 + 2 * 48600, 8780 Da cytochrome subunit and 48600 flavoprotein subunit, amino acid analysis [15]) [14, 15] Additional information (, the subunit dissociation is strongly dependent on ionic strength in the oxidized form of the enzyme but not in the reduced form [12]) [12]

 2 %$ %' %

3#

cytoplasm (, low activity [8]) [8] periplasm (, highest activity [8]; , the 9200 Da c-type cytochrome subunit from Pseudomonas putida NCIMB 9869, overexpressed in recombinant form in Pseudomonas aeruginosa PAO1-LAC [23]) [8, 23] $"

(recombinant enzyme from Escherichia coli [13]; the 9200 Da c-type cytochrome subunit from Pseudomonas putida NCIMB 9869, overexpressed in recombinant form in Pseudomonas aeruginosa PAO1-LAC [23]) [1, 12, 13, 17, 20, 23] (partial [18]) [18]

531

4-Cresol dehydrogenase (hydroxylating)

1.17.99.1


 #

(crystals prepared by free interface diffusion method in 8% polyethylene glycol 8000 [10]; X-ray crystal structure at 2.5 resolution [19]) [10, 19]
 

(expression in Escherichia coli JM109 and Pseudomonas putida RA4007 [13]; overexpression of the flavoprotein in Escherichia coli [20]; the 9200 Da c-type cytochrome subunit from Pseudomonas putida NCIMB 9869 is overexpressed in recombinant form in Pseudomonas aeruginosa PAO1-LAC. Efforts to produce the cytochrome in Escherichia coli using a pET vector, with or without ist signal peptide, are unsuccessful, yielding relatively low levels of the protein [23]) [13, 20, 23] 

 

apo-PchF[Y384F] (, the mutant Y384F of the flavoprotein subunit displays stoichiometric noncovalent FAD binding. The mutant flavoprotein subunit associates with the cytochrome subunit, although not as avidly as the wild-type flavoprotein subunit containing covalently bound FAD [22]) [22]

4     , -20 C, stable for a few days [1]

"  [1] Hopper, D.J.; Taylor, D.G.: The purification and properties of p-cresol-(acceptor) oxidoreductase (hydroxylating), a flavocytochrome from Pseudomonas putida. Biochem. J., 167, 155-162 (1977) [2] Hopper, D.J.: Incorporation of [18O]water in the formation of p-hydroxybenzyl alcohol by the p-cresol methylhydroxylase from Pseudomonas putida. Biochem. J., 175, 345-347 (1978) [3] McIntire, W.; Edmondson, D.E.; Singer, T.P.; Hopper, D.J.: 8 a-O-TyrosylFAD: a new form of covalently bound flavin from p-cresol methylhydroxylase. J. Biol. Chem., 255, 6553-6555 (1980) [4] McIntire, W.; Edmondson, D.E.; Hopper, D.J.; Singer, T.P.: 8 a-(O-Tyrosyl)flavin adenine dinucleotide, the prosthetic group of bacterial p-cresol methylhydroxylase. Biochemistry, 20, 3068-3075 (1981) [5] Hopper, D.J.: Redox potential of the cytochrome c in the flavocytochrome p-cresol methylhydroxylase. FEBS Lett., 161, 100-102 (1983) [6] Causer, M.J.; Hopper, D.J.; McIntire, W.S.; Singer, T. P.: Azurin from Pseudomonas putida: an electron acceptor for p-cresol methylhydroxylase. Biochem. Soc. Trans., 12, 1131-1132 (1984) [7] McIntire, W.; Hopper, D.J.; Craig, J.C.; Everhart, E. T.; Webster, R.V.; Causer, M.J.; Singer, T.P.: Stereochemistry of 1-(4-hydroxyphenyl)ethanol produced 532

1.17.99.1

[8] [9] [10] [11] [12] [13]

[14]

[15] [16]

[17] [18] [19]

[20] [21]

4-Cresol dehydrogenase (hydroxylating)

by hydroxylation of 4-ethylphenol by p-cresol methylhydroxylase. Biochem. J., 224, 617-621 (1984) Hopper, D.J.; Jones, M.R.; Causer, M.J.: Periplasmic location of p-cresol methylhydroxylase in Pseudomonas putida. FEBS Lett., 182, 485-488 (1985) Bhattacharyya, A.; Tollin, G.; McIntire, W.; Singer, T.P.: Laser-flash-photolysis studies of p-cresol methylhydroxylase. Electron-transfer properties of the flavin and haem components. Biochem. J., 228, 337-345 (1985) Shamala, N.; Lim, L.W.; Mathews, F.S.: Preliminary X-ray study of p-cresol methylhydroxylase (flavocytochrome c) from Pseudomonas putida N.C.I.B. 9869. J. Mol. Biol., 183, 517-518 (1985) McIntire, W.; Hopper, D.J.; Singer, T.P.: p-Cresol methylhydroxylase. assay and general properties. Biochem. J., 228, 325-335 (1985) Koerber, S.C.; McIntire, W.; Bohmont, C.; Singer, T. P.: Resolution of the flavocytochrome p-cresol methylhydroxylase into subunits and reconstitution of the enzyme. Biochemistry, 24, 5276-5280 (1985) Kim, J.; Fuller, J.H.; Cecchini, G.; McIntire, W.S.: Cloning, sequencing, and expression of the structural genes for the cytochrome and flavoprotein subunits of p-cresol methylhydroxylase from two strains of Pseudomonas putida. J. Bacteriol., 176, 6349-6361 (1994) Shamala, N.; Lim, L.W.; Mathews, F.S.; McIntire, W.; Singer, T.P.; Hopper, D.J.: Structure of an intermolecular electron-transfer complex: p-cresol methylhydroxylase at 6.0-A resolution. Proc. Natl. Acad. Sci. USA, 83, 4626-4630 (1986) McIntire, W.; Singer, T.P.; Smith, A.J.; Mathews, F.S.: Amino acid and sequence analysis of the cytochrome and flavoprotein subunits of p-cresol methylhydroxylase. Biochemistry, 25, 5975-5981 (1986) McIntire, W.S.; Hopper, D.J.; Singer, T.P.: Steady-state and stopped-flow kinetic measurements of the primary deuterium isotope effect in the reaction catalyzed by p-cresol methylhydroxylase. Biochemistry, 26, 4107-4117 (1987) Keat, M.J.; Hopper, D.J.: p-Cresol and 3,5-xylenol methylhydroxylases in Pseudomonas putida N.C.I.B. 9896. Biochem. J., 175, 649-658 (1978) Bossert, I.D.; Whited, G.; Gibson, D.T.; Young, L.Y.: Anaerobic oxidation of p-cresol mediated by a partially purified methylhydroxylase from a denitrifying bacterium. J. Bacteriol., 171, 2956-2962 (1989) Cunane, L.M.; Chen, Z.W.; Shamala, N.; Mathews, F.S.; Cronin, C.N.; McIntire, W.S.: Structures of the flavocytochrome p-cresol methylhydroxylase and its enzyme-substrate complex: Gated substrate entry and proton relays support the proposed catalytic mechanism. J. Mol. Biol., 295, 357-374 (2000) Engst, S.; Kuusk, V.; Efimov, I.; Cronin, C.N.; McIntire, W.S.: Properties of p-cresol methylhydroxylase flavoprotein overproduced by Escherichia coli. Biochemistry, 38, 16620-16628 (1999) Kim, J.; Fuller, J.H.; Kuusk, V.; Cunane, L.; Chen, Z.; Mathews, F.S.; McIntire, W.S.: The cytochrome subunit is necessary for covalent FAD attachment to the flavoprotein subunit of p-cresol methylhydroxylase. J. Biol. Chem., 270, 31202-31209 (1995) 533

4-Cresol dehydrogenase (hydroxylating)

1.17.99.1

[22] Efimov, I.; Cronin, C.N.; McIntire, W.S.: Effects of noncovalent and covalent FAD binding on the redox and catalytic properties of p-cresol methylhydroxylase. Biochemistry, 40, 2155-2166 (2001) [23] Cronin, C.N.; McIntire, W.S.: Heterologous expression in Pseudomonas aeruginosa and purification of the 9.2-kDa c-type cytochrome subunit of pcresol methylhydroxylase. Protein Expr. Purif., 19, 74-83 (2000)

534

  #    

8::

   
 1.17.99.2    ethylbenzene:(acceptor) oxidoreductase    ethylbenzene hydroxylase    ethylbenzene dehydrogenase
    249614-72-0

     Azoarcus sp. (EbN1 [1]; EB1 [2]) [1, 2]

!    "
 # 

ethylbenzene + H2 O + acceptor = (S)-1-phenylethanol + reduced acceptor    oxidation redox reaction reduction       ethylbenzene + H2 O + acceptor ( anaerobic degradation of ethylbenzene in bacteria [1,2]) (Reversibility: ir [1]; ? [2]) [1, 2] $ (S)-1-phenylethanol + reduced acceptor [1, 2]      3-methyl-2-pentene + molybdopterin (Reversibility: ? [2]) [2] $ ?  4-fluoro-ethylbenzene + molybdopterin (Reversibility: ? [2]) [2] $ ?

535

Ethylbenzene hydroxylase

1.17.99.2

 ethylbenzene + H2 O + [Fe(III) (C5 H5 )2 ]+ (Reversibility: ir [1]) [1] $ (S)-1-phenylethanol + [Fe(II) (C5 H5 )2 ] [1]  ethylbenzene + H2 O + molybdopterin (Reversibility: ? [2]) [2] $ (S)-1-phenylethanol + molybdopterin (reduced form) [2]  ethylidenecyclohexane + molybdopterin (Reversibility: ? [2]) [2] $ ?  n-propylbenzene + H2 O + [Fe(III) (C5 H5 )2 ]+ (Reversibility: ? [1]) [1] $ (S)-1-phenylpropanol + [Fe(II) (C5 H5 )2 ] [1] 2  O2 ( does not effect enzyme in cell extracts, inhibitory for purified enzyme [1]) [1]
"%  molybdopterin [2] '(   [Fe(III) (C5 H5 )2 ]+ [1] iron sulfur cluster ( located in b-subunit [2]) [2] " & *-% , 0.0035 ( activity towards n-propylbenzene of cell extract from cells grown on ethylbenzene [1]) [1] 0.0224 ( activity towards ethylbenzene of cell extract from cells grown on ethylbenzene [1]) [1] . / *', 0.002 (ethylbenzene) [1] 0  7 [1] 0 

 5.5-9 ( about maximum activity at both pH [1]) [1]

  #   ' 1  70000 ( gel filtration of purified enzyme [2]) [2] 155000 ( gel filtration, native PAGE [1]) [1] 160000 ( calculated from DNA-sequence [2]) [2] 545000 ( native PAGE [2]) [2]   trimer ( abg 1 * 96000 + 1 * 43000 + 1 * 23000, SDS-PAGE [1]; a,b,g 1 * 100000 + 1 * 35000 + 1 * 25000, SDS-PAGE, 1 * 104226 + 1 * 39643 + 1 * 23061, calculated from DNA-sequence [2]) [1, 2]

536

1.17.99.2

Ethylbenzene hydroxylase

 2 %$ %' %

3#

membrane [2] periplasm [1] $"

[1, 2]
 

(in Escherichia coli DH5a [2]) [2]

"  [1] Kniemeyer, O.; Heider, J.: Ethylbenzene dehydrogenase, a novel hydrocarbon-oxidising molybdenum/iron-sulfur/heme enzyme. J. Biol. Chem., 276, 21381-21386 (2001) [2] Johnson, H.A.; Pelletier, D.A.; Spormann, A.M.: Isolation and characterisation of anaerobic ethylbenzene dehydrogenase, a novel Mo-Fe-S enzyme. J. Bacteriol., 183, 4536-4542 (2001)

537

 9D 

   
 1.18.1.1    rubredoxin:NAD+ oxidoreductase    rubredoxin-NAD+ reductase    (flavo)rubredoxin reductase DPNH-rubredoxin reductase EC 1.6.7.2 (formerly) FIRd-reductase NADH-rubredoxin oxidoreductase NADH-rubredoxin reductase NADH: rubredoxin oxidoreductase NOR dihydronicotinamide adenine dinucleotide-rubredoxin reductase reduced nicotinamide adenine dinucleotide-rubredoxin reductase reductase, rubredoxin-nicotinamide adenine dinucleotide rubredoxin reductase rubredoxin-NAD reductase
    9032-27-3

    



538

Clostridium acetobutylicum [1, 2] Pseudomonas oleovorans [3, 4, 8] Desulfovibrio gigas [5, 7] Acinetobacter calcoaceticus [6] Escherichia coli [9]

7

1.18.1.1

Rubredoxin-NAD+ reductase

!    "
 # 

reduced rubredoxin + NAD+ = oxidized rubredoxin + NADH + H+    oxidation redox reaction reduction       NADH + oxidized rubredoxin (, the induction of rubredoxin reductase, normally observed at pH 4.3 is stopped immediately after the addition of rifampicin. The enzyme could play a role in some deacidification mechanism in relation to proton transport [2]; , enzyme is required for fatty acid and alkane hydroxylation [4]) (Reversibility: ? [2, 4]) [2, 4] $ NAD+ + reduced rubredoxin      NADH + 1,4-naphthoquinone (Reversibility: ? [1]) [1] $ NAD+ + 1,4-naphthoquinol  NADH + 2,6-dichloroindophenol (Reversibility: ? [1, 4, 6]) [1, 4, 6] $ NAD+ + reduced 2,6-dichloroindophenol  NADH + 2-methyl-1,4-naphthoquinone (Reversibility: ? [1]) [1] $ NAD+ + 2-methyl-1,4-naphthoquinol  NADH + Fe(CN)36- (Reversibility: ? [6]) [6] $ NAD+ + Fe(CN)26 NADH + cytochrome c (, weak activity [1]) (Reversibility: ? [1]) [1] $ NAD+ + reduced cytochrome c  NADH + ferricyanide (Reversibility: ? [1, 4]) [1, 4] $ NAD+ + reduced ferricyanide  NADH + metmyoglobin (Reversibility: ? [1]) [1] $ NAD+ + reduced metmyoglobin  NADH + nitroblue tetrazolium (, weak activity [1]) (Reversibility: ? [1]) [1] $ NAD+ + reduced nitroblue tetrazolium  NADH + oxidized rubredoxin (, enzyme catalyzes electron transfer not only to the rubredoxin of Pseudomonas oleovorans but also to the lower molecular weight rubredoxins of the anaerobic bacteria Peptostreptococcus elsdenii, Clostridium pasteurianum and Desulfovibrio gigas [4]; , very specific towards Desulfovibrio gigas rubredoxin [7]; , enzyme mediates electron transfer from NADH to Desulfovibrio gigas rubredoxin as well as to E. coli flavorubredoxin [9]) (Reversibility: ? [1, 2, 3, 4, 5, 6, 7, 8, 9]) [1, 2, 3, 4, 5, 6, 7, 8, 9] $ NAD+ + reduced rubredoxin

539

Rubredoxin-NAD+ reductase

1.18.1.1

NADH + p-benzoquinone (Reversibility: ? [1]) [1] NAD+ + p-benzoquinol NADH + p-iodonitrotetrazolium (Reversibility: ? [1]) [1] NAD+ + reduced p-iodonitrotetrazolium NADH + p-toluoquinone (Reversibility: ? [1]) [1] NAD+ + p-toluoquinol Additional information (, enzyme catalyzes rubredoxindependent reduction of cytochrome c in presence of NADH [1, 3, 4, 5, 8]; , diaphorase activity towards ferricyanide [3]; , no activity with spinach ferredoxin, putidaredoxin and adrenodoxin [4]; , both the nonphysiological 1Fe form of rubredoxin and the physiological 2 Fe form combine with rubredoxin reductase to form functional electron transfer complexes. The reductive half-reaction of the rubredoxin reductase occurs by a simple one-step mechanism in which oxidized enzyme is reduced to an enzyme-NAD+ charge-transfer species [8]) [1, 3, 4, 5, 8] $ ?

 $  $  $ 

2  1,10-phenanthroline [1] 2,4-dinitrophenol [1] 8-hydroxyquinoline [6] AgNO3 [1, 4, 6] BaCl2 [6] CaCl2 [6] HgCl2 [6] NAD+ [4] NADPH [4] NEM [1] Na2 SO3 [7] NaCl (, 70 mM, 50% inhibition [7]) [7] PCMB [5, 6, 7] ZnCl2 [6] deoxycholate [6] dicumarol [1] p-hydroxymercuribenzoate [1, 7] p-mercuribenzoate [4] quinacrine [1] sodium arsenite [1] sodium mersalyl [4] tetrodotoxin [4] thionicotinamide-NAD+ [4]
"%  FAD (, prosthetic group [1, 2, 6, 7]; , 1 mol per mol of enzyme [3, 4]; , enzyme contains FAD [9]) [1, 3, 4, 6, 7, 9] FMN (, prosthetic group [7]) [7] NADH (, NADH is highly superior to NADPH as electron donor [4]) [1, 2, 3, 4, 5, 6, 7, 8, 9] 540

1.18.1.1

Rubredoxin-NAD+ reductase

NADPH (, NADH is highly superior to NADPH as electron donor [4]; , no significant reaction with [1, 6]; , completely inactive as electron donor [5]) [4] &

  FAD (, addition of FAD increases activity [6]) [6] ) &  * +, 505 (Desulfovibrio gigas rubredoxin) [7] " & *-% , 12 [7] 46 (, rubredoxin-dependent reduction of cytochrome c [1]) [1] 82 (, rubredoxin-dependent reduction of cytochrome c [3]) [3] 86 (, rubredoxin-dependent reduction of cytochrome c [8]) [8] Additional information [5] . / *', 0.0025 (2,6-dichloroindophenol) [6] 0.055 (Fe(CN)36- ) [6] 0.11 (NADH, , reaction with 2,6-dichloroindophenol or Fe(CN)36[6]) [6] 0  7.7 (, phosphate buffer [6]) [6] 8.5 (, Tris-HCl buffer [6]) [6]

  #   ' 1  38000 (, gel filtration [1]) [1] 55300 (, calculation from sedimenation and diffusion measurement [3]) [3]   ? (, x * 27000 + x * 32000, SDS-PAGE [7]) [7] monomer (, 1 * 41000, SDS-PAGE [1]; , 1 * 50500, SDS-PAGE [3]) [1, 3]

 2 %$ %' %

$"

[1] [3, 8] (partial [5]) [5, 7]
 

(overexpression in Escherichia coli [8]) [8]

541

Rubredoxin-NAD+ reductase

1.18.1.1

4  )  50 (, 5 min, no inactivation in presence of FAD [6]) [6]

"  [1] Petitdemange, H.; Marczak, R.; Blusson, H.; Gay, R.: Isolation and properties of reduced nicotinamide adenine dinucleotiderubredoxin oxidoreductase of Clostridium acetobutylicum. Biochem. Biophys. Res. Commun., 91, 12581265 (1979) [2] Marczak, R.; Ballongue, J.; Petitdemange, H.; Gay, R.: Regulation of the biosynthesis of NADH-rubredoxin oxidoreductase in Clostridium acetobutylicum. Curr. Microbiol., 10, 165-168 (1984) [3] Ueda, T.; Lode, E.T.; Coon, M.J.: Enzymatic w-oxidation. VI. Isolation of homogeneous reduced diphosphopyridine nucleotide-rubredoxin reductase. J. Biol. Chem., 247, 2109-2116 (1972) [4] Ueda, T.; Coon, M.J.: Enzymatic oxidation. VII. Reduced diphosphopyridine nucleotide-rubredoxin reductase: properties and function as an electron carrier in hydroxylation. J. Biol. Chem., 247, 5010-5016 (1972) [5] Le Gall, J.: Partial purification and study of NAD:rubredoxin oxidoreductase from D. gigas. Ann. Inst. Pasteur, 114, 109-115 (1968) [6] Claus, R.; Asperger, O.; Kleber, H.P.: Properties of rubredoxin reductase from the alkane-assimilating bacterium Acinetobacter calcoaceticus. Z. Allg. Mikrobiol., 19, 695-704 (1979) [7] Chen, L.; Liu, M.Y.; Legall, J.; Fareleira, P.; Santos, H.; Xavier, A.V.: Purification and characterization of an NADH-rubredoxin oxidoreductase involved in the utilization of oxygen by Desulfovibrio gigas. Eur. J. Biochem., 216, 443-448 (1993) [8] Lee, H.J.; Basran, J.; Scrutton, N.S.: Electron transfer from flavin to iron in the Pseudomonas oleovorans rubredoxin reductase-rubredoxin electron transfer complex. Biochemistry, 37, 15513-15522 (1998) [9] Gomes, C.M.; Vicente, J.B.; Wasserfallen, A.; Teixeira, M.: Spectroscopic studies and characterization of a novel electron-transfer chain from Escherichia coli involving a flavorubredoxin and its flavoprotein reductase partner. Biochemistry, 39, 16230-16237 (2000)

