Eicosanoids and Related Compounds in Plants and Animals 9781400873111

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Eicosanoids and Related Compounds in Plants andAnimals

· Eicosanoids and Related Compounds in Plants and Animals Edited by

A. F. Rowley

H.Kuhn T.Schewe

PRINCETON UNIVERSITY PRESS PRINCETON, NEW JERSEY

Published in N o rth A m erica by P rinceton U niversity Press, 41 W illiam S treet, P rinceton, N e w Jersey 08540 Published in th e U nited Kingdom by P ortland Press Ltd, 59 Portland P la ce ,L o n d o n W IN 3AJ,U.K. Tel: (+44) 171 580 5530; e-mail: ed it@ p o rtla n d p ress.co .u k

© 1998 Portland Press Ltd, London

Library of Congress Catalog Card Number 98-87898 ISBN 0-691-00902-3

AU rights reserved A lthough, at the tim e of going to press, the in fo rm atio n contained in this publication is believed to be correct, neither the authors n o r the publisher assume any responsibility for any errors or omissions herein contained. O pinions expressed in this book are those of the authors and are n o t necessarily held by the editors or the publishers.

Typeset by Portland Press Ltd Printed in G reat Britain by Inform ation Press Ltd, Eynsham, U.K. http://pup.princeton.edu 1 3 5 7 9

10 8 6 4 2

1I~~lIUI~i~1 ~I I 32101 0361

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II

Contents

Preface

vii

Abbreviations

ix

Section I - Enzymes and factors involved in the biosynthesis of eicosanoids Eicosanoids and related compounds: structures, nomenclature and biosynthetic pathways H. Kuhn and S. Borngraber

J

~

3

Cyclooxygenases (prostaglandin H synthases): structure and evolutionary aspects H.Toh andT.Tanabe

25

Mammalian lipoxygenases: structure, function and evolutionary aspects N. Ueda, H. Suzuki and S.Yamamoto

47

Plant lipoxygenase: structure and mechanism GAVeldink, M.P. Hilbers,WF. Nieuwenhuizen and J.F.G.Vliegenthart

69

FLAP: structure, function and evolutionary aspects P.J.Yickers

97

Oxylipin production and action in fungi and related organisms R. Peter Herman

115

v

Section 2 - Functional aspects Basic functions of lipoxygenases and th e ir products in higher plants

133

T. S chew e

Lipoxygenases in plant developm ent and senescence

151

D.F. H ildebrand, H. Fukushige, M.A fitlhile an d C .W a n g

Eicosanoids in anim al reproduction: w hat can we learn from invertebrates?

183

D.W. Stanley and J.S. Miller

Cardiovascular effects of eicosanoids in am phibians

197

C A H erm an

T he role of eicosanoids in th e h aem o static m echanism s of fish

209

D.J. Hill an d A.F. Row ley

Subject index

223

Preface

It is n ow o ve r fifty years since von Euler re p o rte d th a t seminal flu id and the prostate contain a factor that causes the contraction o f smooth muscle [Euler, H. ( 1936) J. Physiol. 8 8 ,2 1 3 -2 3 4 ], He named this fa cto r prostaglandin because o f its organ source and hence trig g e re d research in eicosanoid b io c h e m is try and biology. In th e last th re e decades great strides have been made in o u r u n der­ standing o f how prostaglandins and related compounds are synthesized and func­ tio n in humans and mammalian models such as rodents. Some o f the highlights o f this research have included the discovery o f Ieukotrienes and lipoxins.the charac­ te riz a tio n o f the key enzymes involved in eicosanoid biosynthesis, the dem on­ s tra tio n o f th e a ctio n o f aspirin on cyclooxygenase, the c h a ra c te riz a tio n o f eicosanoid receptors and the recent discovery o f an inducible isoform o f cyclo­ oxygenase (C O X -2 ). Four years ago the firs t three-dim ensional s tru c tu re o f a lipid-peroxidizing enzyme (soybean lipoxygenase-1) was published. Since then the X-ray structures o f C O X -I and C O X -2 have become available and preliminary Xray data on ra bbit 15-lipoxygenase have also been presented at various conferences.These structural data may be used in the future fo r the rational drug design o f C O X and lipoxygenase inhibitors. As would be expected, most o f these findings have been made w ith mammals. O th e r organisms, corals in p a rticu la r, have, however, made im p o rta n t contributions in eicosanoid research. Because in some corals prostaglandins (mainly PGA2 methyl ester acetate) constitute 2-3% o f the d ry w e ig h t o f these animals, th e y became a valuable natural source o f th is compound.The stimulus fo r us to produce this volume was the incorporation o f a separate session on ‘ N on-M am m alian Eicosanoids’ at th e 8th In te rn a tio n a l Conference on Prostaglandins and Related Compounds in M ontreal in 1992 and subsequently at the equivalent meetings in Florence and Vienna in 1994 and 1996, respectively. Such sessions marked the ‘coming o f age’ o f this area and the recog­ n itio n th a t studies w ith plants and non-m am m als are o f value to eicosanoid biochemistry as a whole. W e particularly wish to thank all o f o u r authors fo r th e ir patience and forbearance while this volume came to fruition.W e also hope that its publication w ill stimulate o th e r scientists to fu rth e r examine the biosynthesis and functional behaviour o f these fascinating compounds in plants and animals over the coming years.