542

C 9$D 

7

   
 1.18.1.2    ferredoxin:NADP+ oxidoreductase    ferredoxin-NADP+ reductase    DA1 EC 1.6.7.1 (formerly) EC 1.6.99.4 (formerly) FLDR FLXR FNR ferredoxin-NADP(+) reductase flavodoxin reductase NADP:ferredoxin oxidoreductase NADPH:ferredoxin oxidoreductase NFR TPNH-cytochrome P-450 reductase [56] TPNH-ferredoxin reductase adrenodoxin reductase ferredoxin-NADP oxidoreductase ferredoxin-NADP reductase ferredoxin-NADP-oxidoreductase ferredoxin-TPN reductase ferredoxin-nicotinamide-adenine dinucleotide phosphate (oxidized) reductase ferredoxin:NADP+ oxidoreductase reduced nicotinamide adenine dinucleotide phosphate-adrenodoxin reductase reductase, ferredoxin-nicotinamide adenine dinucleotide phosphate
    56367-57-8 9029-33-8

543

Ferredoxin-NADP+ reductase

1.18.1.2

     Spinacia oleracea (spinach, multiple forms [43]; 6 different forms differ in specific activities in various assay systems and affinity for NADPH, interconvertible at 4 C [23]; 5 molecular forms: a, b, c, d, e [25]; 2 forms: P-1, P-2 [28]; strain Atlanta [24,52]; ferredoxin NADP reductase binding protein is not related to CF0II [63]) [1, 7, 11, 13, 14, 18-20, 23-28, 31, 32, 3740, 42, 43, 45, 47, 50-55, 59-61, 63, 64, 67, 68, 70, 81, 82] Spirulina platensis (2 forms: FNRS I and FNRS II [2]) [2] Bos taurus (bovine [4]) [4, 8, 49, 56, 77, 78, 83] Sus scrofa (pig [5]) [5, 35] Synechococcus sp. (thermophilic blue-green algae [6]) [6] Anabaena variabilis (cyanobacterium [34]) [9, 34, 62] Desulfobacter postgatei [10] Spirulina sp. (blue-green algae [15]) [15, 39] Bryopsis corticulans (marine green algae [16]) [16] Anabaena cylindrica (cyanobacterium [17]) [17] Pisum sativum (multiple isoenzymes [21]) [21, 72, 74] Bumilleriopsis filiformis (xanthophycean algae [66]) [22, 66] Raphanus sativus (radish, var acanthiformis cultivar miyashige [29]) [29] Phaseolus vulgaris (bean [30]) [30] Euglena gracilis [33] Anabaena sp. (strain 7119, multiple forms: FNR-I, FNR-II, FNR-III, FNRIV [36]; petH gene product [76]) [12, 36, 41, 58, 71, 73, 76, 79] Nostoc sp. (strain MAC, cyanobacterium [44]) [44] Rattus norvegicus (rat,Wistar,male, H18H [75]) [46, 75] Bacillus polymyxa [48] Ovis aries [3] Azotobacter vinelandii [80] Nostoc muscorum (cyanobacterium strain 7119 [57]) [57] Gallus gallus (chick [65]) [65] Capsicum annuum yolo wonder (paprika [69]) [69] Zea mays (corn root [70]) [70]

!    "
 # 

reduced ferredoxin + NADP+ = oxidized ferredoxin + NADPH + H+ ( interaction between ferredoxin and ferredoxin-NADP+ reductase [32]; catalytic mechanism [38])    oxidation redox reaction reduction

544

1.18.1.2

Ferredoxin-NADP+ reductase

      NADPH + oxidized ferredoxin ( first enzyme in mitochondrial P-450-linked monooxygenase system catalyzing several steps in the biosynthesis of steroid hormones, bile acids or vitamin D3 in various tissues [4]; first enzyme in mitochondrial P-450-linked monooxygenase system catalyzing several steps in the biosynthesis of steroid hormones, bile acids or vitamin D3 in various tissues [5]; first enzyme in mitochondrial P-450-linked monooxygenase system catalyzing several steps in the biosynthesis of steroid hormones, bile acids or vitamin D3 in various tissues [3]; key enzyme catalyzing the electron transport between NADPH generated by pentose phosphate pathway and ferredoxin in plastids of plant heterotrophic tissues [3]; supports in vivo reduction of membrane bound adrenal mitochondrial P-450 [35]) (Reversibility: ? [3, 4, 5, 6, 37]) [3-5, 35] $ NADP+ + reduced ferredoxin  Additional information ( pathway of cyclic electron transport includes both ferredoxin and ferredoxin-NADP+ reductase, but not the NADP+ -binding site of the reductase [26]; ferredoxin-NADP+ reductase not involved in cyclic electron transport [27]; involved in oxidative stress [80]) [26, 27, 80] $ ?      K3 Fe(CN)6 + NADPH (Reversibility: ? [25]) [25] $ ? + NADP+  NADPH + oxidized ferredoxin (Reversibility: r, [1, 10, 47]; ? [3-5, 8, 11, 17, 29, 34, 35]) [1, 3-5, 8, 10, 11, 17, 29, 34, 35, 47] $ NADP+ + reduced ferredoxin [1, 3-5, 7, 17, 29, 34, 35, 47]  aclacinomycin A + NADP+ ( under anaerobic conditions [55]) (Reversibility: ? [55]) [55] $ 7-deoxyaklavinone + NADPH  daunomycin + NADP+ ( under anaerobic conditions [55]) (Reversibility: ? [55]) [55] $ 7-deoxydaunomycinone + NADPH  menogarol + NADP+ ( under anaerobic conditions [55]) (Reversibility: ? [55]) [55] $ 7-deoxynogarol + NADPH  nogalamycin + NADP+ ( under anaerobic conditions [55]) (Reversibility: ? [55]) [55] $ 7-deoxynogalarol + NADPH  oxidized iodonitrotetrazolium violet + NADPH (Reversibility: ? [33]) [33] $ reduced iodonitrotetrazolium violet + NADP+  oxidized rubredoxin + NADP+ ( Clostridium pasteurianum rubredoxin [54]) (Reversibility: ? [54]) [54]

545

Ferredoxin-NADP+ reductase

1.18.1.2

$ reduced rubredoxin + NADPH  Additional information ( enzyme has also little NADP-2,6-dichlorophenol indophenol diaphorase activity [2, 17]; enzyme has also NADPH-diaphorase activity [1, 2, 70, 74]; enzyme has also NADPH-NAD transhydrogenase activity [1, 2, 33]; enzyme has also NADPHcytochrome c reductase activity [6, 12, 21, 46, 70, 74, 75]; enzyme has also ferredoxin dependent cytochrome c reductase activity [17, 33]; enzyme has also low diaphorase activity [30]; enzyme has also indonitrotetrazolium-violet diaphorase activity [33]; enzyme has also irreversible NADPH-NAD+ transhydrogenase activity [48]; enzyme contains no FAD but shows NADP-specific diaphorase activity [45]; after cross-linking ferredoxin to ferredoxin NADP+ -reductase the enzyme maintains most of the diaphorase activity and gains capacity to catalyze the NADPH-cytochrome c reaction without addition of free ferredoxin [53]; truncated enzyme has no capacity to catalyze the ferredoxin-dependent reaction [60]) [1, 2, 6, 12, 17, 21, 30, 33, 35, 45, 46, 48, 53, 60, 70, 74, 75] $ ? 2  2',5'-ADP ( competitive inhibition [41]; competitive inhibition, but there could also be a non-competitive component caused by binding at a weak secondary NADP+ binding site [64]) [41, 64] 2',5'-ATP-ribose ( competitive inhibition, competitive in forming complexes with reductase [64]) [64] 2'-AMP [47] 5,5'-dithiobis(2-nitrobenzoate) ( NAD+, NADP+ prevent inhibition [49]) [49] H2 CO3 [42] MgCl2 ( inhibits at lower salt concentrations [29]) [29] N-ethyl-3(3-dimethylaminopropyl)carbodiimide ( inactivates ferredoxin-NADP+ reductase [53]) [53] N6 -(6-aminohexyl)-2',5'-ADP ( competitive inhibition [41]) [41] NaCl ( high concentration [6]) [6] [Cr[CN]6 ]4- ( binds to the enzyme [11]) [11] butanedione ( inhibitor of transhydrogenase and diaphorase activity, reacts with arginine residue involved in binding of pyridine nucleotides [66]) [66] diethyl dicarbonate ( NAD+, NADP+ prevent inhibition [49]) [49] diphosphate ( inhibitor of ferredoxin-dependent photoreduction [43]) [43] disulfodisalicylidenepropane-1,1-diamine ( inhibits all reactions except photoreduction of cytochrome c [26]) [26] ferredoxin ( competitive inhibitor with NADPH in dichlorophenolindophenol reductase reaction [43]; oxidized ferredoxin inhibits both the first and second one-electron reduction [38]) [38, 43]

546

1.18.1.2

Ferredoxin-NADP+ reductase

heparin ( binds to the enzyme, inhibits ferredoxin and NADPH binding to the enzyme [14]) [14] mercurials [43] sorbitol ( 0.25M [16]) [16] triazine dyes ( interaction with the enzyme, competitive inhibitor of NADPH in ferricyanide reduction assays [31]; competitive inhibition of diaphorase activity [40]) [31, 40] Additional information ( enzyme is activated by light and inactivated by dark [16]; inhibited by specific antibodies [9]; association of ferredoxin inhibits binding of NADPH [61]) [9, 16, 61]
"%  FAD ( flavoprotein [1, 3, 4, 33-35, 43, 46, 47, 48, 75]; 1 mol FAD per mol of enzyme [3, 47]; in contrast to stromal reductase, the solubilized and purified membrane-bound enzyme contains no FAD [45]) [1, 3, 4, 24, 30, 33-35, 43, 45-48, 52, 58, 68, 74-76] NAD+ ( enzyme reduces NADP+ and NAD+, specific for NADP+ reduction under physiological conditions [47]) [47] NADP+ ( enzyme reduces NADP+ and NAD+, specific for NADP+ reduction under physiological conditions [47]; requirement for NADPH [29, 72]; reductase is covalently cross linked to Azotobacter vinelandii flavodoxin [62]) [1, 29, 47, 62, 72, 78] ferredoxin ( activation of diaphorase and transhydrogenase [43]) [43] flavin ( flavoprotein [29]) [1, 17, 20, 29] Additional information ( no activity with NADH [29]) [16, 29] &

  ferredoxin ( enhances diaphorase reaction with NADPH, but not with NADH [57]) [57] polylysine ( activator of ferredoxin-NADP+ reductase [43]) [43] '(   NH+4 ( activator of ferredoxin-NADP+ reductase [43]) [43] NaCl ( stimulation at 0.1M [24]) [24] ) &  * +, 222 (NADPH, NADPH cytochrome c reductase activity, flavodoxin [62]) [62] 6000 (NADPH, NADPH cytochrome c reductase activity, ferredoxin [62]) [62] 6300 (NADPH, NADPH-dichlorophenol indophenol diaphorase activity [34]) [34] 14000 (NADPH, NADPH-ferredoxin-cytochrome c reductase activity [34]) [34] 16020 (NADPH, diaphorase activity, FNR-flavodoxin complex [62]) [62] 31000 (NADPH, NADPH-ferricyanide diaphorase activity [34]) [34] 31020 (NADPH, diaphorase activity [62]) [62] 547

Ferredoxin-NADP+ reductase

1.18.1.2

" & *-% , 0.73 ( NADPH/2,6-dichlorophenol indophenol diaphorase activity [30]) [30] 1.01 ( NADPH-oxidase activity at 25 C [4]) [4] 8.8 ( NADPH-cytochrome c reductase acticvity [4]) [4] 9.25 [46] 9.255 ( cytochrome c reduction [46]) [46] 9.4 ( fraction 1 [17]) [17] 10 [21] 16.3 ( reduction of cytochrome c [35]) [35] 20 ( NADPH-ferricyanide reductase activity at 25 C [4]) [4] 22.27 [37] 22.8 ( NADPH-ferricyanide reductase activity, purification method 2 [5]) [5] 23.05 ( NADPH-ferricyanide reductase activity, purification method 1 [5]) [5] 28 ( fraction 4 [17]) [17] 39.4 ( fraction 2 [17]) [17] 53.9 ( fraction 3 [17]) [17] 76 ( diaphorase activity of the 32 kDa enzyme [7]) [7] 79 ( diaphorase activity of the 35 kDa enzyme [7]) [7] 120 [41] 128 ( NADPH/cytochrome c activity [30]) [30] 136.7 [25] 160.8 ( fusion protein with GST, ferricyanide reduction [74]) [74] 563 [45] Additional information [3, 5, 36, 40, 44, 56, 72] . / *', 0.00094 (NADPH) [35] 0.0012 (NADPH, intact enzyme [77]) [77] 0.0012 (ferredoxin) [29] 0.0027 (NADPH, proteolyzed 22.8 kDa enzyme [77]) [77] 0.0038 (ferredoxin, enzyme II [2]) [2] 0.0043 (ferredoxin, enzyme I [2]) [2] 0.0045-0.0046 (ferredoxin, ferredoxin-dependent cytochrome c reductase activity [17]) [17] 0.005 (NADPH) [9] 0.005 (ferredoxin) [72] 0.00722 (NADP+ ) [47] 0.0092 (NADPH) [29] 0.01 (NADPH, INT as electron acceptor [70]) [70] 0.01-0.016 (2,6-dichlorophenol-indophenol, NADPH-2,6-dichlorophenol-indophenol diaphorase activity [17]) [17] 0.011-0.035 (NADPH, ferredoxin-dependent cytochrome c reductase activity [17]) [17] 0.012 (NADPH, K3 Fe(CN)6 as electron acceptor [70]) [70]

548

1.18.1.2

Ferredoxin-NADP+ reductase

0.028 (NADPH) [72] 0.03 (NADPH) [38] 0.03-0.0547 (iodonitrotetrazolium violet, 4 different fractions after ferredoxin-Sepharose chromatography [33]) [33] 0.033-0.062 (NADPH, NADPH-2,6-dichlorophenol indophenol diaphorase activity [17]) [17] 0.036 (2,6-dichlorophenol indophenol, diaphorase activity enzyme I [2]) [2] 0.036-0.043 (NADPH, multiple forms of ferredoxin-NADP+ reductase [25]) [25] 0.047 (2,6-dichlorophenol indophenol, diaphorase activity enzyme II [2]) [2] 0.049 (ferredoxin, 35 kDa enzyme [7]) [7] 0.053 (ferredoxin, 32 kDa enzyme [7]) [7] 0.059 (NADPH, diaphorase activity enzyme I [2]) [2] 0.067 (NADPH, diaphorase activity enzyme II [2]) [2] 0.097-0.1 (K3 Fe(CN)6 ) [25] 3.77 (NAD+ ) [47] Additional information ( increasing light intensity reduces Km [18]; increasing NH4 Cl concentration enhances Km [18]; Km value increases with pH [18]; enzyme covalently cross-linked to flavodoxin [62]; Km value of different mutants [71]; enzyme reacts slower with different mutants of ferredoxin than with wild type ferredoxin [73]) [2, 18, 23, 30, 34, 36, 42, 43, 47, 48, 53, 55, 62, 71-76] 0  7 ( reduced ferredoxin + NADP+ [48]) [48] 7.1 ( ferredoxin dependent cytochrome c reducing activity [2]) [2] 7.4-7.8 ( ferredoxin-dependent cytochrome c reductase activity [17]) [17] 7.5 [21] 7.9 ( NADP+ photoreduction activity [2]) [2] 8 ( cytochrome c reduction [72]) [72] 8.2 [25] 8.4 (100 mM Tris-HCl [29]) [29] 8.9 ( NADPH-NAD+ transhydrogenase [48]) [48] 9 ( diaphorase activity at 55 C [6]; diaphorase activity decreases at lower pH [18]) [6, 18] 9-9.1 [42] 9.4 ( at 25 C, diaphorase activity is largely independent of pH, slight optimum at pH 9.3 [6]) [6] 9.5 ( diaphorase activity [2]) [2] 10 ( NADPH + methyl viologen [48]; diaphorase activity [76]) [48, 76] 10.1 ( NADPH-2,6-dichlorophenol-indophenol diaphorase activity [17]) [17]

549

Ferredoxin-NADP+ reductase

1.18.1.2

)  *
, 60 ( cytochrome c reductase and diaphorase activity [6]) [6]

  #   ' 1  22800 ( proteolysed enzyme [77]) [77] 31000 ( truncated form after limited proteolysis with proteases, deletion of N-terminal region [60]) [60] 31500 ( strain MAC, sedimentation equilibrium [44]) [44] 31970 ( calculated from amino acid composition, enzyme I [2]) [2] 32000 ( lack of the first 28 amino acid residues after expression in E. coli [7]) [7] 32240 ( calculated from amino acid composition, enzyme II [2]) [2] 33000 ( gel filtration and SDS-PAGE, ultracentrifugation [2]; SDS-PAGE [19]) [2, 19] 33000-35000 ( gel filtration, SDS-PAGE [29]) [29] 33000-36000 ( ultracentrifugation [19]) [19] 33000-38000 ( strain 7119, multiple forms [36]) [36] 33180 ( mature protein calculated from amino acid sequence [69]) [69] 33300-37000 ( SDS-PAGE, gel filtration, monomer [17]) [17] 33500-42000 ( multiple forms: a, b, c, d, e, SDS-PAGE, disc gel electrophoresis [25]) [25] 33800 ( gel filtration [72]) [72] 34000 ( SDS-PAGE, monomer [20]; more intense band of two bands after SDS-PAGE [9]) [9, 20] 34140 ( amino acid sequence [15]) [15] 35000 ( after expression in E. coli [7]; gel filtration [30]; native reductase, SDS-PAGE [60]; limited proteolysis to 33 kDa is suppressed by pH 9.3 [67]) [7, 30, 60, 67] 36000 ( SDS-PAGE [37,72]; at least 5 molecular forms [34]) [34, 37, 72, 73] 37000 ( SDS-PAGE, Western blot [13]; SDS-PAGE [59]) [13, 24, 59] 38000 ( gel filtration, monomer [22,66]; SDS-PAGE [30,66]) [22, 30, 66] 39000 ( gel filtration [4]) [4] 40000 [47] 40410 ( calculated molecular mass from amino acid analysis of FNR including amino-terminal transit peptide [69]) [69] 45000 ( analytical ultracentrifugation [5]) [5] 48000 ( SDS-PAGE [35]) [35] 49000 ( ultracentrifugation, gel filtration, SDS-PAGE [5]; predicted according to protein sequence [76]) [5, 76] 50000 ( gel filtration, P-2 [28]; gel filtration [5]) [5, 28] 550

1.18.1.2

Ferredoxin-NADP+ reductase

50320 ( calculated from cDNA sequence [75]) [75] 51000 ( sedimentation equilibrium [4]; SDS-PAGE [75]) [4, 75] 51500 ( gel filtration [35]) [35] 52000 ( minimum MW, SDS-PAGE [3]; gel filtration, SDS-PAGE [46]) [3, 46] 52000 ( SDS-PAGE [4]; weaker band of two bands after SDSPAGE [9]) [4, 9] 52300 ( calculated from amino acid analysis [3]) [3] 53000 ( SDS-PAGE [5]) [5] 60000 ( gel filtration [48]; fusion protein with glutathione S-transferase [74]) [48, 74] 70000 ( SDS-PAGE, dimer [20]; SDS-PAGE, dimer [17]) [17, 20] 72000 ( SDS-PAGE [30]) [30] 80000 ( gel filtration [22]; SDS-PAGE, occurs in higher concentrations when enzyme is purified with protease inhibitors [36]) [22, 36] 117000 ( gel filtration, P-1 [28]) [28] Additional information [43, 45, 53, 62]   dimer ( composed of two different subunits, 1 * 15000 + 1 * 36000, SDS-PAGE [33] two different subunits, SDS-PAGE, occurs in higher concentrations when enzyme is purified with protease inhibitors [36]) [17, 20, 22, 33, 36] monomer ( 1 * 38000 gel filtration [22,66]) [22, 66]

 2 %$ %' %

% adrenal gland [3, 4, 8, 49, 56, 75, 83] commercial preparation [31] kidney [35, 65] leaf [20, 25, 27, 37, 40, 42, 52, 53, 67, 68] liver [46] root [29, 70, 72] seedling [21] sprout ( non-photosynthetic plant tissue [30]) [30] 3#

chloroplast [16, 18, 25, 27, 52] mitochondrion [3, 4, 35, 46, 49, 56, 65, 83] plastid [72] thylakoid membrane [9, 12, 14, 20, 33, 37, 43, 45, 53, 59, 67]

551

Ferredoxin-NADP+ reductase

1.18.1.2


 #

[51, 81, 83] [4, 49, 78, 83] [5] (mutant [79]) [71, 79] [80] [69] [70]
 

(two forms, one lacks the first 28 amino acid residues, has full diaphorase activity but reduced NADPH/cytochrome-c activity [7]) [7] [74] [75] [69] 

 