A.F. Rowley, H . Kiihn andT. Schewe /997

Abbreviations

AA ΑΒΑ ACC ADP CO X cyt P-4S0 DE DHA DMSO DOX EGF Ep EPA EPR ER FLAP HE

Arachidonic acid Abscisic acid I -AminocycIopropane-1-carboxylic acid Adenosine diphosphate Cyclooxygenase Cytochrome P-450 Dienoic Docosahexaenoic acid Dimethyl sulphoxide Dioxygenase Epidermal growth factor Epoxy Eicosapentaenoic acid Electron paramagnetic resonance Endoplasmic reticulum 5-Lipoxygenase-activating protein Hexaenoic

Hp HPLC IL LDL LOX LT LX

Hydroperoxy

ME NDGA NE NSAID PE PG PL psi RT-PCR TE TrE TX XAFS

Monoenoic

High-performance liquid chromatography Interleukin Low-density lipoprotein Lipoxygenase Leukotriene Lipoxins Nordihydroguaiaretic acid Nuclear envelope Non-steroidal anti-inflammatory drug Pentaenoic Prostaglandin Phospholipase Precocious sexual inducer Reverse transcriptase PCR Tetraenoic Trienoic Thromboxane X-ray-absorption fine structure

Section 1 - Enzymes and factors involved in the biosynthesis of eicosanoids

T his section deals prim arily w ith the m echanism s o f b iosyn th esis o f eicosanoids and related com pou n d s, in particular the nature o f the enzym es and other factors involved. The first chapter briefly surveys the biosynthetic routes involved and the structure o f the com pounds produced. The proceeding chapters each deal w ith the d iversity and ev o lu tio n o f cy clo o xy g e n a ses, lip oxygen ases and 5-lip o x y g en a seactivating p rotein (F L A P ). Each author w as given a brief to produce a tim ely review o f their particular subject, avoiding repetition o f those areas already reviewed elsewhere. There have been a number o f excellent volumes and reviews on eicosanoid b iosyn th esis in the last few years. A num ber o f these are listed below . This list is not meant to be exhaustive but is designed to point the reader to som e areas not dealt w ith in great detail in this first section o f the present volume.

Further Reading Books Vane, J. and B ottin g , J. (1988) Selective C O X -2 In h ib ito rs, Pharm acology, C linical E ffects and T herapeutic Potential, Kluwer and William H arvey Press, D ordecht Piomelli, D . (1996) Arachidonic Acid in Cell Signalling, Springer-Verlag, Berlin Sam uelsson, B., Ram w ell, P.W., P aoletti, R., Folco, G ., G ranstrom , E. and N icosia, S. (1995) A dvances in P rostaglandin, T hrom boxane and L eukotriene Research, vol. 23, Raven Press, N ew York (the latest in a series by these and o ther editors dealing w ith tim ely advances in the field) C u n n in g h am , F.M. (1994) T he H an d b o o k of Im m unopharm acology, L ipid M ediators, A cademic Press, London C rooke, S.T. and Wong, A. (1991) Lipoxygenases and their Products, Academic Press, San Diego

C hapters/review s General Serhan, C .N ., H a e g g s tro m J .Z . and Leslie, C .C . (1996) L ipid m ediator netw orks in cell signalling: update and im pact of cytokines, FASEB J. 10,1147-1158 Y am am oto, S. and Sm ith, W.L. (1996) M olecular biology of the arachidonate cascade, J. Lipid M ediators Cell Signal. 12, 83-454 (a series of timely reviews by various authors)

2

Section 1— Enzymes and factors involved in the biosynthesis o f eicosanoids

Phospholipases D ennis, E.A. (1994) D iversity of group types, regulation, and function of phospholipase A2, J. Biol. Chem. 269,13057-13060

Lipoxygenases, and Ieukotriene and Iipoxin generation Funk, C .D . (1996) The molecular biology of mammalian lipoxygenases and the quest for eicosanoid functions using lipoxygenase-deficient mice, Biochim. Biophys. Acta 1304,65-84 Kiihn, H. (1996) Biosynthesis, m etabolization and biological im portance of the prim ary 15-lipoxy­ genase m etabolites 1 5 -h y d ro (p ero )x y -(5 Z ,8 Z ,llZ ,1 3 £ )-eico satetra en o ic acid and 13hydro(pero)xy-(9Z, 11 £)-octadecadienoic acid, Prog. Lipid Res. 35,203-226 F o rd -H u tc h in so n , A .W., G resser, M. and Young, R.M . (1994) 5-L ipoxygenase, A n n u . Rev. Biochem. 63,383—417 Serhan, C .N . (1994) L ipoxin biosynthesis and its im pact in inflam m atory and vascular events, Biochim. Biophys. Acta 1212,1-25 Y am am oto, S. (1992) M am m alian lipoxygenases: m olecular stru ctu res and fu n ctio n s, B iochim . Biophys. Acta, 1128,117-131 K dnig, W., Schonfeld, W., Raulf, M ., Koller1J., Schefferj J. and Brom , J. (1990) T he neu tro p h il and leukotrienes-role in health and disease, Eicosanoids 3,1 -2 2

Cyclooxygenases H erschm an, H .R. (1996) Prostaglandin synthase 2, Biochim. Biophys. Acta 1299, 125-140 L uong, C ., Miller, A., B arnettj J., C how , j., Ramesha, C. and Browner, M.F. (1996) Flexibility o f the N S A ID b in d in g site in th e stru c tu re of hum an cyclooxygenase-2, N a t. S truct. Biol. 3, 927-933 Vane,J.R. (1996) Introduction: mechanism of action of N SA ID s, Br.J. Rheum atol. 35(suppl. I), 1-3 (brief review on action of N S A ID s as inhibitors of C O X - 1/C O X -2 ) DeW itt, D. and Smith, W.L. (1995) Yes, b ut do they still get headaches?, Cell 83, 345-348 (review of C O X -2 biosynthesis, structure and function)

Biosynthetic pathways in plants H am berg, M . (1993) P athw ays in the biosynthesis of oxylipins in plants, J. L ipid M ediators 6, 375-384 H am b erg , M . and G ardner, H.W . (1992) O x y lip in p ath w ay to jasm onates: b io ch em istry and biological significance, Biochim. Biophys. A c u 1165,1-18

Eicosanoids and related compounds: structures, nomenclature and biosynthetic pathways H artm ut Kiihn* and Sabine Borngraber

Institute of Biochemistry, University Clinics (Charite), H um boldt University, H essische Str. 3-4, I OI 15 Berlin, G erm any