E139? ( different conformation [79]) [79] E301A ( no significant differences to wild type enzyme [79]) [79] R100A ( amino acid replacement removes the positive charge and the ability to form hydrogen bonds, enzyme interacts weakly with CibacronBlue Sepharose, increased Km for NADPH [71]) [71] R264E ( altered behavior with ferredoxin and flavodoxin [71]) [71] S96G ( shows 2% of wild type activity [82]) [82] S96V ( shows only 0.05% of wild type activity [82]) [82]

4  )  65 ( no loss of activity at 65 C after incubation for 5 min [6]) [6] 76 ( loss of 50% activity at 76 C for 5 min [6]) [6] 5    "

, instability [65]    , -20 C, stable for 2 months in nitrogen or in air [53] , 4 C, stable for 1 week at 4 C when bound to ADP-agarose column [35] , 4 C, rapid loss of activity [10]

"  [1] Shin, M.; Tagawa, K.; Arnon, D.I.: Crystallization of ferredoxin-TPN reductase and its role in the photosynthetic apparatus of chloroplasts. J. Biochem., 338, 84-96 (1963)

552

1.18.1.2

Ferredoxin-NADP+ reductase

[2] Masaki, R.; Wada, K.; Matsubara, H.: Isolation and characterization of two ferredoxin-NADP+ reductases from Spirulina platensis. J. Biochem., 86, 951-962 (1979) [3] Yamazaki, M.; Ichikawa, Y.: Crystallization and comparative characterization of reduced nicotinamide adenine dinucleotide phosphate-ferredoxin reductase from sheep adrenocortical mitochondria. Comp. Biochem. Physiol. B, 96, 93-100 (1990) [4] Hiwatashi, A.; Ichikawa, Y.; Maruya, N.; Yamano, T.; Aki, K.: Properties of crystalline reduced nicotinamide adenine dinucleotide phosphate-adrenodoxin reductase from bovine adrenocortical mitochondria. I. Physicochemical properties of holo- and apo-NADPH-adrenodoxin reductase and interaction between non-heme iron proteins and the reductase. Biochemistry, 15, 3082-3090 (1976) [5] Hiwatashi, A.; Ichikawa, Y.; Yamano, T.: Crystallization and properties of reduced nicotinamide adenine dinucleotide phosphate-adrenodoxin reductase of pig adrenocortical mitochondria. FEBS Lett., 82, 201-205 (1977) [6] Koike, H.; Katoh, S.: Thermal properties of NADP:ferredoxin oxidoreductase and ferredoxin isolated from a thermophilic blue-green alga. Photosynth. Res., 1, 163-170 (1980) [7] Aliverti, A.; Jansen, T.; Zanetti, G.; Ronchi, S.; Herrmann, R.G.; Curti, B.: Expression in Escherichia coli of ferredoxin:NADP+ reductase from spinach. Bacterial synthesis of the holoflavoprotein and of an active enzyme form lacking the first 28 amino acid residues of the sequence. Eur. J. Biochem., 191, 551-555 (1990) [8] Hanukoglu, I.; Gutfinger, T.; Haniu, M.; Shively, J. E.: Isolation of a cDNA for adrenodoxin reductase (ferredoxin-NADP+ reductase). Implications for mitochondrial cytochrome P-450 systems. Eur. J. Biochem., 169, 449-455 (1987) [9] Scherer, S.; Alpes, I.; Sadowski, H.; Böger, P.: Ferredoxin-NADP+ oxidoreductase is the respiratory NADPH dehydrogenase of the cyanobacterium Anabaena variabilis. Arch. Biochem. Biophys., 267, 228-235 (1988) [10] Möller-Zinkhan, D.; Thauer, R.K.: Membrane-bound NADPH dehydrogenase- and ferredoxin:NADP oxidoreductase activity involved in electron transport during acetate oxidation to CO2 in Desulfobacter postgatei. Arch. Microbiol., 150, 145-154 (1988) [11] Armstrong, F.A.; Corbett, S.G.: Inhibition of ferredoxin: NADP+ reductase activity by the hexacyanochromate (III) ion. Biochem. Biophys. Res. Commun., 141, 578-583 (1986) [12] Serrano, A.; Soncini, F.C.; Vallejos, R.H.: Localization and quantitative determination of ferredoxin-NADP+ oxidoreductase, a thylakoid-bound enzyme in the cyanoacterium Anabaena sp. strain 7119. Plant Physiol., 82, 499-502 (1986) [13] Matthijs, H.C.P.; Coughlan, S.J.; Hind, G.: Removal of ferredoxin:NADP+ oxidoreductase from thylakoid membranes, rebinding to depleted membranes, and identification of the binding site. J. Biol. Chem., 261, 1215412158 (1986)

553

Ferredoxin-NADP+ reductase

1.18.1.2

[14] Hosler, J.P.; Yocum, C.F.: Heparin inhibition of ferredoxin-NADP reductase in chloroplast thylakoid membranes. Arch. Biochem. Biophys., 236, 473-478 (1985) [15] Yao, Y.; Tamura, T.; Wada, K.; Matsubara, H.; Kodo, K.: Spirulina ferredoxin-NADP+ reductase. The complete amino acid sequence. J. Biochem., 95, 1513-1516 (1984) [16] Satoh, K.: Fluorescence induction and activity of ferredoxin-NADP+ reductase in Bryopsis chloroplasts. Biochim. Biophys. Acta, 638, 327-333 (1981) [17] Rowell, P.; Diez, J.; Apte, S.K.; Stewart, W.D.P.: Molecular heterogeneity of ferredoxin:NADP+ oxidoreductase from the cyanobacterium Anabaena cylindrica. Biochim. Biophys. Acta, 657, 507-516 (1981) [18] Carrillo, N.; Lucero, H.A.; Vallejos, R.H.: Light modulation of chloroplast membrane-bound ferredoxin-NADP+ oxidoreductase. J. Biol. Chem., 256, 1058-1059 (1981) [19] Sherriff, S.; Teller, D.C.; Herriott, J.R.: Ferredoxin-NADP+ oxidoreductase is active as a monomer with molecular weight 33000-36000. Arch. Biochem. Biophys., 205, 499-502 (1980) [20] Zanetti, G.; Arosio, P.: Solubilization from spinach thylakoids of a higher molecular weight form of ferredoxin-NADP+ reductase. FEBS Lett., 111, 373-376 (1980) [21] Dutton, J.E.; Rogers, L.J.: Multiple isoenzymes of ferredoxin-nicotinamideadenine dinucleotide phosphate (oxidized) reductase from Pisum sativum. Biochem. Soc. Trans., 7, 1262-1264 (1979) [22] Bookjans, G.; Böger, P.: Algal ferredoxin-NADP+ reductase with different molecular weight forms. Z. Naturforsch. C, 34, 637-640 (1979) [23] Ellefson, W.L.; Krogmann, D.W.: Studies of the multiple forms of ferredoxin-NADP oxidoreductase from spinach. Arch. Biochem. Biophys., 194, 593599 (1979) [24] Bookjans, G.; San Pietro, A.; Böger, P.: Resolution and reconstitution of spinach ferredoxin-NADP+ reductase. Biochem. Biophys. Res. Commun., 80, 759-765 (1978) [25] Gozzer, C.; Zanetti, G.; Galliano, M.; Sacchi, G.A.; Minchiotti, L.; Curti, B.: Molecular heterogeneity of ferredoxin-NADP+ reductase from spinach leaves. Biochim. Biophys. Acta, 485, 278-290 (1977) [26] Shahak, Y.; Crowther, D.; Hind, G.: The involvement of ferredoxin-NADP+ reductase in cyclic electron transport in chloroplasts. Biochim. Biophys. Acta, 636, 234-243 (1981) [27] Böhme, H.: On the role of ferredoxin and ferredoxin-NADP+ reductase in cyclic electron transport of spinach chloroplasts. Eur. J. Biochem., 72, 283289 (1977) [28] Fredricks, W.W.; Gehl, J.M.: Multiple forms of ferredoxin-nicotinamide adenine dinucleotide phosphate reductase from spinach. Arch. Biochem. Biophys., 174, 666-674 (1976) [29] Morigasaki, S.; Takata, K.; Suzuki, T.; Wada, K.: Purification and characterization of a ferredoxin-NADP+ oxidoreductase-like enzyme from radish root tissues. Plant Physiol., 93, 896-901 (1990)

554

1.18.1.2

Ferredoxin-NADP+ reductase

[30] Hirasawa, M.; Chang, K.T.; Knaff, D.B.: Characterization of a ferredoxin:NADP+ oxidoreductase from a nonphotosynthetic plant tissue. Arch. Biochem. Biophys., 276, 251-258 (1990) [31] Levy, L.M.; Bets, G.F.: Interaction of NADPH and triazine dyes with ferredoxin-NADP+ oxidoreductase. Biochim. Biophys. Acta, 955, 236-242 (1988) [32] Zanetti, G.; Morelli, D.; Ronchi, S.; Negri, A.; Aliverti, A.; Curti, B.: Structural studies on the interaction between ferredoxin and ferredoxin-NADP+ reductase. Biochemistry, 27, 3753-3759 (1988) [33] Spano, A.J.; Schiff, J.A.: Purification, properties, and cellular localization of Euglena ferredoxin-NADP reductase. Biochim. Biophys. Acta, 894, 484-498 (1987) [34] Sancho, J.; Peleato, M.L.; Gomez-Moreno, C.; Edmondson, D.E.: Purification and properties of ferredoxin-NADP+ oxidoreductase from the nitrogen-fixing cyanobacteria Anabaena variabilis. Arch. Biochem. Biophys., 260, 200207 (1988) [35] Gnanaiah, W.; Omdahl, J.L.: Isolation and characterization of pig kidney mitochondrial ferredoxin:NADP+ oxidoreductase. J. Biol. Chem., 261, 12649-12654 (1986) [36] Serrano, A.: Characterization of cyanobacterial ferredoxin-NADP+ oxidoreductase molecular heterogeneity using chromatofocusing. Anal. Biochem., 154, 441-448 (1986) [37] Apley, E.C.; Wagner, R.; Engelbrecht, S.: Rapid procedure for the preparation of ferredoxin-NADP+ oxidoreductase in molecularly pure form at 36 kDa. Anal. Biochem., 150, 145-154 (1985) [38] Batie, C.J.; Kamin, H.: Electron transfer by ferredoxin:NADP+ reductase. Rapid-reaction evidence for participation of a ternary complex. J. Biol. Chem., 259, 11976-11985 (1984) [39] Wada, K.; Tamura, T.; Matsubara, H.; Kodo, K.: Spirulina ferredoxin-NADP+ reductase. Further characterization with an improved preparation. J. Biochem., 94, 387-393 (1983) [40] Carrillo, N.; Vallejos, R.H.: Interaction of ferredoxin-NADP+ oxidoreductase with triazine dyes. A rapid purification method by affinity chromatography. Biochim. Biophys. Acta, 742, 285-294 (1983) [41] Serrano, A.; Rivas, J.: Purification of ferredoxin-NADP+ oxidoreductase from cyanobacteria by affinity chromatography on 2,5-ADP-sepharose 4B. Anal. Biochem., 126, 109-115 (1982) [42] Zanetti, G.: The reduction of iodonitrotetrazolium chloride by ferredoxinNADP+ reductase: a new tool for the characterization of the spinach chloroplast flavoprotein. Plant Sci. Lett., 23, 55-61 (1981) [43] Zanetti, G.; Curti, B.: Ferredoxin-NADP+ oxidoreductase. Methods Enzymol., 69, 250-255 (1980) [44] Hutber, G.N.; Smith, A.J.; Rogers, L.J.: Isolation of ferredoxin-nicotinamideadenine dinucleotide phosphate (oxidized) reductase from a prokaryote. Biochem. Soc. Trans., 6, 1214-1216 (1978) [45] Suss, K.H.: Isolation and partial characterization of membrane-bound ferredoxin-NADP+ -reductase from chloroplasts. FEBS Lett., 101, 305-310 (1979) 555

Ferredoxin-NADP+ reductase

1.18.1.2

[46] Pedersen, J.I.; Godager, H.K.: Purification of NADPH-ferredoxin reductase from rat liver mitochondria. Biochim. Biophys. Acta, 525, 28-36 (1978) [47] Shin, M.: Ferredoxin-NADP reductase from spinach. Methods Enzymol., 23, 440-447 (1971) [48] Yoch, D.C.: Purification and characterization of ferredoxin-nicotinamide adenine dinucleotide phosphate reductase from a nitrogen-fixing bacterium. J. Bacteriol., 116, 384-391 (1973) [49] Hiwatashi, A.; Ichikawa, Y.; Yamano, T.; Maruya, N.: Properties of crystalline reduced nicotinamide adenine dinucleotide phosphate-adrenodoxin reductase from bovine adrenocortical mitochondria. II. Essential histidyl and cysteinyl residues at the NADPH binding site of NADPH-adrenodoxin reductase. Biochemistry, 15, 3091-3097 (1976) [50] Batie, C.J.; Kamin, H.: The relation of pH and oxidation-reduction potential to the association state of the ferredoxin-ferredoxin:NADP+ reductase complex. J. Biol. Chem., 256, 7756-7763 (1981) [51] Sherriff, S.; Heriott, J.R.: Structure of ferrdoxin-NADP+ oxidoreductase and the location of the NADP binding site. J. Mol. Biol., 145, 441-451 (1981) [52] Böhme H.: Quantitative determination of ferredoxin, ferredoxin-NADP+ reductase and plastocyanin in spinach chloroplasts. Eur. J. Biochem., 83, 137141 (1978) [53] Zanetti, G.; Aliverti, A.; Curti, B.: A cross-linked complex between ferredoxin and ferredoxin-NADP+ reductase. J. Biol. Chem., 259, 6153-6157 (1984) [54] Przysiecki, C.T.; Bhattacharyya, A.K.; Tollin, G.; Cusanovich, M.A.: Kinetics of reduction of clostridium pasteurianum rubredoxin by laser photoreduced spinach ferredoxin:NADP+ reductase and free flavins. J. Biol. Chem., 260, 1452-1458 (1985) [55] Fisher, J.; Abdella, B.R.J.; McLane, K.E.: Anthracycline antibiotic reduction by spinach ferredoxin-NADP+ reductase and ferredoxin. Biochemistry, 24, 3562-3571 (1985) [56] Omura, T.; Sanders, E.; Estabrook, R.W.; Cooper, D.Y.; Rosenthal, O.: Isolation from adrenal cortex of a nonheme iron protein and a flavoprotein functional as a reduced triphosphopyridine nucleotide-cytochrome P-450 reductase. Arch. Biochem. Biophys., 117, 660-672 (1966) [57] Melamed-Harel, H.; Tel-Or, E.; San Pietro, A.: Effect of ferredoxin on the diaphorase activity of cyanobacterial ferredoxin-NADP reductase. Plant Physiol., 77, 229-231 (1985) [58] Walker, M.C. Pueyo, J.J.; Gomez-Moreno, C.; Tollin, G: Comparison of the kinetics of reduction and intramolecular electron transfer in electrostatic and covalent complexes of ferredoxin-NADP+ reductase and flavodoxin from Anabaena PCC 7119. Arch. Biochem. Biophys., 281, 76-83 (1990) [59] Clark, R.D.; Hawkesford M.J.; Coughlan, J.B.; Hind, G.: Association of ferredoxin-NADP+ oxidoreductase with the chloroplast cytochrome b-f complex. FEBS Lett., 174, 137-142 (1984) [60] Gadda, G.; Aliverti, A.; Ronchi, S.; Zanetti, G.: Structure-function relationship in spinach ferredoxin-NADP+ reductase as studied by limited proteolysis. J. Biol. Chem., 265, 11955-11959 (1990)

556

1.18.1.2

Ferredoxin-NADP+ reductase

[61] Batie, C.J.; Kamin, H.: Ferredoxin-NADP+ reductase. J. Biol. Chem., 259, 8832-8839 (1984) [62] Pueyo, J.J.; Sancho, J.; Edmondson, D.E.; Gomez-Moreno, C.: Preparation and properties of a cross-linked complex between ferredoxin-NADP+ reductase and flavodoxin. Eur. J. Biochem., 183, 539-544 (1989) [63] Berzborn, R.J.; Klein-Hitpa´, L.; Otto, J.; Schunemann, S.; Oworah-Nkruma, R.; Meyer, H.E.: The ªadditionalª CF0II of the photosysthetic ATP-synthase and the thylakoid polypeptide, binding ferredoxin-NADP+ reductase: are they different?. Z. Naturforsch. C, 45, 772-784 (1990) [64] Batie, C.J.; Kamin, H.: Association of ferredoxin-NADP+ reductase with NADP(H) specificity and oxidation-reduction properties. J. Biol. Chem., 261, 11214-11223 (1986) [65] Yoon, P.S.; DeLuca, H.F.: Purification and properties of vitamin D hydroxylases. Methods Enzymol., 67, 430-440 (1980) [66] Bookjans, G.; Böger,P.: Complex-forming properties of butanedione-modified ferredoxin-NADP+ reductase with NADP+ and ferredoxin. Arch. Biochem. Biophys., 194, 387-393 (1979) [67] Shin, M.; Tsujita, M.; Tomizawa, H.; Sakihama, N.; Kamei, K.; Oshino R.: Proteolytic degradation of ferredoxin-NADP+ reductase during purification from spinach. Arch. Biochem. Biophys., 279, 97-103 (1990) [68] Shin M.: Complex formation by ferredoxin-NADP+ reductase with ferredoxin or NADP+. Biochim. Biophys. Acta, 292, 13-19 (1973) [69] Dorowski, A.; Hofmann, A.; Steegborn, C.; Boicu, M.; Huber, R.: Crystal structure of paprika ferredoxin-NADP+ reductase. J. Biol. Chem., 276, 9253-9263 (2001) [70] Aliverti, A.; Faber, R.; Finnerty, C.M.; Ferioli, C.; Pandini, V.; Negri, A.; Karplus, P.A.; Zanetti, G.: Biochemical and crystallographic characterization of ferredoxin-NADP+ reductase from nonphotosynthetic tissues. Biochemistry, 40, 14501-14508 (2001) [71] Martinez-Julvez, M.; Hermoso, J.; Hurley, J.K.; Mayoral, T.; Sanz-Aparicio,J.; Tollin, G.; Gomez-Moreno,C.; Medina M.: Role of Arg100 and Arg 264 from Anabaena PCC 7119 ferredoxin-NADP+ reductase for optimal NADP+ binding and electron transfer. Biochemistry, 37, 17680-17691 (1998) [72] Bowsher, C.G.; Dunbar, B.; Emes, M.J.: The purification and properties of ferredoxin-NADP+ oxidoreductase from roots of Pisum sativum L.. Protein Expr. Purif., 4, 512-518 (1993) [73] Hurley, J.K.; Weber-Main, A.M.; Stankovich, M.T.; Benning, M.M.; Thoden, J.B.; Vanhooke,J.L.; Holden H.M.; Chi Y.K.; Xia, B.; Cheng, H.; Markley,J.L.; Martinez-Julvez, M.; Gomez-Moreno, C.; Schmeits J.L.; Tollin, G.: Structurefunction relationships in Anabaena ferredoxin: correlations between X-ray crystals structures, reduction potentials, and rate constants of electron transfer to ferredoxin-NADP+ reductase for specific ferredoxin mutants. Biochemistry, 36, 11100-11117 (1997) [74] Serra, E.C.; Carillo, N.; Krapp, A.R.; Ceccarelli, E.A.: One step purification of plant ferredoxin-NADP+ oxidoreductase expressed in Escherichia coli as fusion with glutathione S-transferase. Protein Expr. Purif., 4, 539-546 (1993)

557

Ferredoxin-NADP+ reductase

1.18.1.2

[75] Sagara Y.; Watanabe, Y.; Kodama, H.; Aramaki, H.: cDNA cloning, overproduction and characterization of rat adrenodoxin reductase. Biochim. Biophys. Acta, 1434, 284-295 (1999) [76] Martinez-Julvez, M.; Hurley, J.K.; Tollin, G.; Gomez-Moreno, C.; Fillat M.F.: Overexpression in E. coli of the complete petH gene product from Anabaena: purification and properties of a 49 kDa ferredoxin-NADP+ reductase. Biochim. Biophys. Acta, 1297, 200-206 (1996) [77] Warburton, R.J.; Seybert D.W.: Structural and functional characterization of bovine adrenodoxin reductase by limited proteolysis. Biochim. Biophys. Acta, 1246, 39-46 (1995) [78] Ziegler, G.A.; Schulz, G.E.: Crystal structures of adrenodoxin reductase in complex with NADP+ and NADPH suggesting a mechanism for the electron transfer of an enzyme family. Biochemistry, 39, 10986-10995 (2000) [79] Mayoral, T.; Medina, M.; Sanz-Aparicio, J.; Gomez-Moreno, C.; Hermoso J.A.: Structural basis of the catalytic role of Glu301 in Anabaena PCC 7119 ferredoxin-NADP+ reductase revealed by X-ray crystallography. Proteins, 38, 60-69 (2000) [80] Sridhar Prasad, G.; Kresge, N.; Muhlberg, A.B.; Shaw, A.; Jung, Y.S.; Burgess, B.K.; Stout, C.D.: The crystal structure of NADPH:ferredoxin reductase from Azotobacter vinelandii. Protein Sci., 7, 2541-2549 (1998) [81] Bruns C.M.; Karplus P.A.: Refined crystal structure of spinach ferredoxin reductase at 1.7A resolution: oxidized, reduced and 2'-phospho-5'AMP bound states. J. Mol. Biol., 247, 125-145 (1995) [82] Aliverti, A.; Bruns, C.M.; Pandini, V.E.; Karplus P.A.; Vanoni, M.A.; Curti, B.; Zanitti, G.: Involvement of serine 96 in the catalytic mechanism of ferredoxin-NADP+ -reductase: structure-function relationship as studies by site-directed mutagenesis and X-ray crystallography. Biochemistry, 34, 8371-8379 (1995) [83] Kuban R.J.; Marg, A.; Resch, M.; Ruckpaul, K.: Crystallization of bovine adrenodoxin reductase in a new unit cell and its crystallographic characterization. J. Mol. Biol., 234, 245-248 (1993)

558

C 9D 

7!

   
 1.18.1.3    ferredoxin:NAD+ oxidoreductase    ferredoxin-NAD+ reductase    NAD-ferredocinTOL reductase (, component of toluene dioxygenase [5]) [5] NAD-ferredoxin reductase NADH flavodoxin oxidoreductase NADH-ferredoxin oxidoreductase NADH-ferredoxin reductase NADH-ferredoxinNAP reductase (, component of naphthalene dioxygenase multicomponent enzyme system [1]) [1] NADH2 -ferredoxin oxidoreductase ferredoxin-NAD reductase ferredoxin-linked NAD reductase reductase, ferredoxin reductase, ferredoxin-nicotinamide adenine dinucleotide reductase, reduced nicotinamide adenine dinucleotide-ferredoxin
    39369-37-4

     Pseudomonas sp. (strain NCIB 9816 [1]; strain KKS102 [11]; strain LB400 [12]; NADH-ferredoxinNAP reductase component of naphthalene dioxygenase [1]; NADH-ferredoxin oxidoreductase component of biphenyl 2,3dioxygenase [12]) [1, 11, 12] Clostridium kluyveri [2] Clostridium pasteurianum [2, 6, 9, 10] Clostridium butyricum [2, 10] Methylosinus trichosporium OB3b [3]

559

Ferredoxin-NAD+ reductase

1.18.1.3

Clostridium thermohydrosulfuricum (activity is present in wild-type strain, alcohol-adapted strain lacks detectable levels of reduced ferredoxin-linked NAD reductase [4]) [4] Pseudomonas putida (NAD-ferredocinTOL reductase component of toluene dioxygenase [5]) [5] Clostridium tyrobutyricum (strain CNRZ 510 [6]) [6, 9] Clostridium thermocellum [7] Clostridium brockii [7] Clostridium acetobutylicum [8, 9] Butyribacterium methylotrophicum [13]

!    "
 # 

reduced ferredoxin + NAD+ = oxidized ferredoxin + NADH + H+    oxidation redox reaction reduction       oxidized ferredoxin + NADH (, essential step in glucose fermentation [2]; , the enzyme couples electron flow from formate dehydrogenase (NAD+ requiring) to ferredoxin [3]; , catabolic enzyme [10]; , the enzyme is required for the mechanism of CO tolerance by the CO-adapted strain [13]) (Reversibility: ? [2, 3, 10, 13]) [2, 3, 10, 13] $ reduced ferredoxin + NAD+  Additional information (, the enzyme can, depending on cellular conditions, produce or oxidize NADH. NADH-ferredoxin reductase can control the level of NAD+ and NADH in the cell [9]) [9] $ ?      NADH + 2,6-dichlorophenolindophenol (Reversibility: ? [1, 5, 12]) [1, 5, 12] $ NAD+ + reduced 2,6-dichlorophenolindophenol  NADH + cytochrome c (Reversibility: ? [1, 5, 12]) [1, 5, 12] $ NAD+ + reduced cytochrome c  NADH + ferricyanide (Reversibility: ? [1, 5, 12]) [1, 5, 12] $ NAD+ + ?  NADH + nitro blue tetrazolium (Reversibility: ? [1, 5, 12]) [1, 5, 12] $ NAD+ + reduced nitro blue tetrazolium  NADPH + 2,6-dichlorophenolindophenol (Reversibility: ? [12]) [12] $ NADP+ + reduced 2,6-dichlorophenolindophenol

560

1.18.1.3

Ferredoxin-NAD+ reductase

 NADPH + cytochrome c (, 39% of the activity with NADH [1]) (Reversibility: ? [1]) [1] $ NADP+ + reduced cytochrome c  oxidized ferredoxin + NADH (, ferredoxin from Clostridium tyrobutyricum and Clostridium acetobutylicum [6]) (Reversibility: r [8, 9]; ? [5, 6]) [5, 6, 8, 9] $ reduced ferredoxin + NAD+  Additional information (, the enzyme also has transhydrogenase activity which transfers electrons and protons from NADH to thionicotinamide adenine dinucleotide phosphate and from NADPH to acetylpyridine adenine dinucleotide [3]) [3] $ ? 2  1,10-phenanthroline (, 10 mM, 30% inhibition [1]) [1, 5] 2,2'-dipyridyl [5] NADH (, competitive inhibitor of ferredoxin-NAD+ reductase activity [8, 9]; , in cells growing on pyruvate/acetate NADH does not control enzyme activity [9]) [8, 9] NEM (, 10 mM, 67% inhibition [1]) [1] PCMB (, 0.0005 mM, 94% inhibition [1]) [1, 5] iodoacetate (, 10 mM, 50% inhibition [1]) [1] sodium azide (, 40 mM, 46% inhibition [1]) [1]
"%  FAD (, 1 mol of FAD is bound per mol of enzyme [1]; , can bind one mol of FAD per mol of enzyme, Km : 2.5 nM [5]; , contains 0.89 mol of FAD per mol of enzyme [12]) [1, 5, 12] NADH (, specific for NADH [3]; , the CO-adapted strain is a metabolic mutant having higher levels of ferredoxin-NAD oxidoreductase activity, which is not inhibited by NADH [13]) [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13] NADPH (, 39% of the activity with NADH in the reaction with cytochrome c [1]) [1, 12] &

  acetyl Co-A (, obligate activator [8, 9]) [8, 9] '(   iron (, iron-sulfur flavoprotein, protein contains 1.8 gatom of iron and 2.0 gatom of acid-labile sulfur [1]) [1] " & *-% , 0.75 [3] 12.3 [5] 397 [1] . / *', 0.0046 (cytochrome c) [5] 0.0105 (NADH, , reaction with cytochrome c [5]) [5] 561

Ferredoxin-NAD+ reductase

1.18.1.3

0.058 (NADH, , reaction with 2,6-dichlorophenolindophenol [12]) [12] 0.125 (NADH) [3] 0.156 (NADPH, , reaction with 2,6-dichlorophenolindophenol [12]) [12] Additional information (, Km -values for transhydrogenase reaction [3]) [3] 0  6.6-7.5 [5] 7.2 (, reduction of 2,6-dichlorophenolindophenol [12]) [12] )  *
, 32 (, reduction of 2,6-dichlorophenolindophenol [12]) [12] ) 

 *
, Additional information (, no activity detected at 5 C or at 55 C. Activity at 50 C is 15.2% of maximal activity [12]) [12]

  #   ' 1  34900 (, non-denaturing PAGE [1]) [1] 36000 (, gel filtration [3]) [3] 37000 (, gel filtration [1]) [1] 41500 (, NADH-ferredoxin oxidoreductase component of biphenyl 2,3-dioxygenase, gel filtration [12]) [12] 46500 (, gel filtration [5]) [5]   monomer (, 1 * 36000, SDS-PAGE [1]; , 1 * 43600, NADHferredoxin oxidoreductase component of biphenyl 2,3-dioxygenase, SDSPAGE [12]; , 1 * 46000, SDS-PAGE [5]) [1, 5, 12]

 2 %$ %' %

$"

(NADH-ferredoxinNAP reductase component of naphthalene dioxygenase [1]; NADH-ferredoxin oxidoreductase component of biphenyl 2,3-dioxygenase [12]) [1, 12] [3] [5]
 #

(ferredoxin reductase component of biphenyl dioxygenase [11]) [11]

562

1.18.1.3

Ferredoxin-NAD+ reductase

4  )  0.5 (, 5 d, 30% loss of activity [1]) [1] 21 (, room temperature, 8 h, 50% loss of activity [1]) [1]    , -20 C, 1 month, minimal loss of activity [1] , 0-5 C, 30% loss of activity after 5 days [1] , -20 C, 3 months, little loss of activity [5] , 0-4 C, stable for up to 30 h [5]

"  [1] Haigler, B.E.; Gibson, D.T.: Purification and properties of NADH-ferredoxinNAP reductase, a component of naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816. J. Bacteriol., 172, 457-464 (1990) [2] Jungermann, K.; Leimenstoll, G.; Rupprecht, E.; Thauer, R.K.: Demonstration of NADH-ferredoxin reductase in two saccharolytic Clostridia. Arch. Mikrobiol., 80, 370-372 (1971) [3] Chen, Y.P.; Yoch, D.C.: Isolation, characterization, and biological activity of ferredoxin-NAD+ reductase from the methane oxidizer Methylosinus trichosporium OB3b. J. Bacteriol., 171, 5012-5016 (1989) [4] Lovitt, R.W.; Shen, G.J.; Zeikus, J.G.: Ethanol production by thermophilic bacteria: biochemical basis for ethanol and hydrogen tolerance in Clostridium thermohydrosulfuricum. J. Bacteriol., 170, 2809-2815 (1988) [5] Subramanian, V.; Liu, T.N.; Yeh, W.K.; Narro, M.; Gibson, D.T.: Purification and properties of NADH-ferredoxinTOL reductase. A component of toluene dioxygenase from Pseudomonas putida. J. Biol. Chem., 256, 2723-2730 (1981) [6] Blusson, H.; Petitdemange, H.; Gay, R.: A new, fast, and sensitive assay for NADH-ferredoxin oxidoreductase detection in Clostridia. Anal. Biochem., 110, 176-181 (1981) [7] Lamed, R.; Zeikus, J.G.: Ethanol production by thermophilic bacteria: relationship between fermentation product yields of and catabolic enzyme activities in Clostridium thermocellum and Thermoanaerobium brockii. J. Bacteriol., 144, 569-578 (1980) [8] Petitdemange, H.; Cherrier, C.; Bengone, J.M.; Gay, R.: Study of the NADH and NADPH-ferredoxin oxidoreductase activities in Clostridium acetobutylicum. Can. J. Microbiol., 23, 152-160 (1977) [9] Petitdemange, H.; Cherrier, C.; Raval, G.; Gay, R.: Regulation of the NADH and NADPH-ferredoxin oxidoreductases in Clostridia of the butyric group. Biochim. Biophys. Acta, 421, 334-347 (1976) [10] Jungermann, K.; Thauer, R.K.; Leimenstoll, G.; Decker, K.: Function of reduced pyridine nucleotide-ferredoxin oxidoreductases in saccharolytic Clostridia. Biochim. Biophys. Acta, 305, 268-280 (1973)

563

Ferredoxin-NAD+ reductase

1.18.1.3

[11] Senda, T.; Yamada, T.; Sakurai, N.; Kubota, M.; Nishizaki, T.; Masai, E.; Fukuda, M.; Mitsui, Y.: Crystal structure of NADH-dependent ferredoxin reductase component in biphenyl dioxygenase. J. Mol. Biol., 304, 397-410 (2000) [12] Broadus, R.M.; Haddock, J.D.: Purification and characterization of the NADH:ferredoxinBPH oxidoreductase component of biphenyl 2,3-dioxygenase from Pseudomonas sp. strain LB400. Arch. Microbiol., 170, 106-112 (1998) [13] Shen, G.J.; Shieh, J.S.; Grethlein, A.J.; Jain, M.K.; Zeikus, J.G.: Biochemical basis for carbon monoxide tolerance and butanol production by Butyribacterium methylotrophicum. Appl. Microbiol. Biotechnol., 51, 827-832 (1999)

564

 9*$,D 

7

   
 1.18.1.4    rubredoxin:NAD(P)+ oxidoreductase    rubredoxin-NAD(P)+ reductase    NAD(P)-rubredoxin oxidoreductase NAD(P)H-rubredoxin oxidoreductase NROR reductase, rubredoxin-nicotinamide adenine dinucleotide (phosphate) rubredoxin-nicotinamide adenine dinucleotide (phosphate) reductase rubredoxin-nicotinamide adenine dinucleotide phosphate reductase
    114514-31-7

     Pyrococcus furiosus (DSM 3638 [1]) [1]

!    "
 # 

reduced rubredoxin + NAD(P)+ = oxidized rubredoxin + NAD(P)H + H+    oxidation redox reaction reduction       oxidized rubredoxin + NADPH (Reversibility: ? [1]) [1] $ reduced rubredoxin + NADP+      2,6-dichloroindophenol + NADH (Reversibility: ? [1]) [1] $ reduced 2,6-dichloroindophenol + NAD+ + H+

565

Rubredoxin-NAD(P)+ reductase

1.18.1.4

 5,5'-dithiobis(2-nitrobenzoic acid) + NADH (Reversibility: ? [1]) [1] $ ?  FAD + NADH (Reversibility: ? [1]) [1] $ FADH2 + NAD+  FMN + NADH (Reversibility: ? [1]) [1] $ FMNH2 + NAD+  Fe(III) citrate + NADH (Reversibility: ? [1]) [1] $ Fe(II) citrate + NAD+  benzyl viologen + NADH (Reversibility: ? [1]) [1] $ reduced benzyl viologen + NAD+  cytochrome c + NADH (, cytochrome c from horse heart [1]) (Reversibility: ? [1]) [1] $ reduced cytochrome c + NAD+ + H+  methyl viologen + NADH (Reversibility: ? [1]) [1] $ reduced methyl viologen + NAD+ + H+  oxidized rubredoxin + NADH (, rubredoxin is the best substrate [1]) (Reversibility: ? [1]) [1] $ reduced rubredoxin + NAD+  oxidized rubredoxin + NADPH (Reversibility: ? [1]) [1] $ reduced rubredoxin + NADP+
"%  FAD (, enzyme contains FAD [1]) [1] FMN [1] NADH [1] NADPH (, preferred physiological electron donor [1]) [1] " & *-% , 1000 [1] . / *', 0.005 (NADPH) [1] 0.0095 (rubredoxin, , 25 C [1]) [1] 0.034 (NADH) [1] 0.05 (rubredoxin, , 80 C [1]) [1] 0  7-8 [1] )  *
, 80 (, above [1]) [1]

  #   ' 1  45000 (, gel filtration [1]) [1]

566

1.18.1.4

Rubredoxin-NAD(P)+ reductase

  monomer (, 1 * 45000, SDS-PAGE [1]) [1]

 2 %$ %' %

3#

cytoplasm [1] $"

[1]

"  [1] Ma, K.; Adams, M.W.W.: NAD(P)H:rubredoxin oxidoreductase from Pyrococcus furiosus. Methods Enzymol., 334, 55-62 (2001)

567

0   

   
 1.18.3.1 (transferred to EC 1.18.99.1)    hydrogenase

568

7!

  

74

   
 1.18.6.1    reduced ferredoxin:dinitrogen oxidoreductase (ATP-hydrolysing)    nitrogenase    EC 1.18.2.1 (formerly) Additional information (see EC 1.19.6.1: from the literature for EC 1.18.6.1. and EC 1.19.6.1 it is not evident whether these are in fact 2 different enzymes)
    9013-04-1

     Azotobacter vinelandii (2 forms of VFe protein [45, 47]; strain OP [39]; nitrogen fixation complex is encoded on nif gene cluster [35, 36, 38, 41, 45]; contains 3 classes of nitrogenase, the second contains V, the third is encoded by a separate set of genes and is lacking V and Mo and is inhibited by V and Mo [9, 14]) [1-6, 9, 12-15, 17, 22, 25, 31, 33, 34, 37-48] Rhodospirillum rubrum [1-4, 25, 31] Rhodopseudomonas capsulata [1, 30] Klebsiella pneumoniae (nitrogen fixation complex is encoded on nif gene cluster [6, 11]) [1-6, 8, 11, 16, 25, 31-33] Chromatium vinosum [1-4] Azotobacter chroococcum (nitrogen fixation complex is encoded on nif gene cluster [45]) [2-5, 9, 25, 26, 45] Clostridium pasteurianum (nitrogen fixation complex is encoded on nif gene cluster [45]) [1-6, 12, 18, 23, 31, 33, 39, 45] Mycobacterium flavum (301 [4]) [3, 4] Anabaena cylindrica [3, 4, 29, 31] Rhizobium japonicum (nitrogen fixation complex is encoded on nif gene cluster [10]; associated with Glycine max [4]) [2-4, 10] Rhizobium lupini (associated with Lupinus luteus [4]) [2-4, 24] Desulfovibrio desulfuricans (low activity [4]) [4] Escherichia coli (C-M 74 [4]) [4] 569

Nitrogenase

1.18.6.1

Gloeocapsa sp. [4] Plectonema boryanum [4, 31] Rhizobium sp. (ORS571 [31]; associated with Phaseolus aureus or Vigna sinensis [4]; ORS571, associated with Sesbania rostrata [21]) [4, 21, 25, 31, 33] Ornithopus sativus [4] Cyanobacterium sp. [7] Anabaena variabilis (nitrogen fixation complex is encoded on nif gene cluster [11]) [7, 11, 25] Beggiatoa alba (strain B18LD [19]) [19] Rhodospirillum amazonense (strain Y1 [20]) [20] Corynebacterium autotrophicum [27] Bacillus polymyxa [2-6, 28, 31] Chromatium sp. [31, 33] Azospirillum sp. [31] Rhodobacter capsulatus (nitrogen fixation complex is encoded on nif gene cluster [35,36]; strain B10S [35, 36]; iron-only nitrogenase and molybdenum nitrogenase [35, 36]) [35, 36] Azospirillum brasiliense [25] Gloeothece sp. [25, 31] Anabaena sp. [25, 31] Frankia sp. [25, 31] Oscillatoria sp. [25] Rhizobium leguminosarum [31] Azotobacter sp. [25, 31] Desulfovibrio sp. [31] Chlorobium sp. [31] Rhodopseudomonas sphaeroides [31] Rhodopseudomonas sp. [31]

!    "
 # 

8 reduced ferredoxin + 8 H+ + N2 + 16 ATP + 16 H2 O = 8 oxidized ferredoxin + H2 + 2 NH3 + 16 ADP + 16 phosphate ( C2H2 inhibition mechanism, structure model [42]; schematic electron flow from Fe protein to substrate via MoFe protein and MoFe protein-cofactor [38]; MgATP/ MgADP-dependent electron and proton transfer kinetics [34]; structure of V-containing enzyme form [9, 47]; schematic mechanism [9, 25, 33]; catalytic mechanism [1, 3, 58, 33]; iron-only enzyme is composed of 2 components: FeFe protein and Fe protein [35, 36]; enzyme is composed of 2 metalloproteins: component I MoFe protein and component II Fe protein [1-34, 36-46, 48]; enzyme complex dissociation and association kinetics [44]; mechanism, Fe protein cycle [44]; Fe protein and MoFe protein are assumed to associate and dissociate to transfer a single electron to the substrates [44]) 570

1.18.6.1

Nitrogenase

   oxidation redox reaction reduction       reduced ferredoxin + H+ + N2 + ATP ( regulation [7]; biological N2 fixation [7, 31, 45, 47]; ferredoxin normally functions as immediate electron donor to nitrogenase, during iron starvation it is replaced by flavodoxin [7]; ferredoxin is the immediate electron carrier to nitrogenase in all nitrogen-fixing organisms with the exception of Klebsiella pneumoniae, and possibly Azotobacter species, where only flavodoxin is effective in coupling electron flow to nitrogenase [31]) (Reversibility: ? [1-48]) [1-47] $ oxidized ferredoxin + NH3 + ADP + phosphate [33, 47]  reduced ferredoxin + H+ + acetylene + ATP (Reversibility: ? [29, 42]) [29, 42] $ oxidized ferredoxin + ethylene + ADP + phosphate [42]      Ti3+ + H+ + N2 + ATP ( in vitro substrate [39]) (Reversibility: ? [39]) [39] $ ?  dithionite + H+ + N2 + ATP ( SO-2 being the actual nitrogenase reductant, reaction kinetics [46]; in vitro substrate [9, 15, 16, 29, 32-34, 39, 44, 46]) (Reversibility: ? [9, 15, 16, 29, 3234, 39, 44, 46]) [9, 15, 16, 29, 32-34, 39, 44, 46] $ ?  dithionite + H+ + acetylene + ATP (Reversibility: ? [20]) [20] $ ?  reduced ferredoxin + H+ + ATP ( H2 -producing activity is much higher in the iron-only enzyme form than in the molybdenum containing form and is less inhibited by competitive substrates [35]; in absence of other acceptors [4, 5, 17]) (Reversibility: ? [4, 5, 17, 35, 43, 46, 48]) [4, 5, 17, 35, 43, 46, 48] $ oxidized ferredoxin + H2 + ADP + phosphate [35, 43, 46, 48]  reduced ferredoxin + H+ + CH3 NC + ATP (Reversibility: ? [4, 13, 17, 33]) [4, 13, 17, 33] $ oxidized ferredoxin + CH4 + C2 H4 + C3 H6 + C3 H8 + CH3 NH2 + ADP + phosphate [4, 13, 17]  reduced ferredoxin + H+ + CN- + ATP (Reversibility: ? [5, 33, 37, 40, 45]) [5, 33, 37, 40, 45] $ oxidized ferredoxin + CH4 + NH3 + ADP + phosphate [5, 33, 37, 40, 45]  reduced ferredoxin + H+ + N2 + ATP ( slow enzyme [44]; Fe protein and MoFe protein are assumed to associate and dissociate to transfer a single electron to the substrates, termed Fe protein cycle, driven 571