Introduction Eicosanoids and oxylipins com prise a family of structurally related lipid mediators th at exhibit interesting biological activities in animals and in the plant kingdom , respectively [1—6]. Eicosanoids are synthesized from arachidonic acid (AA) that is released upon cell stim ulation from membrane phospholipids. However, AA is not the only substrate for eicosanoid synthesis. Even in mammals, where it is one of the m ajor polyenoic fatty acids, o th e r fatty acids w ith different chain lengths and different degrees of unsaturation may be used as substrate (Fig. I). In higher plants, A A only occurs in small am ounts and thus eicosanoids are usually n ot form ed. Instead, the C -1 8 fatty acids (linoleic acid and ot-Iinolenic acid) w hich occur in plants in large am ounts are converted into oxylipins via several oxidative pathways [5,6]. In the early days of eicosanoid research only a small num ber of bioactive lipids were know n and there was no need for a systematic classification. However, during the last 20 years a large variety of eicosanoids and structurally related lipid m ediators have been identified and for m ost of them the biosynthetic route has been investigated. To manage this structural multiplicity, a systematic classification and a comprehensive nomenclature for eicosanoids and related com pounds is required. In this introductory chapter the basic rules for the currently used classifi­ cation and nom enclature of eicosanoids and related com pounds are sum m arized. F or m ore detailed inform ation the reader is referred to several reviews in w hich the nom enclature of H cosanoids [1-4] and oxylipins [5] is explained. M oreover, the

*To w h o m correspondence sh o u ld be addressed.

H. Kiihn and S. Borngraber

4

Fig. 1

Chemical structures of eicosanoid and oxylipin precursor fatty acids

( 8 2 , 1 1 2 , 1 4 Z ) - e i c o s a - 8 , 1 1 , 1 4 - t r i e n o i c acid (dihomo--v-lir\oler»c acid)

( 5 2 , 8 Z , 1 1 2 , 1 4 2 ) - e i c o s a - 5 , 8 , 1 1 , 1 4 - t e t r a e n o i c acid (arachidonic a c i d )

(52,82,112,142,172)-eicosa-5,8,11,14,17p e n t a e n o i c acid ( e i c o s a p e n t a e n o i c acid)

(42,72,102,132,162,192)-docosa-4,7,10,13,16,19h e x a e n o i c acid ( d o c o s a h e x a e n o i c acid)

( 9 2 , 1 2 2 ) - o c t a d e c a - 9 , 1 2 - d i e n o i c acid (linoleic acid)

( 9 2 , 1 2 2 , 1 5 2 ) - o c t a d e c a - 9 , 1 2 - d i e n o i c acid (a-linoienic acid)

recommendations of the Committee on Eicosanoid Nomenclature may be consulted [7]. In this paper suggestions for the nomenclature of enzymes involved in eicosanoid metabolism are also provided. These suggestions have been considered for revision of the recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology on the Nomenclature and Classification of Enzymes [8].

Structures, nomenclature and biosynthetic pathways

Eicosanoids and related compounds in animals The arachidonic acid cascade As indicated above, the m ajor source of eicosanoids and related com pounds in anim als is the A A cascade (Fig. 2). U p o n cell stim ulation, AA, or u nder certain circum stances o th e r p recursor fatty acids, are liberated from the m em brane phospholipids via activation o f lipid-cleaving enzymes, such as phospholipase A r The free fatty acids are subsequently m etabolized via three different pathways (Fig. 2). (i) T he cyclooxygenase (C O X) pathway, forming prostaglandins, thromboxanes or prostacyclins, (ii) the lipoxygenase (LO X ) pathway, forming leukotrienes, lipoxins, hepoxilins and hydro(pero)xy fatty acids and (iii) the cytochrome P-450 (cyt P450) pathw ay, form ing hydroxylated fatty acids and epoxy derivatives. In these m etabolic routes the initial reaction is an oxygenation of the fatty acid substrate. D u rin g the C O X reaction tw o molecules of dioxygen are introduced, one at C 11 and the second at C 15 of the AA backbone. In contrast, the L O X reaction involves the introduction of one molecule of dioxygen at different positions of the substrate molecule, w hich are determ ined by the positional specificity of the enzymes [9,10]. D uring cyt P-450-catalysed oxygenation, atomic oxygen is introduced, leading to fatty acid hydro x y latio n or to epoxidation of double bonds [11]. Both the C O X and L O X reactions are initiated by hydrogen abstractions from d o u b ly allylic m ethylene groups, form ing fatty acid radicals. This radical form ation may be regarded as fatty acid activation. In contrast, the cyt-P-450-catalysed oxygenation

Fig. 2

A rachidonic acid c a sc a d e ________________________ _______ membrane phospholipids

phospholipase arachidonic acid cyt P-450

COX LOX

hydroxy fatty acids, fatty acid epoxides

prostaglandins, thromboxanes, prostacyclins leukotrienes, hydroxy fatty acids, lipoxins, hepoxilins

H. Kiihn and S. B o rn g rab er

6

involves activation of atm ospheric dioxygen, destabilizing the 0 - 0 b ond. A fter this, one oxygen atom is transferred to the fatty acid su b strate, th e o th e r one is reduced, forming water.

T he cyclooxygenase pathway M ore than 50 years ago a com pound was discovered in the seminal fluid and in the p rostate w hich caused contraction of sm ooth m uscle cells [12]. A lth o u g h the chem ical stru ctu re of this factor rem ained unclear fo r m any years, it was nam ed prostaglandin because of its organ source. Since then the chem ical stru ctu res of a variety of prostanoids have been identified, and w e also k now th a t the pro state is n o t the only, and n o t even the major, source of prostaglandin (PG ) form ation. M oreover, m ost enzym es involved in prostaglandin biosynthesis have been well characterized. The initial enzym e fo r p rostaglandin form atio n is p ro stag lan d in endoperoxide synthase which, for simplicity, is called cyclooxygenase. This enzym e, the three-dim ensional structure of w hich has been reported [13,14] is a h aem oprotein and exhibits bo th cyclooxygenase and peroxidase activity. It in tro d u ces tw o molecules of dioxygen into the fatty acid substrate, forming the cyclic endoperoxide P G G 2 w hich is subsequently reduced to the m ore stable P G H 2 (Scheme I). P G H 2 serves as substrate fo r the form ation of the classical prostaglandins P G D 2, P G E 2 and P G F 2a which exhibit interesting bioactivities. Two isoenzymes of C O X have been show n to exist [15]: C O X -I is constitutively expressed in m any m am m alian cells and tissues and appears to be responsible fo r the form ation of prostaglandins involved in the regulation of physiological events. C O X -2 is an inducible form of the enzym e th at is low -level expressed in inflam m atory cells u nder basal conditions b ut is strongly induced in response to inflam m atory stimuli [16]. This induction suggested an involvem ent of the enzym e in the pathogenesis of inflam m ation and suggested C O X -2 as a m ajor target for the development of non-steroidal anti-inflam m atory drugs. AA, w hich contains four double bonds (Fig. I), is converted via the C O X pathway into the prostaglandins of the 2-series (PG D 2, P G E 2, P G F 2o, P G I2, T X A 2). If (82,1 lZ ,14Z )-eicosa-8,l 1,14-trienoic acid or (5 Z ,8 Z ,llZ ,1 4 Z ,1 7 Z )-eico sa5,8,11,14,17-pentaenoic acid are used as substrate, the prostaglandins of the 1-series (PG E 1, P G F lo etc.) and those of the 3-series (PG E3, P G F 3o, etc.) are form ed, respec­ tively. Polyenoic fatty acids containing only tw o double bonds, such as linoleic acid or (llZ ,14Z )-eicosa-l 1,14-dienoic acid, cannot be converted into prostaglandins. In Scheme I the biosynthesis and the chemical structures of the classical prostaglandins (P G D 2, P G E 2, P G F 2o) are sum m arized. It can be seen th a t these com pounds differ from each oth er w ith respect to the chem ical nature of the substituents at the prostane ring and in their stereochem istry. In Table I the scientific nam es of the biologically m ost relevant p ro stan o id s and of o th er eicosanoids and related com pounds are summarized.

Scheme 1

Cyclooxygenase pathway of the arachidonic acid cascade

Arachidonic acid Prostaglandinen d o p ero x id e s y n th a se E.C. 1.14.99.1

OOH P ro stag lan d in G 2 P ro stag lan d in en d o p ero x id e s y n th a se E.C. 1.14.99.1

OH P ro stag lan d in H2 P rostagland in -D s y n th a s e E.C. 5 .3 .9 9 .2

P rostaglandin-E s y n th a se

ProstagIandinXv e .C . 5.3 .9 9 .3 F synthase E.C. 1 .1.1.188

O

OH

P ro sta g lan d in D,

HO

0H P ro stag lan d in F2o

X

o

HO

6h P rostaglandin E2

The unstable endoperoxide P G H 2 form ed via the C O X -reaction can also be converted in to prostacyclin (P G I2) and throm boxane (TX) A 2 (Scheme 2). In m am m als, T X A 2 is m ainly produced by activated blood platelets and induces vasoconstriction, cell adhesion to the vessel wall and platelet aggregation [17,18], P G I2 is form ed by vascular endothelial cells and antagonizes the T X A 2-induced effects. T hus, the T X A 2- P G I 2 steady state appears to be im p o rtan t for systemic blood pressure regulation and for the pathogenesis of thrombosis. In addition to the classical prostaglandins (P G D 2, P G E 2, P G F 2a, T X A 2, P G I2), the biological effects of w hich have been well investigated, several oth er prostanoids (PG A 2, PG B 2, P G J2) have been detected (Fig. 3), b ut little is know n as to their biological importance [19-21]. T he bioactive PG s are fu rth er m etabolized to decom position products that are subsequently eliminated from the body by excretion in urine. This metabolization increases the structural m ultiplicity of the prostanoids. It should be em phasized th a t som e of the m etabolization products may still be bioactive. The decom position pathw ays fo r various prostanoids are different b u t there are com m on principles for prostanoid m etabolization: (i) oxidation of the O H -g ro u p at C - 15, form ing the corresponding keto derivatives; (ii) β -oxidation of the carboxyl term inus, form ing the d i-n o r prostaglandins and (iii) ω-oxidation of the

Cytochrome P-450

Lipoxygenase

PGE, PGF,A PGL2 TXA 2 l3S-H(p)ODE 9S-H(p)ODE l5S-H(p)ETE l2S-H(p)ETE 5S-H(p)ETE 14S-H(p)DoHE 5S,ISS-DiH(p)ETE 8S,l5S-DiH(p)ETE I4R, ISS-DiH(p)ETE LTA4 LTB4 LXB4 LXA 4 5,6-EpETrE 8,9-EpETrE 11,12-EpETrE 14,15-EpETrE

PGFJa

PGH2 PGE2 PGDJ

rS)-configuration, fo rm in g a v icin al d io l. E x p e rim e n ts w ith d iffe re n t o x y g en iso to p e s revealed th a t b o th oxygens o f th e vicinal diols o rig in ate fro m the h y d ro p e ro x y group. T h u s this ty p e o f h y d ro p e ro x id e iso m erase inv o lv es an in tra m o le c u la r o x ygen tra n sfe r (Schem e 12).

References 1. 2.