Nitrogenase

$  $ 

$  $ 

$



$

1.18.6.1

by MgATP hydrolysis, with the dissociation of the Fe protein-MoFe protein complex being the rate limiting step of the cycle [44]; MgATP-dependent [34, 41, 45]; 1-propyne, 1-butyne and allene are reduced to the corresponding alkenes [13]) (Reversibility: ? [148]) [1-48] oxidized ferredoxin + NH3 + ADP + phosphate [1-48] reduced ferredoxin + H+ + N2 O + ATP (Reversibility: ? [5]) [5] oxidized ferredoxin + H2 O + N2 + ADP + phosphate [5] reduced ferredoxin + H+ + N-3 + ATP ( mutant H195Q shows only about 7.5% activity compared to wild-type [43]) (Reversibility: ? [5, 13, 17, 33, 37, 43, 45]) [5, 13, 17, 33, 37, 43, 45] oxidized ferredoxin + NH3 + N2 + ADP + phosphate [5, 13, 17, 33, 37, 43, 45] reduced ferredoxin + H+ + SCN- + ATP (Reversibility: ? [37]) [37] oxidized ferredoxin + H2 S + HCN + ADP + phosphate [37] reduced ferredoxin + H+ + acetylene + ATP ( anaerobic atmosphere [48]; active site for acetylene reduction interacts not directly with N2 reduction [42]) (Reversibility: ? [1, 4, 5, 7, 13, 16, 17, 27-29, 33, 35-37, 40, 41, 45-48]) [1, 4, 5, 7, 13, 16, 17, 27-29, 33, 35-37, 40, 41, 45-48] oxidized ferredoxin + ethylene + ADP + phosphate ( reduction cycle continues until complete reduction of the substrate to ethane [48]) [1, 4, 5, 7, 13, 16, 17, 27-29, 33, 35-37, 40, 41, 45-48] reduced flavodoxin + H+ + N2 + ATP ( intermediate is a flavodoxin hydroquinone [44]) (Reversibility: ? [1, 9, 44]) [1, 9, 44] oxidized flavodoxin + NH3 + ADP + phosphate [44]

2  1,10-phenanthroline [13] 1,2-dihydroxybenzene 3,5-disulfonate [13] 2,2'-dipyridyl [13] 2,3-dimercaptopropanol [13] ADP ( above 5 mM [17]) [13, 17] ATP [17] C2 H2 ( competitive to N2 [42]; inhibition of H195 mutants [39]; noncompetitive inhibitor of N2 reduction [5]) [5, 39, 42] CO ( inhibition of CH4 and NH3 production from CN[40]; noncompetitive inhibitor of N2 , C2 H2 and N3 - reduction, no inhibition of H+ reduction [5]; inhibition of H+ reduction by about 50% [48]) [5, 6, 13, 17, 40, 42, 48] Ca2+ [17] Cu2+ [13]

572

1.18.6.1

Nitrogenase

H2 ( competitive [42]; competitive inhibitor of N2 , no inhibition of N-3 , C2 H2 , CN- or H+ reduction [5]) [3, 5, 13, 17, 42] MgADP [5] N2 ( inhibits C2 H2 reduction of mutant H195Q [43]; maximal inhibition of H2 production at Fe protein to MoFe protein ration 2.5 [43]; inhibits the C2 H2 reduction [42, 48]) [42, 43, 48] N-3 ( inhibits H2 production competitively and reversibly [43]) [43] NH+4 ( immediate inhibition, repression of induction [1, 19]) [1, 19] NO [13] O2 ( complete reversible inhibition, reversibility decreases by increasing the time of exposure to O2 [32]; irreversibly inactivated [7]) [1, 6-8, 13, 32] SCN- ( above 6 mM occurs substrate inhibition [37]) [37] Zn2+ [13] glutamine [1] hydrazine ( and derivatives [17]) [17] phosphate ( above 30 mM [17]) [17] urea ( immediate inhibition, repression of induction [19]) [19] vanadium ( contains 3 classes of nitrogenase, the second contains V, the third is encoded by a separate set of genes and is inhibited by V and Mo [9]) [9] Additional information ( overview [33]; high ionic strength inhibits [13, 17]; e.g. above 50 mM NaCl [17]) [13, 17, 33]
"%  Additional information ( structure models of Fe protein, MoFe protein, and MoFe-cofactor, and their metal centers, e.g. the [4Fe-4S] cluster based on crystallographic data, substrate binding and electron transfer [45]; structure of MoFe-cofactor [48]) [45, 48] &

  C2 H2 ( enhances the CH4 production but not NH3 production from CN- , wild-type enzyme [40]) [40] homocitrate ( plays role in electron transfer at the [4Fe-4S] cluster to the MoFe-cofactor of the MoFe protein, can be substituted by erythro-fluorohomocitrate but not by threo-fluorohomocitrate [45]) [45] Additional information ( Fe protein contains an adenine-like molecule, a pentose moiety and a phosphate residue covalently attached to the molecule [1, 30]) [1, 30] '(   Ca2+ ( 1.2 gatom per mol of MoFe protein [8]) [8] Co2+ ( can replace Mg2+ , but is less effective [17]; divalent cation requirement is satisfied by Co2+ , is best supported by concentrations of divalent cation one-half the concentration of ATP [13]) [13, 17] Cu2+ ( 1.4 gatom per mol of MoFe protein [8]) [8]

573

Nitrogenase

1.18.6.1

Fe2+ ( can replace Mg2+ , but is less effective [17]; divalent cation requirement is satisfied by Fe2+ , is best supported by concentrations of divalent cation one-half the concentration of ATP [13]) [13, 17] Mg2+ ( 1.8 gatom per mol of MoFe protein [8]; Mg2+ required for MgATP complex [3-6, 8, 16, 17, 23, 25, 33, 34, 37, 39, 41, 43-46]; divalent metal requirement is satisfied by Mg2+ , reaction is best supported by concentration of divalent cation one-half the concentration of ATP [13]) [3-6, 8, 16, 17, 23, 25, 31, 33, 34, 37, 39, 41, 43-46] Mn2+ ( can replace Mg2+ , but is less effective [17]; divalent cation requirement is satisfied by Mn2+ , is best supported by concentrations of divalent cation one-half the concentration of ATP [13]) [13, 17] Ni2+ ( can replace Mg2+ , but is less effective [17]; divalent cation requirement is satisfied by Ni2+ , is best supported by concentrations of divalent cation one-half the concentration of ATP [13]) [13, 17] Zn2+ ( 0.8 gatom per mol of MoFe protein [8]) [8] iron ( VFe protein form 1 is an incomplete form that contains only 1 cofactor and 1 [4Fe-4S] cluster with an additional [Fe4 -S4 ]like cluster [47]; reduction kinetics [39]; contains [4Fe4S] cluster [38, 39, 42, 45, 47]; the iron-only enzyme consists of 2 components: Fe protein and FeFe protein, the latter contains 26 Fe atoms per molecul of protein [35]; 17.5 gatom per mol of MoFe protein [8]; enzyme consists of 2 proteins: a molybdenum and iron-containing protein, MoFe protein, component I, dinitrogenase, and an iron containing protein, Fe protein, component II, dinitrogenase reductase, together they form the active nitrogenase complex [1, 6]; iron content of MoFe protein: 30 [1]; 20 atoms of iron per molecule of MoFe protein [1]; 27.7 atoms of iron per molecule of MoFe protein [1]; 17-19 atoms of iron per molecule of MoFe protein, overview [2]; 18-36 atoms of iron per molecule of MoFe protein , overview [4]; 22.5 atoms of iron per molecule of protein [21]; 24 atoms of iron per molecule of MoFe protein [20]; the MoFe protein contains 2 molybdenum, about 30 iron and 30 inorganic sulfur atoms, 16 of the 30 Fe atoms are associated with S2- in four cubic [4Fe-4S] clusters, the remaining metal atoms are arranged in two copies of a cofactor called FeMo cofactor, FeMoCo, with a minimum stoichiometry of MoFe6S8-9 [1]; characterization of the metal clusters in the nitrogenase molybdenum-iron and vanadium-iron proteins [15]; iron content of the iron protein: 3.5 [4]; 2.7-4.1 mol per mol of Fe protein [1]; 3.1 [21]; 4 atoms of iron per molecule of Fe protein [6, 8, 24, 26]) [1, 2, 4, 5, 6, 8, 15-18, 20, 21, 24-31, 33-35, 38-42, 44, 45, 47] molybdenum ( MoFe-cofactor contains 2 clusters of composition [4Fe-3S] and [1Mo-3Fe-3S] that are brigded by 3 nonprotein ligands [45]; mol Mo per mol MoFe protein: wild-type and mutant H195Q 1.9, mutant H195N and Q191K 0.9 [40]; 23 mol Fe + 1.9 mol Mo per mol of MoFe protein [26]; 1 gatom per mol of MoFe protein [8]; molybdenum metabolism, cofactor synthesis from nif genes , regulation and structure, overview [6, 33]; 6 iron atoms to 1 molybde574

1.18.6.1

Nitrogenase

num atom in MoFe protein [6]; enzyme consists of 2 proteins: a molybdenum and iron-containing protein (MoFe protein, component I, dinitrogenase) and an iron containing protein (Fe protein, component II, dinitrogenase reductase), together they form the active nitrogenase complex [1, 6-7]; 2 gatom per mol of MoFe protein [1, 20]; 1.7 gatom per mol of MoFe protein [1]; 1-2 gatom per mol of MoFe protein, overview [2, 4, 5]; 1.2-1.3 gatom per mol of MoFe protein [1, 21]; review on molybdenum in nitrogenase [6]; also possesses Mo-independent nitrogenases: one vanadium containing nitrogenase and another lacking both molybdenum and vanadium [9, 14]; characterization of the metal clusters in the nitrogenase molybdenum-iron protein [15]) [1, 2, 4-9, 14-18, 20, 21, 24-31, 33, 34, 38, 40-42, 44, 45] vanadium ( VFe protein form 1 is an incomplete form that contains only 1 cofactor and 1 [4Fe-4S] cluster with an additional [Fe4 -S4 ]like cluster [47]; 2 forms of VFe protein: form 1 has V-toFe ratio of 1:19, form 2 of 1:15 [47]; possesses 2 molybdenum-independent nitrogenases: one vanadium-containing nitrogenase and another lacking both molybdenum and vanadium [9,14]; characterization of the metal clusters in the nitrogenase vanadium-iron protein [15, 45]) [9, 14, 15, 45, 47] Additional information ( structure and organization of metal clusters [45, 47, 48]; redox properties of metal clusters [36]; the iron-only nitrogenase form contains no molybdenum, vanadium or any other heterometal atom [35]; contains also an inactive MoFe protein species [33]; metal-sulfur cluster, e.g. [4Fe-4S] [31]; other metal ions, e.g. Cu2+ , Mg2+ , Zn2+ , Ca2+ , at levels of 1-2 atoms per mol detected in the MoFe protein, no evidence for specific requirement, except for Mg2+ in MgATP complex, of any of these metals [3]) [3, 9, 31, 33, 35, 36, 45, 47, 48] ) &  * +, 18 (SCN- ) [37] 24000 (flavodoxine hydroquinone, before and after reduction of the nitrogenase complex relatively slow reactions take place, which limits the rate of the Fe protein cycle [44]) [44] Additional information ( 1200 per min: proton production of the reduced enzyme, MgATP-dependent [34]) [34] " & *-% , 0.024 ( purified mutant H195Q enzyme, anaerobic atmosphere [43]) [43] 0.027 ( reductant dithionite, whole cell assay [32]) [32] 0.066 ( crude extract, 100% Ar atmosphere, H2 production [48]) [48] 0.235 ( substrate N2 , purified Mo nitrogenase [35]) [35] 0.26 ( substrate C2 H2 , iron-only nitrogenase with Fe protein to FeFe protein ratio of 40:1 [35]) [35] 0.27 ( mutant A175G, purified enzyme, substrate C2H2 [41]) [41] 575

Nitrogenase

1.18.6.1

0.35 ( substrate N2 , iron-only nitrogenase with Fe protein to FeFe protein ratio of 40:1 [35]) [35] 0.43 ( purified Fe protein [24]) [24] 0.6-0.8 ( purified Fe protein [30]) [30] 0.7 ( purified enzyme, anaerobic atmosphere [43]; purified MoFe protein [24]) [24, 43] 1.01 ( purified MoFe protein [22]) [22] 1.07 ( purified MoFe protein [27]) [27] 1.2 ( substrate C2 H2 , purified Mo nitrogenase [35]; purified MoFe protein [18]) [18, 35] 1.26 ( purified Fe protein [27]) [27] 1.3 ( substrate H+ , purified Mo nitrogenase [35]) [35] 1.5-1.7 ( purified MoFe protein [30]) [30] 1.52 ( wild-type, purified enzyme, substrate C2 H2 [41]) [41] 1.7-2.2 ( purified Fe protein [18]) [18] 1.8 ( purified Fe protein [20]) [20] 2.01 ( purified Fe protein [22]) [22] 2.082 ( purified component II [16]) [16] 2.16 ( purified component I [16]) [16] 2.27-2.28 ( purified enzyme, substrate acetylene [37]) [37] 2.4 ( substrate H+ , iron-only nitrogenase with Fe protein to FeFe protein ratio of 40:1 [35]; purified MoFe protein [20]) [20, 35] Additional information ( wild-type and diverse a-His195 MoFe protein mutants [48]; assay in anaerobic atmosphere required [41,43,48]; overview [33]; activity of Fe proteins and MoFe proteins [2, 33]) [2, 3, 8, 13, 17, 21, 28-30, 33, 35, 41, 43, 48] . / *', 0.1 (N2 , with C2 H2 [17]) [13, 17] 0.3 (ATP, with C2 H2 [17]) [13, 17] 0.3 (C2 H2 ) [17] 0.4-1.2 (C2 H2 ) [13] 0.45 (CH4 , mutant H195Q [40]) [04] 0.9 (SCN- ) [37] 1.6 (CH4 , wild-type [40]) [40] 4.5 (CH4 , mutant H195N [40]) [40] 12 (CH4 , mutant Q191K [40]) [40] Additional information ( overview, different substrates [33]) [3, 13, 29, 33, 35, 42, 43, 48] . / *', 10.1 (SCN- ) [37] Additional information [43,48] 0  6.5 ( SCN- reduction [37]) [37] 6.5-8 [19]

576

1.18.6.1

Nitrogenase

7.1-7.3 ( with substrates: N2 , C2 H2 [27]) [27] 7.3 [17] 7.4 ( assay at [16,22,40,41]) [16, 22, 40, 41] 0 

 6.5-8.3 ( pH 6.5: no activity below, pH 8.3: about 70% of activity maximum [27]) [27] 6.5-8.5 ( below and above no remaining activity [35]) [35] )  *
, 15-40 ( no maximum with Ti3+ as reductant [39]) [39] 29 [19] 30 ( assay at [16, 17, 20-22, 39-41]) [16, 17, 20-22, 39-41] Additional information [43] ) 

 *
, 13-45 [43] 15-40 ( no maximum with Ti3+ as reductant [39]) [39]

  #   ' 1  35000-40000 ( Fe protein [33]) [33] 40000 ( Fe protein, ultracentrifugation [3]) [3] 40000-74000 ( Fe protein: various methods, overview [2, 3]) [2, 3] 51000 ( Fe protein, gel filtration [3]) [3] 55000-60000 ( component II Fe protein [6, 33]) [6, 33] 56000 ( Fe protein, gel filtration [3, 23]) [3, 23] 60000 ( about [45]; Fe protein [7,45]) [7, 45] 61500 ( Fe protein [1]) [1] 62000 ( Fe protein, gel filtration [3, 8]) [3, 8] 63000 ( Fe protein [1, 30, 44, 46]) [1, 30, 44, 46] 64000 ( Fe protein, gel filtration [3, 26]) [3, 26] 65000 ( Fe protein, gel filtration [3, 24]) [3, 24] 66800 ( Fe protein [33]) [33] 68200 ( Fe protein, ultracentrifugation [3]) [3] 74000 ( Fe protein, gel filtration [21]) [21] 160000 ( MoFe protein, estimation from Mo content, ultracentrifugation [3]) [3] 160000-270000 ( MoFe protein: various methods, overview [2,3]) [2, 3] 168000 ( MoFe protein, ultracentrifugation [2]) [2] 180000 ( MoFe protein, gel filtration [2]) [2] 194000 ( MoFe protein, gel filtration [2, 24]) [2, 24]

577

Nitrogenase

1.18.6.1

200000 ( MoFe protein, ultracentrifugation [3]; MoFe protein, gel filtration [2]) [2, 3] 210000 ( MoFe protein, gel filtration [23]) [23] 216000 ( nitrogenase complex [7]; MoFe protein, gel filtration [2]) [2, 7] 219000 ( MoFe protein, gel filtration [21]) [21] 220000 ( MoFe protein, gel filtration [8,29]) [8, 29] 220000-250000 ( component I MoFe protein [6]) [6] 226000 ( MoFe protein, gel filtration [2]) [2] 227000 ( MoFe protein, gel filtration [26]) [26] 230000 ( MoFe protein [1,30,44,46]) [1, 30, 44, 46] 270000 ( MoFe protein, ultracentrifugation [2,3]) [2, 3] Additional information ( comparison of amino acid composition [33]; enzyme consists of 2 proteins: a molybdenum and iron-containing protein, MoFe protein, component I, dinitrogenase, and an iron containing protein, Fe protein, component II, dinitrogenase reductase, together they form the active nitrogenase complex [1,8]) [1, 4, 5, 8, 18, 20, 24, 26-30, 33]   ? ( MoFe protein: 2 * 59000 + 2 * 57000, a2 b2 , SDS-PAGE [35]; FeFe protein of iron-only enzyme: 2 * 59000 + 2 * 51000 + 2 * 13500, a2 b2 g2 , SDS-PAGE [35]; Fe protein of both enzyme forms: 2 * 3000031000, g2 , SDS-PAGE [35]; MoFe protein 4 * 60000 + Fe protein 2 * 30800, SDS-PAGE [26]) [26, 35] dimer ( Fe protein is a dimer of 2 identical subunits, MW 27500-34600, various methods, overview [2,4]; 2 * 36000, SDS-PAGE [21]; 2 * 27500, SDS-PAGE [23]) [1-5, 21, 23] tetramer ( MoFe protein 1 * 55000 + 1 * 59000 + Fe protein 2 * 33500, SDS-PAGE [30]; component I Fe protein 2 * 27500 + component II MoFe protein 1 * 60000 + 1 * 51000, SDS-PAGE [23]; MoFe protein is an a2 b2 -tetramer [1]; a: 58500, b: 58500 [1]; a: 55000, b: 59500 [1]; a: 56000, b: 59000 [21]) [1, 3-5, 7, 21, 23, 30, 35] Additional information ( VFe protein form 1 has ab2 conformation, VFe protein form 2 has a2 b2 conformation [47]; a-bmonomer of the FeMo protein consists of the FeMo cofactor FeMo-co with the substrate reduction site and the [4Fe-4S] cluster [5]; enzyme is composed of 2 metalloproteins: Fe protein and MoFe protein which are assumed to associate and dissociate to transfer a single electron to the substrates [44]; apo-MoFe protein has a a2 b2 subunit composition and interacts with Fe protein, can be rebuilt by addition of FeMo-cofactor [38]; enzyme consists of 2 proteins: a molybdenum and iron-containing protein (MoFe protein, component I, dinitrogenase) and an iron containing protein, Fe protein, component II, dinitrogenase reductase, together they form the active nitrogenase complex [1]) [1, 4, 18, 20, 27-30, 38, 44, 47]

578

1.18.6.1

Nitrogenase

 2 %$ %' %

% heterocyst ( in heterocysteous cyanobacteria exclusive site of N2 fixation during aerobic growth [7]) [7] root nodule ( bacteroid [2,24]) [2, 24] 3#

Additional information ( not established, whether the nitrogenase exists in vivo in a specific particle or whether the nitrogenase proteins are bound nonspecifically to the membranes of some cells [3]) [3] $"