Smith, W (1989)Prostaglandins 38, 125-133 A ndersen, N .H ., H artzell, C .J. and De, B. (1985) Adv. Prostaglandin T hrom boxane Lcukot. Res. 14,1-43 3. Serhan, C .N ., Wong, P.Y. and Samuelsson, B. (1987) Prostaglandins 34,201-204 4. Samuelsson, B. and H am m arstrom , S. (1980) Prostaglandins 19,645-648 5. H am berg, M. ¢1993) J. Lipid M ediators 6,375-384 6. Blee, E. (1996) Lipoxygenase and L ipoxygenase P athw ay E nzym es (Piazza, G., ed.), pp. 13 8-161, A O C S Press, Cham paign, IL 7. Sm ith, W.L., B orgeat, P., H am berg, M., Jackson-R oberts II, L., W illis, A .L., Yamam oto, S., Ram w ell, P., R ockach, J., Samuelsson, B., Corey, E.J. and Pace-Asciak, C.R. (1980) M ethods Enzym ol. 187,1-9 8. W ebb, E .C . (1992) E nzym e N om enclature. R ecom m endations of the N om enclature C o m m ittee of the Intern atio n al U n io n of B iochem istry and M olecular Biology on the N om enclature and Classification of Enzymes, Academic Press, San Diego 9. Kuhn, H ., Schewe, T. and R apoport, S.M. (1986) Adv. Enzym ol. 58,273-311 10. Yamamoto, S. (1992) Biochim. Biophys. Acta 1128,117-131 11. Capdevila, J.H . (1992) FASEB J. 6,713-736 12. Euler, H . (1936) J. Physiol. 88,213-216 13. Picot, D ., Loll, P.J. and Garavito, R.M. (1992) N ature (London) 367,243-249 14. Loll, P.J., Picot, D., Ekabo, O . and Garavito, R.M. (1996) Biochemistry 35, 7330-7340 15. Smith, W.L. and D ew itt, D.L. (1996) Adv. Immunol. 62,167-215 16. H w an g , D ., Fischer, N .H ., Jang, B .C ., Tak, H ., Kim, J.K. and Lee, W. (1996) Biochem. Biophys. Res. C om m un. 226, 810-818 17. Smith, W.L. ¢1992) Am. J. Physiol. 263, F181-F191 18. Am brosioni, E. and Degli-Esposti, D. (1993) Ann. ItaL Med. Int. 8 ,59S-65S 19. H am berg, M. and Samuelsson, B. (1966) J. Biol. Chem. 241,257-260 20. Polet, H . and Levine, L. (1975) J. Biol. Chem. 250,351-357 21. F u k u sh im a, M., K ato, T., O ta, K., A rai, Y., N arum iya, S. and H ayaishi, O . (1982) Biochem. Biophys. Res. Com m un. 109,626-633 22. Jiang, H ., Yamamoto, S. and Kato, R. (1994) Carcinogenesis 15,807-812 23. M urphy, R .C ., H am m arstrom , S. and Samuelsson, B. (1979) Proc. N atl. Acad. Sci. U.S.A. 76, 4275—4279 24. Bryant, R.W., Schewe, T , R apoport, S.M. and Bailey, J.M. (1985) J. Biol. Chem. 260,3548-3555 25. Serhan, C .N ., H am berg, M. and Sam uelsson, B. (1984) Proc. N atl. Acad. Sci. U.S.A. 81, 5335-5339 26. Serhan, C .N . (1991) J. Bioenerg. Biomembr. 23,105-121 27. Fitzsim m ons, B.J., Adam sj J., Evans, J.F., Leblanc, Y. and Rokach, J. (1985) J. BioL Chem. 260, 13008-13012 28. Kiihn, H ., Wiesner, R., Alder, L., Fitzsim m ons, B.J., Rokach5J. and Brash, A.R. (1987) Eur. J. Biochem. 169, 593-601

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29. 30. 30a.

31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59.

Pace-Asciak1J. (1984) J. Biol. Chem. 259, 8332-8337 Pace-Asciak, C R ., Lee, S.P. and M artin, J.M . (1987) Biochem . B iophys. Res. C om m un. 147, 881-884 Pace-A sciak, C .R ., D em in, P. and N igam , S. (1998) in L ipoxygenases and lipoxygenase m etabolites. Biological A ctions (N igam , S. and Pace-Asciak, C .R ., eds.), Plenum , L ondon, in the press L aneuville, O ., C hang, M ., R eddy, C .C ., C orey, E.J. and Pace-A sciak, C.R . (1990) J. Biol. Chem. 265,21415-21418 Pace-Asciak, C.R., Laneuville, O ., Su, W.G., Corey, E.]., Gurevich, N ., Wu, P. and Carlen, P.L. (1990) Proc. N atl. Acad. Sci. U.S.A. 87,3037-3041 Spiteller, G. (1993) J. Lipid M ediators 7,199-221 C a p d e v ila ,].H ., K arara, A., W axm an, D .J., M artin, M.V., F alck1J.R. and G uenguerich, F.P. (1990) J. Biol. Chem. 265,10865-10871 Karara, A., D ishm an, E., Jacobson, H ., FaIck1J.R. and Capdevila1J .H . (1990) FEBS L ett. 268, 227-230 Karara, A., Dishm an, E., Falck1J.R. and Capdevila, J.H . (1991) J. Biol. Chem. 266, 7561-7569 Capdevila1J., Yadagiri, P., M anna, S. and Falck1J.R. (1986) Biochem. B iophys. Res. C om m un. 141,1007-1011 D eW itt1D.L. and Smith, W.L. (1983) J. Biol. Chem. 258,3285-3293 Shen, R.F. and Tai, H .H . (1986) J. Biol. Chem. 261,11592-11599 Vick, B.A. (1993) Lipid M etabolism in Plants (M oore, T.S., ed.), pp. 167-191, C R C Press, Boca R aton, FL Zim m erm ann, D .C . and C oudron, C.A. (1979) Plant Physiol. 63,536-541 K olattukudy, P.E. (1981) Annu. Rev. Plant Physiol. 32, 539-567 Siedow .J.N . (1991) Annu. Rev. Physiol. Plant Mol. Biol. 42,145-188 Regdel, D., Schewe, T. and Kuhn, H . (1995) Biochemistry (M oscow) 60,715-721 Axelrod, B. (1974) Adv. Chem . Ser. 136,324-348 Gardner, H.W. (1989) Biochim. Biophys. A cta 1001,274-281 Feussner, I. and Kiihn, H . (1995) FEBS Lett. 367,12-14 Feussner, I., W asternack, C ., K indi, H . and K iihn, Li. (1995) Proc. N atl. Acad. Sci U.S.A. 92, 11849-11853 Brodowsky, I.D. and Oliw, E .H . (1992) Biochim. Biophys. Acta 1124,59-65 Brodow sky, I.D ., H am berg, M. and Oliw, E.FL (1992) J. Biol. Chem . 267, 14738-14745 H am berg, M., Zhang, L.Y., B rodow sky, I.D . and Oliw, E .H . (1994) Arch. Biochem. Biophys. 309, 77-80 H am berg, M. and Gardner, H.W. (1992) Biochim. Biophys. Acta 1165,1-18 Farmer, E.E. (1994) Plant Mol. Biol. 26,1423-3147 Zim m erm ann, C .D . (1966) Biochem. Biophys. Res. C om m un. 23,398—402 H am berg, M. (1988) Biochem. Biophys. Res. C om m un. 156,543-550 Brash, A .R., B aertschi, S.W., Ingram , C .D . and H arris, T.M. (1988) Proc. N a tl. A cad. Sci. U.S.A. 85, 3382-3386 Song, W C . and Brash, A.R. (1991) Science 253,781-784 Ishim ura, A. and Yamazaki, I, (1977) J. Biol. Chem. 252,199-204 H am berg, M. (1995) J. Lipid M ediators Cell Signal. 12,283-292

Cyclooxygenases (prostaglandin H synthases): structure and evolutionary aspects Hiroyuki Toh* and Tadashi Tanabef Biom oiecular Engineering Research Institute 6-2-3, Furuedai, Suita, O saka 5 6 5 ,Japan, and J-National Cardiovascular C en ter Research Institute, Fujishirodai1Suita1Osaka 565, Japan

Introduction P rostaglandins (PG s) are bioactive com pounds involved in various sym ptom s associated w ith inflam m ation. T he first step in prostaglandin biosynthesis is catalysed by an enzym e called cyclooxygenase or prostaglandin endoperoxide synthase (C O X ; E C 1.14.99.1). C O X is a bifunctional enzyme w ith both cyclooxy­ genase activity and P G hydroperoxidase activity [1,2]. T he enzym e catalyses the conversion o f arachidonic acid into P G G 2 by the form er activity. Subsequently, P G H 2 is yielded fro m the P G G 2 by the latter activity. T he enzym e show s its catalytic activities as a h om odim er of a 70 kD a polypeptide [3-5], w hich contains iro n (III)-p ro to p o rp h y rin IX w ith a stoichiom etry of one haem group p er 70 kD a m onom er. This haem group is required for b oth catalytic activities of the enzym e

[1,6 - 8], Two isoforms of C O X have been described, w hich are here referred to as C O X -I and C O X -2 . C O X -I is a constitutive enzym e w ith a role in basal or housekeeping prostaglandin synthesis, w hereas C O X -2 is an inducible enzym e, w hich is associated w ith inflam m atory processes. C O X -2 is strongly induced by p ro -in flam m ato ry o r m itogenic agents such as cytokines, endotoxins, tum our prom otors, m itogens and lipids. Both isoforms have similar Vmax and K m values for arachidonate, and undergo suicide inactivation [8-11]. The tw o isoforms, despite th e overall enzym ic sim ilarities, show some different characteristics. T he C O X isoform s are encoded by genes located on different chromosomes. It has long been k n ow n th a t C O X -I requires a hydroperoxide initiator, and it is proposed that cellular prostaglandin synthesis is regulated by the level of intracellular hydroperoxide [12]. C O X -2 is, however, initiated by considerably low er levels of hydroperoxide than C O X -1, w hich w ould provide a biochemical basis for differ­ ential co n tro l of prostaglandin synthesis by the tw o isoform s [13]. F atty acid *7o w hom correspondence should be addressed.

26

H.Toh andT.Tanabe

derivatives such as eicosapentanoate, dihom o-y-lin o len ate, linoleate and a linolenate, serve as substrates for both isoforms. T he fatty acid substrate specificities of the tw o isoform s are, how ever, slightly different [14]. T he cyclooxygenase activity of C O X is inhibited by various non -stero id al an ti-in flam m ato ry drugs (NSA ID s) such as aspirin, ibuprofen, naproxen, indom ethacin and sulindac [15]. It is thought that the therapeutic anti-inflam m atory effects of N SA ID s are ow ing to inhibition of C O X -2, w hile inhibition of C O X -I by these drugs induces m ost of the unw anted side-effects. Therefore, selective inhibition of C O X -2 activity w ould provide a significant im provem ent over therapeutics fo r inflam m atory diseases. Several arom atic com pounds w ith a m ethyl sulph o n y l group, such as N S-398, N imesulide, T-614, FK3311, Floslide, L-745337, D up-697 and SC-58125 have been developed as C O X -2-specific inhibitors [16]. R ecent studies have identified N S A ID s th a t preferentially inhibit C O X -I o r C O X -2 , o r in h ib it b o th isoform s equally [17-19], T he different affinities tow ard som e fatty acid substrates and N S A ID s suggested a subtle difference betw een the active sites of C O X -I and C O X -2. T hat is, the cyclooxygenase active site of C O X -2 is considered to be som ew hat larger and m ore accom m odating than that of C O X - I . Both C O X -I and C O X -2 are located w ithin the endoplasm ic reticulum (ER) and the nuclear envelope (N E ) [20-23]. C O X -2 is, how ever, m ore concentrated in the N E than C O X -I [24]. T he observation suggests th a t C O X -I and C O X -2 in the E R and C O X -2 in the N E constitute independent prostanoid biosynthetic systems. M ost of the current know ledge on the biological contributions of the tw o C O X isoform s has come from studies by N SA ID inhibition, C O X -2 induction or both. Both gene d isruption and overexpression of the C O X isoform s have recently been developed as alternative approaches to investigate the problem . It has been dem onstrated th at C O X -I gene d isru p tio n reduces arachidonic acid-induced inflam m ation and indom ethacin-induced gastric ulceration in mice [25], O n the o th er hand, renal abnorm alities and an altered inflam m atory response w ere observed in mice w ith C O X -2 gene disruption [26,27], Rat intestinal epithelial cells p erm anently infected w ith a C O X -2 expression vector show ed n o t o nly the elevated expression level of C O X -2 p rotein, b u t also b o th increased adhesion to extracellular matrix and inhibition of apoptosis [28]. T he nucleotide sequences of cD N A s fo r C O X -I from sheep [29-31], hum ans [32-34], mice [35] and rats [36], and those fo r C O X -2 from chickens [37], hum ans [38,39], mice [40-42] and rats [36,43], have been determ ined. T he identity of the deduced am ino acid sequences betw een the tw o isoform s from the same species is about 60% . In addition, the C O X -I gene from hum ans [44] and C O X -2 genes from hum ans [45,46] and mice [47] have also been sequenced. T h e hum an C O X -I gene consists of 11 exons spanning ab o u t 22 kb and m apped to chrom osom e 9q32-q33.3, whereas the hum an C O X -2 gene is about 8 kb, w here 10 exons are encoded and m apped to chrom osom e Iq25.2-q25.3 [45]. In spite of this difference, the organization of the C O X -I gene is quite similar to that of the C O X 2 gene (Fig. I). M oreover, the tertiary structures of ovine C O X -I have been