(2 forms of VFe protein [47]; wild-type and mutant H195Q [43,48]; wildtype and mutants H195Q, H195N, Q191K [40]; FeMo-cofactorless MoFe protein from nifB deletion mutant [38]; strict anaerobic conditions [12,22]; large scale [22]) [2, 13, 17, 22, 33, 37, 38, 40, 43, 47, 48] [30] (both components [16]) [2, 16] [2] (large scale [26]) [2, 26] (strict anaerobic conditions [12,18]; all components [23]) [2, 12, 18, 23] [29] (both components [24]) [24] (ORS571 [21]) [21] (strain Y1 [20]) [20] (both components [27]) [27] [28] (molybdenum containing enzyme form, both components [36]; irononly nitrogenase, both components [35]) [35, 36]
 #

(MoFe protein [12,13]; purified enzyme is diluted at room temperature with 3 volumes of Tris-HCl 0.01 M, pH 7.2, immediate crystal formation [13]; crystallized in presence of approx. 5% w/v polyethylene glycol 6000 and 0.20.4 M MgCl2 under strictly anaerobic conditions, X-ray analysis [12]) [3, 6, 12, 13] (MoFe protein, crystallized in presence of approx. 5% w/v polyethylene glycol 6000 and 0.2-0.4 M MgCl2 under strictly anaerobic conditions, X-ray analysis [12]) [12]
 

(genetic analysis [14]) [14] (expression in Escherichia coli [10]) [10] (amino acid sequence comparison with other species [11]; expression in Escherichia coli, expression in Klebsiella pneumoniae, Anabaena gene library screening with genes of Klebsiella pneumoniae [11]) [11]

579

Nitrogenase

1.18.6.1



 

A175G ( shows in vivo 55% of enzyme activity compared to wildtype, in vitro 20% activity remaining with purified enzyme, slowlier conformational change upon binding of MgATP, model of steric interactions using X-ray crystal structures [41]) [41] A175S ( unable to support substrate reduction because of an inability to undergo required a MgATP-induced conformational change [41]) [41] H195G ( a-His of MoFe protein, site directed mutagenesis, reduced MoFe protein activity, slightly decreased Fe protein activity, altered phenotype [48]) [48] H195L ( a-His of MoFe protein, site directed mutagenesis, reduced MoFe protein activity, increased Fe protein activity, altered phenotype [48]) [48] H195N ( aHis195 of MoFe protein, shows 59% activity compared to wild-type, substrate CN- , NH3 and CH4 production from CN- are decreased by C2 H2 addition, NH3 production decreased much less [40]; a-His of MoFe protein, site directed mutagenesis, reduced MoFe protein activity, altered phenotype [48]) [40, 48] H195Q ( below 2% N2 reducing activity remaining compared to wild-type due to less effective N2 binding [43]; aHis195 of MoFe protein, shows 159% activity compared to wild-type, substrate CN- , NH3 and CH4 production from CN- are decreased by C2 H2 addition [40]; a-His of MoFe protein, site directed mutagenesis, decreased MoFe protein activity, altered phenotype [48]) [40, 43, 48] H195T ( a-His of MoFe protein, site directed mutagenesis, reduced MoFe protein and Fe protein activity, altered phenotype [48]) [48] H195Y ( a-His of MoFe protein, site directed mutagenesis, reduced MoFe protein and Fe protein activity, altered phenotype [48]) [48] Q191K ( aGln191 of MoFe protein, shows 6% activity compared to wild-type, substrate CN- , not affected by addition of C2 H2 [40]) [40] S69G ( a-subunit MoFe protein, resistant to inhibition by C2 H2 , thus acetylene binding/reduction site is not directly relevant to the mechanism of nitrogen reduction [42]) [42] Additional information ( natural nifB deletion mutant, MoFe protein without FeMo-cofactor and with small changes in the electronic properties of the [4Fe-4S] cluster [38]; construction of 2 mutants strain: 1 kanamycin-resistant with a deletion in NifHDK and 1 kanamycin, gentamycin, and molybdenum-resistant with double deletion in nif HDK and modABCD [35]; construction of mutant strain RP114 [14]; transposon insertion mutants of several plasmids [10]) [10, 14, 35, 38, 42]

4  0  5-8 ( MoFe protein stable [26]) [26] 8.7 ( 50% loss of activity after overnight dialysis at pH 8.7 [26]) [26] 580

1.18.6.1

Nitrogenase

)  22 ( Fe protein, half-life: 18 h [17]) [17] Additional information ( Fe protein: cold labile [17]) [17]   , extreme O2 lability, susceptibility to O2 increases with purification, but is retarded in presence of MgCl2 [17] , complete reversible inhibition by O2, reversibility decreases by increasing the time of exposure to O2, after 20 min 60% reversibility of the inhibition remains [32] , extreme O2 lability, t1=2 : 10 min, MoFe protein 45 s, Fe protein [8] , Fe protein is very O2 sensitive [18] , extreme O2 lability, t1=2 : 1 min, Fe protein [24] , stable to O2, no loss in nitrogen fixation activity [7] , t1=2 Fe protein: 45 sec, t1=2 MoFe protein: 10 min [2] , MoFe protein is extremely sensitive to O2 [6] , overview: O2 lability and protection mechanisms against O2 in various organisms in vivo [25] , extreme sensitivity to O2 [1-3, 7] 5    "

, nitrogenase complex is more stable than either the MoFe protein or the Fe protein alone [17] , Fe protein is salt sensitive [18]    , -15 C, anaerobic storage, overnight, complete loss of activity [13] , 0 C, anaerobic conditions, FeMo protein stable [17] , 22 C, O2 -free atmosphere, pH 7-8, stable [13] , 5 C, O2 -free atmosphere, overnight, 80% loss of activity [13]

"  [1] Vignais, P.M.; Colbeau, A.; Willison, J.C.; Jouanneau, Y.: Hydrogenase, nitrogenase, and hydrogen metabolism in the photosynthetic bacteria. Adv. Microb. Physiol., 26, 155-234 (1985) [2] Eady, R.R.: Isolation and characterization of various nitrogenases. Methods Enzymol., 69, 753-778 (1980) [3] Winter, H.C.; Burris, R.H.: Nitrogenase. Annu. Rev. Biochem., 45, 409-426 (1976) [4] Eady, R.R.; Postgate, J.R.: Nitrogenase. Nature, 249, 805-810 (1974) [5] Mortenson, L.E.; Thorneley, R.N.F.: Structure and function of nitrogenase. Annu. Rev. Biochem., 48, 387-418 (1979) [6] Shah, V.K.; Ugalde, R.A.; Imperial, J.; Brill, W.J.: Molybdenum in nitrogenase. Annu. Rev. Biochem., 53, 231-257 (1984) [7] Houchins, J.P.: The physiology and biochemistry of hydrogen metabolism in cyanobacteria. Biochim. Biophys. Acta, 768, 227-255 (1984)

581

Nitrogenase

1.18.6.1

[8] Palmer, G.: Iron-sulfur proteins. The Enzymes, 3rd Ed. (Boyer, P.D., ed.), 12, 1-56 (1975) [9] Pau, R.N.: Nitrogenases without molybdenum. Trends Biochem. Sci., 14, 183-186 (1989) [10] Fuhrmann, M.; Hennecke, H.: Coding properties of cloned nitrogenase structural genes from Rhizobium japonicum. Mol. Gen. Genet., 187, 419425 (1982) [11] Hirschberg, R.; Samson, S.M.; Kimmel, B.E.; Page, K. A.; Collins, J.J.; Myers, J.A.; Yarbrough, L.R.: Cloning and characterization of nitrogenase genes from Anabaena variabilis. J. Biotechnol., 2, 23-37 (1985) [12] Weininger, M.S.; Mortenson, L.E.: Crystallographic properties of the MoFe proteins of nitrogenase from Clostridium pasteurianum and Azotobacter vinelandii. Proc. Natl. Acad. Sci. USA, 79, 379-380 (1982) [13] Burns, R.C.; Hardy, R.W.F.: Purification of nitrogenase and crystallization of its Mo-Fe protein. Methods Enzymol., 24, 480-496 (1972) [14] Pau, R.N.; Mitchenall, L.A.; Robson, R.L.: Genetic evidence for an Azotobacter vinelandii nitrogenase lacking molybdenum and vanadium. J. Bacteriol., 171, 124-129 (1989) [15] Morningstar, J.E.; Johnson, M.K.; Case, E.E.; Hales, B.J.: Characterization of the metal clusters in the nitrogenase molybdenum-iron and vanadium-iron proteins of Azotobacter vinelandii using magnetic circular dichroism spectroscopy. Biochemistry, 26, 1795-1800 (1987) [16] Shah, V.K.: Isolation and characterization of nitrogenase from Klebsiella pneumoniae. Methods Enzymol., 118, 511-519 (1986) [17] Bulen, W.A.; LeComte, J.R.: Nitrogenase complex and its components. Methods Enzymol., 24B, 456-470 (1972) [18] Mortenson, L.E.: Purification aof nitrogenase from Clostridium pasteurianum. Methods Enzymol., 24B, 446-456 (1972) [19] Polman, J.K.; Larkin, J.M.: Properties of in vivo nitrogenase activity in Beggiatoa alba. Arch. Microbiol., 150, 126-130 (1988) [20] Song, S.D.; Hartmann, A.; Burris, R.H.: Purification and properties of the nitrogenase of Azospirillum amazonense. J. Bacteriol., 164, 1271-1277 (1985) [21] Kush, A.; Elmerich, C.; Aubert, J.P.: Nitrogenase of Sesbania Rhizobium strain ORS571: purification, properties, and 'switch-off ' by ammonia. J. Gen. Microbiol., 131, 1765-1777 (1985) [22] Burgess, B.K.; Jacobs, D.B.; Stiefel, E.I.: Large-scale purification of high activity Azotobacter vinelandii nitrogenase. Biochim. Biophys. Acta, 614, 196209 (1980) [23] Tso, M.Y.W.: Some properties of the nitrogenase proteins from Clostridium pasteurianum. Molecular weight, subunit structure, isoelectric point and EPR spectra. Arch. Microbiol., 99, 71-80 (1974) [24] Whitting, M.J.; Dilworth, M. J.: Legume root nodule nitrogenase. Purification, properties, and studies on its genetic control. Biochim. Biophys. Acta, 371, 337-351 (1974) [25] Oelze, J.: Mechanismen zum Schutz der Sauerstoff-labilen Nitrogenase. Forum Mikrobiol., 4, 116-126 (1988) 582

1.18.6.1

Nitrogenase

[26] Yates, M.G.; Planque, K.: Nitrogenase from Azotobacter chroococcum. Purification and properties of the component proteins. Eur. J. Biochem., 60, 467-476 (1975) [27] Berndt, H.; Lowe, D.J.; Yates, M.G.: The nitrogen-fixing system of Corynebacterium autotrophicum. Purification and properties of the nitrogenase components and two ferredoxins. Eur. J. Biochem., 86, 133-142 (1978) [28] Emerich, D.W.; Burris, R.H.: Nitrogenase from Bacillus polymyxa. Purification and properties of the component proteins. Biochim. Biophys. Acta, 536, 172-183 (1978) [29] Hallenbeck, P.C.; Kostel, P.J.; Benemann, J.R.: Purification and properties of nitrogenase from the cyanobacterium, Anabaena cylindrica. Eur. J. Biochem., 98, 275-284 (1979) [30] Hallenbeck, P.C.; Meyer, C.; Vignais, P.M.: Nitrogenase from the photosynthetic bacterium Rhodopseudomonas capsulata: purification and molecular properties. J. Bacteriol., 149, 708-717 (1982) [31] Haaker, H.; Klugkist, J.: The bioenergetics of electron transport to nitrogenase. FEMS Microbiol. Rev., 46, 57-71 (1987) [32] Kavanagh, E.P.; Hill, S.: Oxygen inhibition of nitrogenase activity in Klebsiella pneumoniae. J. Gen. Microbiol., 139, 1307-1314 (1992) [33] Zumft, W.G.; Mortenson, L.E.: The nitrogen-fixing complex of bacteria. Biochim. Biophys. Acta, 416, 1-52 (1975) [34] Duyvis, M.G.; Wassink, H.; Haaker, H.: Pre-steady-state MgATP-dependent proton production and electron transfer by nitrogenase from Azotobacter vinelandii. Eur. J. Biochem., 225, 881-890 (1994) [35] Schneider, K.; Gollan, U.; Drottboom, M.; Selsemeier-Voigt, S.; Muller, A.: Comparative biochemical characterization of the iron-only nitrogenase and the molybdenum nitrogenase from Rhodobacter capsulatus. Eur. J. Biochem., 244, 789-800 (1997) [36] Siemann, S.; Schneider, K.; Drottboom, M.; Muller, A.: The Fe-only nitrogenase and the Mo nitrogenase from Rhodobacter capsulatus: a comparative study on the redox properties of the metal clusters present in the dinitrogenase components. Eur. J. Biochem., 269, 1650-1661 (2002) [37] Rasche, M.E.; Seefeldt, L.C.: Reduction of thiocyanate, cyanate, and carbon disulfide by nitrogenase: kinetic characterization and EPR spectroscopic analysis. Biochemistry, 36, 8574-8585 (1997) [38] Christiansen, J.; Goodwin, P.J.; Lanzilotta, W.N.; Seefeldt, L.C.; Dean, D.R.: Catalytic and biophysical properties of a nitrogenase Apo-MoFe protein produced by a nifB-deletion mutant of Azotobacter vinelandii. Biochemistry, 37, 12611-12623 (1998) [39] Erickson, J.A.; Nyborg, A.C.; Johnson, J.L.; Truscott, S.M.; Gunn, A.; Nordmeyer, F.R.; Watt, G.D.: Enhanced efficiency of ATP hydrolysis during nitrogenase catalysis utilizing reductants that form the all-ferrous redox state of the Fe protein. Biochemistry, 38, 14279-14285 (1999) [40] Fisher, K.; Dilworth, M.J.; Kim, C.H.; Newton, W.E.: Azotobacter vinelandii nitrogenases with substitutions in the FeMo-cofactor environment of the MoFe protein: effects of acetylene or ethylene on interactions with H+ , HCN, and CN- . Biochemistry, 39, 10855-10865 (2000) 583

Nitrogenase

1.18.6.1

[41] Bursey, E.H.; Burgess, B.K.: Characterization of a variant iron protein of nitrogenase that is impaired in its ability to adopt the MgATP-induced conformational change. J. Biol. Chem., 273, 16927-16934 (1998) [42] Christiansen, J.; Cash, V.L.; Seefeldt, L.C.; Dean, D.R.: Isolation and characterization of an acetylene-resistant nitrogenase. J. Biol. Chem., 275, 1145911464 (2000) [43] Dilworth, M.J.; Fisher, K.; Kim, C.H.; Newton, W.E.: Effects on substrate reduction of substitution of histidine-195 by glutamine in the a-subunit of the MoFe protein of Azotobacter vinelandii nitrogenase. Biochemistry, 37, 17495-17505 (1998) [44] Duyvis, M.G.; Wassink, H.; Haaker, H.: Nitrogenase of Azotobacter vinelandii: kinetic analysis of the Fe protein redox cycle. Biochemistry, 37, 1734517354 (1998) [45] Kim, J.; Rees, D.C.: Nitrogenase and biological nitrogen fixation. Biochemistry, 33, 389-397 (1994) [46] Johnson, J.L.; Tolley, A.M.; Erickson, J.A.; Watt, G.D.: Steady-state kinetic studies of dithionite utilization, component protein interaction, and the formation of an oxidized iron protein intermediate during Azotobacter vinelandii nitrogenase catalysis. Biochemistry, 35, 11336-11342 (1996) [47] Blanchard, C.Z.; Hales, B.J.: Isolation of two forms of the nitrogenase VFe protein from Azotobacter vinelandii. Biochemistry, 35, 472-478 (1996) [48] Kim, C.H.; Newton, W.E.; Dean, D.R.: Role of the MoFe protein a-subunit histidine-195 residue in FeMo-cofactor binding and nitrogenase catalysis. Biochemistry, 34, 2798-2808 (1995)

584

 

7:4

   
 1.18.96.1 (transferred to EC 1.15.1.2)    superoxide reductase

585

0   

   
 1.18.99.1 (transferred to EC 1.12.7.2)    hydrogenase

586

7::

   *"& ,

:4

   
 1.19.6.1    reduced flavodoxin:dinitrogen oxidoreductase (ATP-hydrolysing)    nitrogenase (flavodoxin)    EC 1.19.2.1 (formerly) Additional information (see EC 1.18.6.1, from the literature for the enzymes EC 1.19.6.1 and EC 1.18.6.1 it is not obvious whether they are in fact 2 different enzymes)
    9013-04-1

    



Klebsiella pneumoniae (nif gene-specific flavodoxin [1,3]) [1-3, 7] Azotobacter vinelandii (OP [4]; nif-specific flavodoxin [4]) [4, 5] Azotobacter chroococcum [6] Rhodobacter capsulatus (nif gene-specific flavodoxin [8]) [8]

!    "
 # 

6 reduced flavodoxin + 6 H+ + N2 + n ATP = 6 oxidized flavodoxin + 2 NH3 + n ADP + n phosphate ( residues Asn11, Ser68, and Asn72 of the flavodoxin are involved in complex formation between the flavodoxin and the Fe protein of the enzyme, influenced by MgADP- [6]; enzyme complex dissociation and association kinetics [5]; mechanism, Fe protein cycle [5]; enzyme is composed of 2 metalloproteins: Fe protein and MoFe protein which are assumed to associate and dissociate to transfer a single electron to the substrates [5])

587

Nitrogenase (flavodoxin)

1.19.6.1

   oxidation redox reaction reduction       reduced flavodoxin + H+ + N2 + ATP ( enzyme consists of 2 metalloproteins, Fe protein and MoFe protein, which are assumed to associate and dissociate to transfer a single electron to the substrates, termed Fe protein cycle, driven by MgATP hydrolysis, with the dissociation of the Fe protein-MoFe protein complex being the rate limiting step of the cycle [5]; anaerobic conditions are required by the enzyme [1, 2, 7]; ferredoxin is the immediate electron carrier to nitrogenase in all nitrogen-fixing organisms with the exception of Klebsiella pneumoniae, and possibly Azotobacter species, where only flavodoxin, not ferredoxin is effective in coupling electron flow to nitrogenase [1]) (Reversibility: ? [1-5, 7]) [1-5, 7] $ oxidized flavodoxin + NH3 + ADP + phosphate      dithionite + H+ + N2 + ATP (Reversibility: ? [2, 5, 7]) [2, 5, 7] $ ?  reduced flavodoxin + H+ + N2 + ATP ( intermediate is a flavodoxin hydroquinone [5]; enzyme consists of 2 metalloproteins, Fe protein and MoFe protein, which are assumed to associate and dissociate to transfer a single electron to the substrates, termed Fe protein cycle, driven by MgATP hydrolysis, with the dissociation of the Fe protein-MoFe protein complex being the rate-limiting step of the cycle [5]; flavodoxin is not essential but required for maximum in vivo nitrogenase activity [4]; highly specific for flavodoxin as electron donor [3]; flavodoxin is encoded by nifF gene, pyruvate:flavodoxin oxidoreductase by nifJ gene [1, 3, 4, 8]; flavodoxin reduces the Fe protein of the enzyme [1,4]; slow enzyme [1,5]) (Reversibility: ? [1-8]) [18] $ oxidized flavodoxin + NH3 + ADP + phosphate ( i.e. flavodoxin semiquinone [5]) [1-8]  Additional information ( dithionite can serve as reductant in vitro [5]; sodium dithionite and reduced methyl viologen can in vitro reduce the flavodoxin and the enzyme directly [2]; see EC 1.18.6.1, from the literature for the enzymes EC 1.19.6.1 and EC 1.18.6.1 it is not obvious whether they are in fact to different enzymes [1]) [1, 2, 5] $ ? 2  O2 ( complete reversible inhibition, reversibility decreases by increasing the time of exposure to O2 [7]) [7]

588

1.19.6.1

Nitrogenase (flavodoxin)

'(   Mg2+ ( required for ATP hydrolysis [1,5,7]) [1, 5, 7] iron ( enzyme consists of 2 proteins: a molybdenum and iron-containing protein, MoFe protein, component I, dinitrogenase, and an iron containing protein, Fe protein, component II, dinitrogenase reductase, together they form the active nitrogenase complex [5]) [5] molybdenum ( enzyme consists of 2 proteins: a molybdenum and iron-containing protein, MoFe protein, component I, dinitrogenase, and an iron containing protein, Fe protein, component II, dinitrogenase reductase, together they form the active nitrogenase complex [5]) [5] ) &  * +, 24000 (flavodoxin hydroquinone, before and after reduction of the nitrogenase complex relative slow reactions take place, which limits the rate of the Fe protein cycle [5]) [5] " & *-% , 0.027 ( reductant dithionite [7]) [7] Additional information ( coupled assay with pyruvate:flavodoxin oxidoreductase [3]) [3]

  #   ' 1  230000 ( nitrogenase MoFe protein component [5]) [5]   dimer ( 2 * a-b-monomer [5]) [5] Additional information ( a-b-monomer of the FeMo protein consists of the FeMo cofactor FeMoco with the substrate reduction site and the Pcluster [5]; enzyme is composed of 2 metalloproteins: Fe protein and MoFe protein which are assumed to associate and dissociate to transfer a single electron to the substrates [5]) [5]