Cyclooxygenases: prostaglandin H synthases

Fig. 1

27

Comparison of genes, cDNAs and proteins for COX-1 and COX-2

(a)

12

345

67

8

9

10

11

-----41~~-,\,-"---)f.+l,J~I-:-,-I/'~J-)1-:-/....j, / 1936: "AUUUA" motif

12345678910

Exon No.

(b)

COX-1 , MembraneSignal EGF-like binding Peptide Domain Domain

Catalytic Domain

COX-2

H193

Y371

H374

S516

(0) The gene structures of human COX-I [44] and human COX-2 [451 are shown. The thin lines indicate

the flanking sequences and the introns of the genes. The filled boxes on the lines indicate the exons. Schematic diagrams for the cDNAs ore shown by open and shadowed boxes, which indicate non-coding and coding regions respectively. The numbers in the boxes show the numbers of nucleotides comprising the boxes. The boxes are connected to the corresponding exons by broken lines.Asterisks, double-osterisks and arrows indicate the positions of start codons, stop codons and polyadenylation signals respectively. (b) The mosaic structures of COX-I and COX-Z. The thickly shaded, thinly shaded, closed and open boxes indicate the Signal peptide, EGF-like domain, membrane-binding domain and catalytic domain respectively. Arrows indicate the positions of exonlintron junctions. H-206 on COX-I and H 193 on COX-Z correspond to the distal His residues.Y384 on COX-I andY371 on COX-Z are the active sites for the formation of tyrosine radical in the cyclooxygenase reaction. H387 on COX-I and H3 74 on COX-Z are the proximal haem ligands for the enzymes. S529 on COX-I and 5516 on COX-Z are the aspirin-acetylated serine residues. Capital letters indicate amino acid residues, and the attached integers indicate residue numbers in the enzymes.

H.Toh andT.Tanabe

28

recently determ ined by X -ray crystallography (Fig. 2) [48], giving fu rth er insight on the function of the enzyme. In this Chapter, we discuss the structural, functional and evolutionary features of C O X , based on the determ ined sequences and structures of the enzyme.

CycIooxygenase as a m osaic protein C O X is regarded as a mosaic p ro tein form ed by exon-shuffling (Fig. I). E xonshuffling is one of the evolutionary strategies that p roduce new p ro tein s [50]. S tructural o r functional dom ains are exchanged betw een tw o p roteins o r inserted into a p ro tein by exon-shuffling, consequently yielding mosaic o r chim aeric proteins. M any examples of such mosaic proteins have been found, w hich suggests that exon-shuffling is an im portant evolutionary m echanism fo r the creation of a new function. The prim ary structure of C O X is divided into four parts: a signal peptide, an epidermal grow th factor (EGF)-Iike domain, a m em brane-binding dom ain and a catalytic dom ain. T he last three parts co rrespond to distinct folding units in the tertiary structure of the enzyme [48]. The genetic inform ation of each part may have been encoded by different genes, w hich may have been assembled into an ancestral gene for C O X by exon-shuffling during the course of molecular evolution.

Signal peptide Both C O X -I and C O X -2 have a signal peptide at their N -term ini, although the N term inus of the m ature enzym e has only been determ ined for ovine C O X -I and rat C O X -2. C O X -I has a longer N -term inal signal peptide than C O X -2 (Fig. 3). T he signal peptide of C O X -I is 23-26 am ino acid residues in length, which is encoded by the first and second exons of the gene. The first exon of hum an C O X -I gene contains the genetic inform ation for the first and second codons and only the first position of the third codon. Thus m ost of the signal peptide of C O X -I is encoded by the second exon. C ontrary to that, the first exon of mice C O X -2 gene encodes the entire region of the signal peptide of 17 am ino acid residues in length. T he signal peptides are highly diverged, n o t only in length, b u t also in am ino acid residues between the isoforms.