 2 %$ %' %

$"

(partial [2]) [2]

4    , complete reversible inhibition by O2, reversibility decreases by increasing the time of exposure to O2, after 20 min 60% reversibility of the inhibition remains [7]

589

Nitrogenase (flavodoxin)

1.19.6.1

"  [1] Haaker, H.; Klugkist, J.: The bioenergetics of electron transport to nitrogenase. FEMS Microbiol. Rev., 46, 57-71 (1987) [2] Yoch, D.C.: Electron transport carriers involved in nitrogen fixation by the coliform, Klebsiella pneumoniae. J. Gen. Microbiol., 83, 153-164 (1974) [3] Shah, V.K.; Stacey, G.; Brill, W.J.: Electron transport to nitrogenase. Purification and characterization of pyruvate:flavodoxin oxidoreductase. The nifJ gene product. J. Biol. Chem., 258, 12064-12068 (1983) [4] Bennett, L.T.; Jacobson, M.R.; Dean, D.R.: Isolation, sequencing, and mutagenesis of the nifF gene encoding flavodoxin from Azotobacter vinelandii. J. Biol. Chem., 263, 1364-1369 (1988) [5] Duyvis, M.G.; Wassink, H.; Haaker, H.: Nitrogenase of Azotobacter vinelandii: kinetic analysis of the Fe protein redox cycle. Biochemistry, 37, 1734517354 (1998) [6] Peelen, S.; Wijmenga, S.; Erbel, P.J.; Robson, R.L.; Eady, R.R.; Vervoort, J.: Possible role of a short extra loop of the long-chain flavodoxin from Azotobacter chroococcum in electron transfer to nitrogenase: complete 1H, 15N and 13C backbone assignments and secondary solution structure of the flavodoxin. J. Biomol. NMR, 7, 315-330 (1996) [7] Kavanagh, E.P.; Hill, S.: Oxygen inhibition of nitrogenase activity in Klebsiella pneumoniae. J. Gen. Microbiol., 139, 1307-1314 (1992) [8] Yakunin, A.F.; Gennaro, G.; Hallenbeck, P.C.: Purification and properties of a nif-specific flavodoxin from the photosynthetic bacterium Rhodobacter capsulatus. J. Bacteriol., 175, 6775-6780 (1993)

590

$     

=

   
 1.20.1.1    phosphonate:NAD+ oxidoreductase    phosphonate dehydrogenase    NAD-dependent phosphite dehydrogenase NAD:phosphite oxidoreductase phosphite dehydrogenase
    9031-35-0

     Pseudomonas stutzeri [1, 2]

!    "
 # 

phosphonate + NAD+ + H2 O = phosphate + NADH + H+    redox reaction       phosphite + H2 O + NAD+ ( use of phosphite as energy source, regeneration of NADH [1]) (Reversibility: ir [1]) [1, 2] $ phosphate + NADH + H+ [1, 2]      hydroxypyruvate + NAD+ ( very poor substrate [1]) (Reversibility: ? [1]) [1] $ pyruvate + NADH [1]  phosphite + H2 O + NAD+ (Reversibility: ir [1]) [1, 2] $ phosphate + NADH + H+ [1, 2]

591

Phosphonate dehydrogenase

1.20.1.1

 phosphite + H2 O + NADP+ ( NADP poorly substitutes for NAD [1,2]) (Reversibility: ? [1, 2]) [1, 2] $ phosphate + NADPH + H+ [1, 2] 2  dl-hydroxyisocapronate [1] d-2-hydroxy-4-methylvalerate [1] d-glycerate [1] NADH ( competitive [1]) [1] NaCl [1] arsenite [1] formate [1] nitrite [1] sulfite ( dead end inhibitor, competitive [1]) [1] ) &  * +, 440 (phosphite) [1] " & *-% , 0.01 ( crude extract from Pseudomonas stutzeri [2]) [2] 0.21 ( crude extract from Escherichia coli clone [2]) [2] 5.2 ( purified enzyme from Pseudomonas stutzeri [2]) [2] 6.52 ( purified enzyme from Escherichia coli clone [2]) [2] 12.2 [1] . / *', 0.053 (phosphite) [1] 0.055 (NAD+ ) [1] 0  8 [1] 0 

 4.5-10 ( low activity [1]) [1] )  *
, 35 [2] ) 

 *
, 15-50 ( 25% of activity at 15 C, no activity at 50 C [2]) [2]

  #   ' 1  69000 ( gel filtration [1]) [1]   homodimer ( gel filtration [1]) [1]

592

1.20.1.1

Phosphonate dehydrogenase

 2 %$ %' %

$"

(recombinant protein [1]; recombinant and native protein [2]) [1, 2]
 

(in Escherichia coli [1,2]) [1, 2]

"  [1] Costas, A.M.G.; White, A.K.; Metcalf, W.W.: Purification and characterization of a novel phosphorus-oxidizing enzyme from Pseudomonas stutzeri WM88. J. Biol. Chem., 276, 17429-17436 (2001) [2] Vrtis, J.M.; White, A.K.; Metcalf, W.W.; van der Donk, W.A.: Phosphite dehydrogenase: An unusual phosphoryl transfer reaction. J. Am. Chem. Soc., 123, 2672-2673 (2001)

593

   *  ,

=

   
 1.20.4.1    glutharedoxin:arsenate oxidoreductase    arsenate reductase (glutaredoxin)    arsenical pump modifier EC 1.97.1.5 (formerly)

    



Staphylococcus aureus (plasmid PI258 [4]) [4, 7] Chrysiogenes arsenatis [5] Homo sapiens [8] Bacillus subtilis [9] Escherichia coli [1-3, 6, 10]

!    "
 # 

arsenate + glutaredoxin = arsenite + glutaredoxin disulfide (A molybdoenzyme. The glutaredoxins catalyse glutathione-disulfide oxidoreductions and have a redox-active disulfide/dithiol in the active site (-Cys-Pro-Tyr-Cys-) that forms a disulfide bond in the oxidized form [2,10]. Glutaredoxins have a binding site for glutathione, which is required to reduce them to the dithiol form [3, 6]. Thioredoxins reduced by NADPH2 and thioredoxin reductase can act as alternative substrates. The enzyme [1, 4, 7, 9] is a part of a system for detoxifying arsenate. Although the arsenite formed is more toxic than arsenate, it can be extruded from some bacteria by EC 3.6.3.16, arsenite-transporting ATPase; in other organisms, arsenite can be methylated by EC 2.1.1.137, arsenite methyltransferase, in a pathway to non-toxic organoarsenical compounds)

594

1.20.4.1

Arsenate reductase (glutaredoxin)

   oxidation reduction      arsenate + reduced glutaredoxin (Reversibility: ? [4, 5, 7-10]) [4, 5, 7-10] $ arsenite + oxidized glutaredoxin

"  [1] Gladysheva, T.; Liu, J.Y.; Rosen, B.P.: His-8 lowers the pKa of the essential Cys-12 residue of the ArsC arsenate reductase of plasmid R773. J. Biol. Chem., 271, 33256-33260 (1996) [2] Gladysheva, T.B.; Oden, K.L.; Rosen, B.P.: Properties of the arsenate reductase of plasmid R773. Biochemistry, 33, 7288-7293 (1994) [3] Holmgren, A.; Aslund, F.: Glutaredoxins. Methods Enzymol., 252, 283-292 (1995) [4] Ji, G.Y.; Garber, E.A.E.; Armes, L.G.; Chen, C.M.; Fuchs, J.A.; Silver, S.: Arsenate reductase of Staphylococcus aureus plasmid PI258. Biochemistry, 33, 7294-7299 (1994) [5] Krafft, T.; Macy, J.M.: Purification and characterization of the respiratory arsenate reductase of Chrysiogenes arsenatis. Eur. J. Biochem., 255, 647653 (1998) [6] Martin, J.L.: Thioredoxin - a fold for all reasons. Structure, 3, 245-250 (1995) [7] Messens, J.; Hayburn, G.; Desmyter, A.; Laus, G.; Wyns, L.: The essential catalytic redox couple in arsenate reductase from Staphylococcus aureus. Biochemistry, 38, 16857-16865 (1999) [8] Radabaugh, T.R.; Aposhian, H.V.: Enzymatic reduction of arsenic compounds in mammalian systems: reduction of arsenate to arsenite by human liver arsenate reductase. Chem. Res. Toxicol., 13, 26-30 (2000) [9] Sato, T.; Kobayashi, Y.: The ars operon in the skin element of Bacillus subtilis confers resistance to arsenate and arsenit. J. Bacteriol., 180, 1655-1661 (1998) [10] Shi, J.; Vlamis-Gardikas, V.; Aslund, F.; Holmgren, A.; Rosen, B.P.: Reactivity of glutaredoxins 1, 2, and 3 from Escherichia coli shows that glutaredoxin 2 is the primary hydrogen donor to ArsC-catalyzed arsenate reduction. J. Biol. Chem., 274, 36039-36042 (1999)

595

'   

=

   
 1.20.4.2    gluthathione:methylarsonate oxidoreductase    methylarsonate reductase    MMA(V) reductase reductase, methylarsonate
    254889-62-8

     Oryctolagus cuniculus [1] Homo sapiens [1] Mesocricetus auratus [2]

!    "
 # 

methylarsonate + 2 glutathione = methylarsonite + glutathione disulfide    oxidation redox reaction reduction       methylarsonate + glutathione (, rate-limiting enzyme in biotransformation of inorganic arsenite in rabbit liver [1]) (Reversibility: ? [1]) [1] $ methylarsonite + oxidized glutathione      arsenate + glutathione (Reversibility: ? [1]) [1] $ arsenite + reduced glutathione

596

1.20.4.2

Methylarsonate reductase

 dimethylarsonate + glutathione (Reversibility: ? [1]) [1] $ dimethylarsonous acid  monomethylarsonate + glutathione (Reversibility: ? [1, 2]) [1, 2] $ monomethylarsonous acid + oxidized glutathione [1] &

  dithiothreitol (, stimulates [1]) [1] " & *-% , Additional information [1] . / *', 2.1 (monomethylarsonate) [1] 20.9 (dimethylarsonate) [1] 109 (arsenate) [1] 0  8 [1]

 2 %$ %' %

% bladder [2] brain [2] heart [2] kidney [2] liver [1, 2] lung [2] skin [2] spleen [2] testis [2] $"

(partial [1]) [1]

"  [1] Zakharyan, R.A.; Aposhian, H.V.: Enzymatic reduction of arsenic compounds in mammalian systems: the rate-limiting enzyme of rabbit liver arsenic biotransformation is MMA(V) reductase. Chem. Res. Toxicol., 12, 1278-1283 (1999) [2] Sampayo-Reyes, A.; Zakharyan, R.A.; Healy, S.M.; Aposhian, H.V.: Monomethylarsonic acid reductase and monomethylarsonous acid in hamster tissue. Chem. Res. Toxicol., 13, 1181-1186 (2000)

597

   *# ,

=:7

   
 1.20.98.1    arsenite:azurin oxidoreductase    arsenate reductase (azurin)    arsenite oxidase
    144638-82-4

     Alcaligenes faecalis [1, 2] Chrysiogenes arsenatis [3]

!    "
 # 

arsenite + H2 O + azurinox = arsenate + azurinred    oxidation redox reaction reduction      arsenate + azurinred (Reversibility: ? [3]) [3] $ arsenite + H2 O + azurinox  arsenite + H20 + azurinox (Reversibility: ? [1]; ir [2]) [1, 2] $ arsenate + azurinred [1, 2] '(   Fe ( contains 3 to 4 mol of Fe per 85 kD protein [1]; contains a [3Fe-4S] cluster and a Rieske-type [2Fe-2S] site [2]; contains [3]) [1-3]

598

1.20.98.1

Arsenate reductase (azurin)

Mo ( contains 0.75 mol Mo per mol of protein [1]; contains one Mo binding site [1]; contains [3]) [1, 3] Zn ( contains [3]) [3] ) &  * +, 1620 (azurin) [1] 862600 (arsenate) [3] " & *-% , 2.88 [1] 7013 [3] . / *', 0.008 (azurin) [1] 0.07 (arsenite) [1] 0.3 (arsenate) [3] 0  6 [1]

  #   ' 1  85000 ( gel filtration [1]) [1] 123000 ( gel filtration [3]) [3]   dimer ( 1 * 87000 + 1 * 29000, SDS-PAGE [3]) [3] monomer ( SDS-PAGE [1]) [1] Additional information ( heterodimer [2]) [2]

 2 %$ %' %

3#

periplasm [3] $"

[1] [3]
 #

[2]

599

Arsenate reductase (azurin)

1.20.98.1

"  [1] Anderson, G.L.; Williams, J.; Hille, R.: The purification and characterization of arsenite oxidase from Alcaligenes faecalis, a molybdenum-containing hydroxylase. J. Biol. Chem., 267, 23674-23682 (1992) [2] Ellis, P.J.; Conrads, T.; Hille, R.; Kuhn, P.: Crystal structure of the 100 kDa arsenite oxidase from Alcaligenes faecalis in two crystal forms at 1.64 A and 2.03 A. Structure, 9, 125-132 (2001) [3] Krafft, T.; Macy, J.M.: Purification and characterization of the respiratory arsenate reductase of Chrysiogenes arsenatis. Eur. J. Biochem., 255, 647-653 (1998)

600

   * ,

=::

   
 1.20.99.1    arsenate:(acceptor) oxidoreductase    arsenate reductase (donor)    EC 1.97.1.6 (formerly)

     Chrysiogenes arsenatis [1] Homo sapiens [2]

!    "
 # 

arsenite + acceptor = arsenate + reduced acceptor (Benzyl viologen can act as an acceptor. Unlike EC 1.97.1.5 arsenate reductase (glutaredoxin), reduced glutaredoxin cannot serve as a reductant)    oxidation reduction      arsenate + donor (Reversibility: ? [1, 2]) [1, 2] $ arsenite + oxidized donor

"  [1] Krafft, T.; Macy, J.M.: Purification and characterization of the respiratory arsenate reductase of Chrysiogenes arsenatis. Eur. J. Biochem., 255, 647-653 (1998) [2] Radabaugh, T.R.; Aposhian, H.V.: Enzymatic reduction of arsenic compounds in mammalian systems: reduction of arsenate to arsenite by human liver arsenate reductase. Chem. Res. Toxicol., 13, 26-30 (2000)

601

2    

!

   
 1.21.3.1    N-[(5S)-5-amino-5-carboxypentanoyl]-l-cysteinyl-d-valine:oxygen oxidoreductase (cyclizing)    isopenicillin-N synthase    IPN synthase ( ATP is not required, and therefore the enzyme should be named IPN synthase rather than IPN synthetase [1]) [1] IPNS [1-17] isopenicillin N synthase isopenicillin N synthase (cyclase) ( ATP is not required, and therefore the enzyme should be named IPN synthase rather than IPN synthetase [1]) [1] isopenicillin N synthetase isopenicillin N-synthase [3, 16] synthetase, isopenicillin N (9Cl)
    78642-31-6

     Streptomyces lactamdurans (i.e. Nocardia lactamdurans, NRRL 3802 var. JC 1843, cephamycin producing strain, Amy+ mutant [1]) [1] Penicillium chrysogenum [1, 5, 17] Cephalosporium acremonium (i.e. Acremonium chrysogenum [1, 4, 5]) [1, 4-6, 10, 11, 16] Streptomyces calvuligerus [2] Flavobacterium sp. (strain SC 12.154 [3]) [3] Aspergillus nidulans [7, 10] Streptomyces clavuligerus [8] Streptomyces jumonjinensis [10, 15] Aspergillus nidulans [9, 10, 12, 14] Streptomyces lipmanii (strain NRRL 3584 [13]) [13]

602

1.21.3.1

Isopenicillin-N synthase

!    "
 # 

N-[(5S)-5-amino-5-carboxypentanoyl]-l-cysteinyl-d-valine + O2 = isopenicillin N + 2 H2 O (forms part of the penicillin biosynthesis pathway; catalytic reaction is under steric regulation [12]; structure and mechanism [7, 9, 10, 12]; ligation of substrate to the iron centre [9]; active site with conserved jelly-roll motif, cysteine residues are not directly involved in the coordination of the metal ion [7]; removal of 4 hydrogen atoms to form the 4-membered b-lactam and the 5-membered thiazolidine ring [1]; Cys106 is involved in substrate binding, Cys255 is involved in maintaining the protein structure [4])    oxidation oxidative cyclization [1-16] redox reaction reduction       N-[(5S)-5-amino-5-carboxypentanoyl]-l-cysteinyl-d-valine + O2 ( catalytic reaction is under steric regulation [12]; modifications of the l-cysteine residue in the second position of the enzyme resulted in tripeptides that were unable to serve as substrates [5]; evaluation of culture conditions for penicillin and cephalosporin C production [2]; key enzyme of the biosynthetic pathway [10]; common step in the biosynthesis of penicillins, cephalosporins and cephamycins [1-14, 16]) (Reversibility: ? [1-17]) [1-17] $ isopenicillin N + H2 O ( product has antibiotic activity [1-3, 7-10, 12]) [1-5, 7-12]      N-[(5S)-5-amino-5-carboxypentanoyl]-l-cysteinyl-d-valine + O2 ( catalytic reaction is under steric regulation [12]) (Reversibility: ? [1-17]) [1-17] $ isopenicillin N + H2 O [1-17]  d-(L-a-aminoadipoyl)-l-cysteinyl-d-a-aminobutyrate + O2 ( wild-type and mutants, reaction mechanism [12]) (Reversibility: ir [12]) [12] $ ?  Additional information ( d-(L-a-aminoadipoyl)-l-cysteinyl-d-aaminobutyrate as substrate is converted to 3 different products: an a- and a b-methyl-penam, and a cepham [12]) [12] $ ? 2  Co2+ ( moderate inhibition [3]; inhibition [1]) [1, 3] Cu2+ ( slight inhibition [1]) [1]

603

Isopenicillin-N synthase

1.21.3.1

d-glucose 6-phosphate ( strong inhibition [1]) [1] GSH ( 70% inhibition at 1 mM [3]) [3] Mn2+ ( moderate inhibition [1, 3]) [1, 3] N-[(5S)-5-amino-5-carboxypentanoyl]-l-cysteinyl-d-valine ( substrate inhibition above 5 mM [4]) [4] N-ethylmaleimide ( wild-type and mutants, binds to Cys104 in the active site [8]; only Cys106 can be modified, partly blocked by substrate analogue inhibitors [4]) [4, 8] NH+4 ( inhibition of enzyme formation in vivo, no inhibitory effect in vitro [1]) [1] Ni2+ ( moderate inhibition [3]) [3] Triton X-100 ( inhibits at concentration of 0.5% [3]) [3] Zn2+ ( moderate inhibition [1, 3]) [1, 3] a-aminoadipic(-Cys-Gly) [5] bis[H-Cys-d-Val] [5] bis[a-aminoadipic(-Cys-d-Phe)] [5] bis[a-aminoadipic(-Cys-d-Trp)] [5] bis[a-aminoadipic(-Cys-d-Tyr)] [5] bis[a-aminoadipic(-Cys-d-chloroalanine)] [5] bis[a-aminoadipic(-Cys-d-hexafluorovaline)] [5] bis[a-aminoadipic(-Cys-dl-hexafluorovaline)] [5] bis[a-aminoadipic(-Cys-hexafluorovaline)] [5] cystamine ( 100% inhibition at 0.5 mM [3]) [3] cysteamine ( 43% inhibition at 1.5 mM [3]) [3] cysteine ( 60% inhibition at 1 mM [3]) [3] cystine ( 81% inhibition at 1.5 mM [3]) [3] glutathione ( slight inhibition [1]) [1] pyruvate ( slight inhibition [1]) [1] Additional information ( conversion of the d-valine residue in third position of the enzyme to an aromatic amino acid or to a highly electronegative residue such as trifluorovaline results in elimination of substrate activity and creation of an inhibitor [5]; ATP, NADPH and FAD: no effect [3]; anions F-, I- , Br-, Cl- , NO-3, H2 PO-4, (AsO3 )3- , (SO4 )2- do not affect the activity [1]; cations Na+ , K+ , Mg2+ , Ca2+ , Fe3+ do not affect the activity [1, 3]) [1, 3, 5] &

  O2 ( required [1-12]) [1-12] Triton X-100 ( leads to 50% stimulation at concentration of 0.01% [3]) [3] ascorbic acid ( 5fold stimulation [3]; required [1]; great stimulation [1]) [1, 3, 8, 12, 14] dithioerythritol ( absolutely required, highly stimulating [3]) [3] dithiothreitol ( required, stimulating [1]) [1, 8, 12] polyethylene glycol 1500 ( 20% stimulation up to concentration of 5% [3]) [3]

604

1.21.3.1

Isopenicillin-N synthase

'(   Fe2+ ( iron-binding motif [10, 15]; structure of the Fe2+ active site and endogenous ligands [10, 15]; His216 and His272 are ligands to bind the non-heme iron in the active site [6]; low concentration [3]; stimulates [2, 3]; required [1, 6, 8-10]) [1-3, 6-12, 14, 15] ) &  * +, 246 (N-[(5S)-5-amino-5-carboxypentanoyl]-l-cysteinyl-d-valine) [14] " & *-% , 0.0000000125 ( mutant C104S, recombinant, partially purified [8]) [8] 0.000000093 ( purified recombinant enzyme [13]) [13] 0.000000177 ( mutant C251S, recombinant, partially purified [8]) [8] 0.000000257 ( mutant C142S, recombinant, partially purified [8]) [8] 0.000000276 ( mutant C37S, recombinant, partially purified [8]) [8] 0.000000338 ( wild-type, recombinant, partially purified [8]) [8] 0.00000224 ( mutant H49L [6]) [6] 0.0000027 ( wild-type [11]) [11] 0.00000664 ( purified recombinant enzyme [14]) [14] 0.0000103 ( mutant H126L [6]) [6] 0.0000112 ( mutant H137L [6]) [6] 0.0000116 ( mutant H116L [6]) [6] 0.0000127 ( mutant H64L [6]) [6] 0.0000136 ( wild-type [6]) [6] 0.00129 ( about, purified enzyme [3]) [3] Additional information ( development of spectrophotometric assay [14]) [6, 14] . / *', 0.08 (d-(l-a-aminoadipyl)-l-cysteinyl-d-valine) [3] 0.12 (N-[(5S)-5-amino-5-carboxypentanoyl]-l-cysteinyl-d-valine) [14] 0.18 (d-(l-a-aminoadipyl)-l-cysteinyl-d-valine) [1] . / *', 0.9 (N-ethylmaleimide, about [4]) [4] 0  7 ( assay at [14]; pI 6.55 [1]) [1, 14] 7-7.5 [3] 0 

 5.8-9 [1] 6-9 ( 30% activity remaining above pH 9.0, highly reduced activity below pH 6.0 [3]) [3] )  *
, 15-20 [3] 25 [1]

605

Isopenicillin-N synthase

1.21.3.1

26 ( assay at [8, 11, 13]) [8, 11, 13] 27 ( assay at [12]) [12] 30 ( assay [14]) [14] ) 

 *
, 15-35 [1]

  #   ' 1  26500 ( gel filtration [1]) [1] 36500-38000 ( gel filtration and SDS-PAGE [3]) [3]   ? ( x * 37800, DNA sequence determination [13]; x * 38000, wild-type and mutants, SDS-PAGE [6]) [6, 13] monomer ( 1 * 36500-38000, SDS-PAGE and gel filtration [3]; 1 * 26500, SDS-PAGE [1]) [1, 3]

 2 %$ %' %

% mycelium [1] 3#

cytosol [1, 3, 11] membrane ( inactive associated form [3]) [3] $"

[1] [5] (wild-type and mutants recombinant from Escherichia coli [6]; recombinant from Escherichia coli [5]; wild-type and mutants are purified as catalytically latent apoenzymes [4]) [4-6] [3] (partially, wild-type and mutants recombinant from Escherichia coli [8]) [8] (recombinant wild-type and mutants from Escherichia coli [12]) [12]
 #

(vapour diffusion method under anaerobic condition in a crystallization tray, complexed with Fe2+ and substrate with and without NO in addition, Xray diffraction [9]; structure analysis [7,9,10]; enzyme complexed with manganese instead of iron in the active site, more stable [7]) [7, 9, 10] (crystal structure, molecular modeling of the active site structure and the Fe2+ -binding motif [15]) [15]

606

1.21.3.1

Isopenicillin-N synthase


 

(expression in Escherichia coli [17]) [17] (expression of wild-type and mutant in Escherichia coli, evaluation of growth temperature for expression of the soluble mutant and wild-type in the Escherichia coli host BL21 (DE3) [16]; overexpression of recombinant enzyme mutants in Cephalosporium acremonium [11]; expression of wildtype and mutants in Escherichia coli, amino acid sequence comparison [6]; expression in Escherichia coli [5]) [5, 6, 11, 16] (expression of wild-type and mutants in Escherichia coli [8]) [8] (expression of wild-type and mutants as maltose-binding fusion proteins in Escherichia coli [12]) [12] (overexpression in Escherichia coli, DNA sequence analysis [13]) [13] 

 

C104S ( single-strand-site-directed mutagenesis, loss of more than 96% activity [8]) [8] C106S ( site-directed mutagenesis, 63% reduced activity, 14fold increased Km for N-[(5S)-5-amino-5-carboxypentanoyl]-l-cysteinyl-d-valine [4]) [4] C106S/C255S ( site-directed mutagenesis, 63% reduced activity, 14fold increased Km for N-[(5S)-5-amino-5-carboxypentanoyl]-l-cysteinyld-valine [4]) [4] C142S ( single-strand-site-directed mutagenesis, loss of 24% activity [8]) [8] C251S ( single-strand-site-directed mutagenesis, loss of 47.7% activity [8]) [8] C255S ( site-directed mutagenesis, 33% reduced activity, 1.4fold increased Km for N-[(5S)-5-amino-5-carboxypentanoyl]-l-cysteinyl-d-valine [4]) [4] C37S ( single-strand-site-directed mutagenesis, loss of 18.3% activity [8]) [8] C37S/C142S/C251S ( triple mutant, conformationally different from wild-type, prepared by recombining fragments of IPNS-encoding gene pcbC from each of the single mutants, loss of more than 90% activity [8]) [8] D214C ( site-directed mutagenesis, active site mutant, complete loss of activity [15]) [15] D214E ( site-directed mutagenesis, active site mutant, retains 1% of activity compared to wild-type [15]) [15] D214H ( site-directed mutagenesis, active site mutant, complete loss of activity [15]) [15] D214H ( site-directed mutagenesis, complete inactive enzyme [10]) [10] H116L ( site-directed mutagenesis, reduced activity [6]) [6] H126L ( site-directed mutagenesis, reduced activity [6]) [6] H137L ( site-directed mutagenesis, reduced activity [6]) [6] H212D ( site-directed mutagenesis, active site mutant, complete loss of activity [15]) [15]

607

Isopenicillin-N synthase

1.21.3.1

H212N ( site-directed mutagenesis, active site mutant, complete loss of activity [15]) [15] H212Q ( site-directed mutagenesis, active site mutant, complete loss of activity [15]) [15] H262L ( site-directed mutagenesis, complete loss of activity [6]) [6] H268D ( site-directed mutagenesis, active site mutant, complete loss of activity [15]) [15] H268N ( site-directed mutagenesis, active site mutant, complete loss of activity [15]) [15] H268Q ( site-directed mutagenesis, active site mutant, complete loss of activity [15]) [15] H272L ( site-directed mutagenesis, complete loss of activity [6]) [6] H49L ( site-directed mutagenesis, complete loss of activity [6]) [6] H64L ( site-directed mutagenesis, reduced activity [6]) [6] L223A ( reduced activity with N-[(5S)-5-amino-5-carboxypentanoyl]-l-cysteinyl-d-valine, d-(L-a-aminoadipoyl)-l-cysteinyl-d-a-aminobutyrate is a poor substrate [12]) [12] L223I ( reduced activity with N-[(5S)-5-amino-5-carboxypentanoyl]-l-cysteinyl-d-valine, product spectrum differs from that of the wildtype with d-(L-a-aminoadipoyl)-l-cysteinyl-d-a-aminobutyrate as substrate [12]) [12] L223V ( reduced activity with N-[(5S)-5-amino-5-carboxypentanoyl]-l-cysteinyl-d-valine, product spectrum differs from that of the wildtype with d-(L-a-aminoadipoyl)-l-cysteinyl-d-a-aminobutyrate as substrate [12]) [12] P285L ( site-directed mutagenesis, complete loss of activity, increased soluble expression in Escherichia coli host [16]) [16] Q227L ( site-directed mutagenesis, 55.10% remaining activity compared to wild-type [11]) [11] Q337L ( site-directed mutagenesis, slightly reduced activity compared to wild-type [11]) [11] Additional information ( exchange of Asp214 and His268, and exchange of Asp214 and His 212 by site-directed mutagenesis leads to completely inactive enzymes [10, 15]) [10, 15] 

pharmacology ( model system for study of endogenous functions of b-lactams in bacteria [3]) [3] synthesis ( direct enzymic synthesis of antibiotics [1]) [1]

4    , enzyme is unstable to oxidizing oxygen species in the reaction solution [14]

608

1.21.3.1

Isopenicillin-N synthase

5    "

, unstable to 20 mM dithiothreitol during storage at -20 C, 10% activity remaining [3]

"  [1] Castro, J.M.; Liras, P.; Laiz, L.; Cortes, J.; Martin, J.F.: Purification and characterization of the isopenicillin N synthase of Streptomyces lactamdurans. J. Gen. Microbiol., 134, 133-141 (1988) [2] Rollins, M.J.; Jensen, S.E.; Westlake, D.W.S.: Isopenicillin N synthase and desacetoxycephalosporin C synthase activities during defined medium fermentations of Streptomyces clavuligerus: effect of oxygen and iron supplements. Can. J. Microbiol., 35, 1111-1117 (1989) [3] Palissa, H.; Von Doehren, H.; Kleinkauf, H.; Ting, H.H.; Baldwin, J.E.: bLactam biosynthesis in a Gram-negative eubacterium: purification and characterization of isopenicillin N synthase from Flavobacterium sp. strain SC 12.154. J. Bacteriol., 171, 5720-5728 (1989) [4] Kriauciunas, A.; Frolik, C.A.; Hassell, T.C.; Skatrud, P.L.; Johnson, M.G.; Holbrook, N.L.; Chen, V.J.: The functional role of cysteines in isopenicillin N synthase. Correlation of cysteine reactivities toward sulfhydryl reagents with kinetic properties of cysteine mutants. J. Biol. Chem., 266, 1177911788 (1991) [5] Huffman, G.W.; Gesellchen, P.D.; Turner, J.R.; Rothenberger, R.B.; Osborne, H.E.; Miller, F.D.; Chapman, J.L.; Queener, S.W.: Substrate specificity of isopenicillin N synthase. J. Med. Chem., 35, 1897-1914 (1992) [6] Tan, D.S.H.; Sim, T.S.: Functional analysis of conserved histidine residues in Cephalosporium acremonium isopenicillin N synthase by site-directed mutagenesis. J. Biol. Chem., 271, 889-894 (1996) [7] Roach, P.L.; Clifton, I.J.; Fulop, V.; Harlos, K.; Barton, G.J.; Hajdu, J.; Andersson, I.; Schofield, C.J.; Baldwin, J.E.: Crystal structure of isopenicillin N synthase is the first from a new structural family of enzymes. Nature, 375, 700-704 (1995) [8] Durairaj, M.; Leskiw, B.K.; Jensen, S.E.: Genetic and biochemical analysis of the cysteinyl residues of isopenicillin N synthase from Streptomyces clavuligerus. Can. J. Microbiol., 42, 870-875 (1996) [9] Roach, P.L.; Clifton, I.J.; Hensgens, C.M.H.; Shibta, N.; Schofield, C.J.; Hajdu, J.; Baldwin, J.E.: Structure of isopenicillin N synthase complexed with substrate and the mechanism of penicillin formation. Nature, 387, 827-830 (1997) [10] Kreisberg-Zakarin, R.; Borovok, I.; Yanko, M.; Aharonowitz, Y.; Cohen, G.: Recent advances in the structure and function of isopenicillin N synthase. Antonie Leeuwenhoek, 75, 33-39 (1999) [11] Loke, P.; Sim, T.S.: Analysis of glutamines in catalysis in Cephalosporium acremonium isopenicillin N synthase by site-directed mutagenesis. Biochem. Biophys. Res. Commun., 252, 472-475 (1998)

609

Isopenicillin-N synthase

1.21.3.1

[12] Rowe, C.J.; Shorrock, C.P.; Claridge, T.D.W.; Sutherland, J.D.: Analysis of the conversion of d-(l-a-aminoadipoyl)-l-cysteinyl-d-a-aminobutyrate by active-site mutants of Aspergillus nidulans isopenicillin N synthase. Chem. Biol., 5, 229-239 (1998) [13] Loke, P.; Ng, C.P.; Sim, T.S.: PCR cloning, heterologous expression, and characterization of isopenicillin N synthase from Streptomyces lipmanii NRRL 3584. Can. J. Microbiol., 46, 166-170 (2000) [14] Dubus, A.; Sami, M.; Brown, T.J.N.; Schofield, C.J.; Baldwin, J.E.; Frere, J.M.: Studies of isopenicillin N synthase enzymatic properties using a continuous spectrophotometric assay. FEBS Lett., 485, 142-146 (2000) [15] Kreisberg-Zakarin, R.; Borovok, I.; Yanko, M.; Frolow, F.; Aharonowitz, Y.; Cohen, G.: Structure-function studies of the non-heme iron active site of isopenicillin N synthase: some implications for catalysis. Biophys. Chem., 86, 109-118 (2000) [16] Loke, P.; Sim, T.S.: Site-directed mutagenesis of proline-285 to leucine in Cephalosporium acremonium isopenicillin N-synthase affects catalysis and increases soluble expression at higher temperatures. Z. Naturforsch. C, 56, 413-415 (2001) [17] Carr, L.G.; Skatrud, P.L.; Scheetz, M.E 2nd.; Queener, S.W.; Ingolia, T.D.: Cloning and expression of the isopenicillin N synthetase from Penicillium chrysogenum. Gene, 48, 257-266 (1986)

610


  

!

   
 1.21.3.2    columbamine:oxygen oxidoreductase (cyclizing)    columbamine oxidase    EC 1.1.3.26 (formerly) berberine synthase synthase, berberine
    95329-18-3

     Berberis stolonifera [1]

!    "
 # 

2 columbamine + O2 = 2 berberine + 2 H2 O (oxidation of the O-methoxyphenol structure forms the methylenedioxy group of berberine)    oxidation redox reaction reduction       columbamine + O2 ( berberine biosynthesis [1]) (Reversibility: ir [1]) [1] $ ?      columbamine + O2 (Reversibility: ir [1]) [1] $ berberine + H2 O

611

Columbamine oxidase

1.21.3.2

 Additional information [1] $ ? 2  cyanide [1] o-phenathroline [1] p-phenanthroline [1] '(   Fe2+ ( an iron protein, oxidation of the O-methoxyphenol structure forms the methylenedioxy group of berberine, Fe2+ restores activity after dialysis against o-phenanthroline [1]) [1] . / *', 0.002 (columbamine) [1] 0  8.9 [1] )  *
, 70 [1]

 2 %$ %' %

$"

[1]

"  [1] Rueffer, M.; Zenk, M.H.: Berberine synthase, the methylenedioxy group forming enzyme in berberine synthesis. Tetrahedron Lett., 26, 201-202 (1985)

612

  

!!

   
 1.21.3.3    (S)-reticuline:oxygen oxidoreductase (methylene-bridge-forming)    reticuline oxidase    BBE EC 1.5.3.9 (formerly) berberine bridge enzyme berberine-bridge-forming enzyme tetrahydroprotoberberine synthase
    152232-28-5

     Papaver somniferum (three genes, only one is expressed, Botrytis elicitor induces mRNA expression [3]) [1, 3] Eschscholzia californica (two genes [2]) [2, 4, 5] Chelidonium majus [6] Berberis beaniana [7, 8] plants (overview of occurence of BBE in Berberidaceae, Ranunculaceae, Menispermaceae, Papaveraceae and Fumariaceae [7]) [7]

!    "
 # 

(S)-reticuline + O2 = (S)-scoulerine + H2 O2    oxidation redox reaction reduction

613

Reticuline oxidase

1.21.3.3

      (S)-reticuline + O2 ( first committed step in sanguinarine biosynthesis [1, 3]; reticuline is the biogenic precursor of the protoberine skeleton [7]) (Reversibility: ? [1-8]) [1-8] $ (S)-scoulerine + H2 O2      (R,S)-6-O-methyllaudanosoline + O2 (Reversibility: ? [4]) [4] $ ? + H2 O 2  (R,S)-crassifoline + O2 (Reversibility: ? [4]) [4] $ ? + H2 O 2  (R,S)-laudanosoline + O2 ( specific for the (S)-enantiomer [4,7,8]) (Reversibility: ? [4, 7, 8]) [4, 7, 8] $ ? + H2 O 2  (S)-N-methylcoclaurine + O2 (Reversibility: ? [4]) [4] $ (S)-coclaurine + H2 O2  (S)-protosinomenine + O2 (Reversibility: ? [4, 7, 8]) [4, 7, 8] $ coramin + H2 O2  (S)-reticuline + O2 ( first committed step in sanguinarine biosynthesis [1,3]; reticuline is the biogenic precursor of the protoberine skeleton [7]) (Reversibility: ? [1-8]) [1-8] $ (S)-scoulerine + H2 O2 2  (R)-norreticuline ( 50% inhibition at 0.02 mM [7]) [7] (S)-coreximine ( 50% inhibition at 0.2 mM [7]) [7] (S)-norreticuline ( 50% inhibition at 0.001 mM [7]) [7] (S)-scoulerine ( 50% inhibition at 0.01 mM [4,7]) [4, 7] H2 O2 ( 50% inhibition at 0.7 M [7]) [7] Na2 EDTA ( 50% inhibition at 6 mM [7]) [7] berberine ( 50% inhibition at 0.004 mM [7]) [7] diethyldithiocarbamate ( 50% inhibition at 0.4 mM [7]) [7] dithioerythritol ( 50% inhibition at 4 mM [8]) [8] jatrorrhizine ( 50% inhibition at 0.03 mM [7]) [7] o-phenanthroline ( 50% inhibition at 0.006 mM [7,8]) [7, 8]
"%  FAD ( 1 mol per mol protein [4]; covalently attached to His104 [4]) [4] . / *', 0.00014 ((S)-reticuline) [8] 0.003 ((S)-reticuline) [5] 0  8.9 [8] 10.5 [4] )  *
, 45 [5] 614

1.21.3.3

Reticuline oxidase

  #   ' 1  49000 ( gel filtration [7,8]) [7, 8]   monomer ( 1 * 54000, SDS-PAGE [7]) [7] $   "

glycoprotein ( undergoes N-glycosylation [1]) [1]

 2 %$ %' %

% root ( soluble in the lumen [1]) [1, 3] stem ( soluble in the lumen [1]) [1, 3] 3#

vacuole ( vacuolar pH is below the functional range of BBE, it is active only before the entry into the vacuole, enters vacuole via a sorting determinant [1]) [1] $"

(expressed in Sf9 cells [5]) [5] [7]
 

(expressed in Spodoptera frugiperda Sf9 cells [4,5]) [4, 5] 

 

H104T ( no activity [4]) [4] H308S ( 5% activity of wild-type [4]) [4] H39G ( 40% activity of wild-type [4]) [4] R100T ( no activity [4]) [4]

4     , -20 C, 320 d, pH 7.4, 50% activity [7] , 25 C, 18 d, pH 7.4, 50% activity [7] , 37 C, 6 d, pH 7.4, 50% activity [7] , 4 C, 150 d, pH 7.4, 50% activity [7]

615

Reticuline oxidase

1.21.3.3

"  [1] Bird, D.A.; Facchini, P.J.: Berberine bridge enzyme, a key branch-point enzyme in benzylisoquinoline alkaloid biosynthesis, contains a vacuolar sorting determinant. Planta, 213, 888-897 (2001) [2] Hauschild, K.; Pauli, H.H.; Kutchan, T.M.: Isolation and analysis of a gene bbe1 encoding the berberine bridge enzyme from the California poppy Eschscholzia californica. Plant Mol. Biol., 36, 473-478 (1998) [3] Facchini, P.J.; Penzes, C.; Johnson, A.G.; Bull, D.: Molecular characterization of berberine bridge enzyme genes from opium poppy. Plant Physiol., 112, 1669-1677 (1996) [4] Kutchan, T.M.; Dittrich, H.: Characterization and mechanism of the berberine bridge enzyme, a covalently flavinylated oxidase of benzophenanthridine alkaloid biosynthesis in plants. J. Biol. Chem., 270, 24475-24481 (1995) [5] Kutchan, T.M.; Bock, A.; Dittrich, H.: Heterologous expression of the plant proteins strictosidine synthase and berberine bridge enzyme in insect cell culture. Phytochemistry, 35, 353-360 (1994) [6] Frenzel, T.; Beale, J.M.; Kobayashi, M.; Zenk, M.H.; Floss, H.G.: Stereochemistry of enzymatic formation of the berberine bridge in protoberine alkaloids. J. Am. Chem. Soc., 110, 7878-7880 (1988) [7] Steffens, P.; Nagakura, N.; Zenk, M.H.: Purification and characterization of the berberine bridge enzyme from Berberis beaniana cell cultures. Phytochemistry, 24, 2577-2583 (1985) [8] Steffens, P.; Nagakura, N.; Zenk, M.H.: The berberine bridge forming enzyme in tetrahydroprotoberberine synthesis. Tetrahedron Lett., 25, 951-952 (1984)

616

  ;*D,   "