EGF-Iike dom ain The third exon of the C O X -I gene and the second exon of the C O X -2 gene encode an E G F-Iike dom ain of ab o u t 40 am ino acid residues in length (Fig. I), w hich corresponds to the first folding u n it of C O X - 1. T he presence of an E G F-Iike

Cyclooxygenases: prostaglandin H synthases

Fig. 2

T h e te rtia ry structure of the 70 kDa m onom er of ovine C O X-1 Catalytic domain EGF-Iike domain

Membrane-binding domain

(b) Catalytic domain Hydrophobic channel haem

EGF-Iike domain

Membranebinding domain

Lipid bilayer

(a) The figure was produced by MOLSCRIPT [4 9 ] with the coordinates o f the ovine C O X-I [4 8 ] th a t are registered as IP R H in the Protein D ata BankThe haem group in the enzyme is shown In the balhandstick representation, (b) Schematic diagram fo r the structure o f COX._______________ ___ _____________

H.Toh andT.Tanabe

30

Fig. 3

T he alignm ents of th e signal peptides of COX-1 and COX-2

C 0 X 1 _ H U MAN COXl_M O U SE C 0 X1_RAT COXl SHEEP

C 0 X 2 _ C H ICK C 0 X 2 _ H U M AN C0X2_M 0 USE C0X2 RAT

M S R -SLLLRF -L L L L L L L P P L P -V L L A D PGAPTPV M S R R S L S L W F P L L L L L L L P P T P S V L L A D PGVPSPV MSRRSLSLQF P L L L L L L L L P P P P V L L T D A G V P S P V M S R Q S I S L R F P L L L L L L -SPSP -V F S A D P G A P A P V

MLLPCAL L A A L L A A G H A A M L A R A L L LCAVLALSHTA M L F R A V L LCAALGLSQAA M L F R A V L LCAALALSHAA

The amino add sequences o f the signal peptides o f COX-I are highly diverged from those o f COX-2. Therefore, the alignment among COX-I and that among COX-2 were separately produced.The abbreviated names for the sequences are shown at the left side o f the sequence; COXI _HUMAN, C0XI_M 0U SE, COXI_RAT, and C0XI_SHEEP indicate COX-I from humans, mice, rats and sheep respectively. Similarly, C0X2_CHICK, C0X2_HUMAN, C0X2_M 0USE and C0X2_RAT indicate COX-2 from chicken, humans, mice, and rats respectively.The reference o f each sequence is given in the introduction o f the texlT he asterisks over the alignments indicate the C-termini o f signal peptides for ovine COX-1 and rat COX-2.

dom ain in C O X at the N -term inal region was first suggested by database searching [51], and its presence was proven by X -ray crystallo g rap h y [48]. T he dom ain consists of tw o small, tw o -stran d ed β -sheets held to g eth er by three disulphide bridges (Fig. 4). T he stru ctu re is quite sim ilar to those of E G F and th eir relatives [52,53], T he E G F-Iike dom ain of C O X is p resent at the interface region of the C O X homodimer. E G F-Iike dom ains are often used as the building blocks fo r various mosaic proteins such as tissue plasm inogen activator and p ro u ro k in ase [54]. T he functional roles of the dom ains rem ain unknow n, although they are considered to act as structural or functional units for p rotein-protein interactions. T he EG F-Iike dom ains are classified into three types based on the size and sequence sim ilarity [55], E G F itself, tum our grow th factor-α, EG F-Iike dom ain of tissue plasm inogen activator, and others, are classified into type I. Type 2 includes EG F-Iike dom ains present in various coagulation factors such as IX, X and X II. Seven repeats in E G F p rec u rso r and E G F-Iike dom ains in low -density lip o p ro tein (LD L ) receptors belong to type 3. Sequence sim ilarity suggests that the E G F-Iike dom ain in C O X belongs to type I o r type 2 [51]. Fig. 5 show s an alignm ent of eight E G F-Iike dom ains from C O X s w ith three m ammalian EG Fs. As show n in the figure, all the Cys residues involved in the disulphide-bridge form ation are conserved. The G ly at the alignm ent sites 34 and 37 is considered to be conserved ow ing to the structural constraint to maintain the EGF-Iike fold, while conservation of aromatic residues at

Cyclooxygenases: prostaglandin H synthases

F|g ·4________ T ertiary stru c tu re of th e EGF-Iike dom ain of ovine COX-1

Cys69

Cys59

Cys36 Cys47

N

The figure was produced by MOLSCRIPT [49] with the coordinates o f IPRH [48] in the Protein Data BankThe three disulphide bridges are drawn in the baii-and-stick style.The numbers identify the positions o f the Cys residues in ovine COX-1.The Cys residues at the positions 3 6 ,4 1 ,4 7 ,5 7 ,5 9 and 69 correspond to those at the aligment sites 3 ,1 1 ,1 7 ,2 8 ,3 0 and 40 in Fig. 5 respectively.

the alignment sites 26 and 35 also characterizes the EGF-Iike domains [55], The Cys residue at the alignm ent site 6, w hich is specifically conserved among the EG F-like dom ains fro m C O X s, form s a disulphide bridge w ith the Cys residue in the catalytic dom ain of the enzym e (alignm ent site 37 in Fig. 10a). The Asn residue at the alignm ent site 39 is subjected to the JV-glycosylation that is considered to be essential for the catalytic activity of the enzyme. Flowever, the relationship between the glycosylation and the catalytic activity remains unknown.

The membrane-binding domain The second folding unit of C O X is a membrane-binding domain of about 50 amino acid residues in length (Fig. 6), w hich does n o t show any significant sequence sim ilarity w ith o ther proteins. As described above, C O X is a membrane-associated p ro tein function ing at the ER and the N E . T he dom ain is involved in the

9 H . T o h andT.Tanabe

32

Fig. 5

A l i g n m e n t of EGF-like domains of C O X and m a m m a l i a n EGF 1

C0X1_HUMAN COXl_MOUSE COXl_RAT COXl_SHEEP COX2_CHICK COX2_HUMAN COX2_MOUSE COX2_RAT EGF_RAT EGF_PIG EGF_HUMAN

+

+

+

+ ...

NPC--CYYP-CQHQGICVRF-GLDRYQCDCTRTGYSGPNCTIP NPC- - C Y Y P - C Q N Q G V C V R F - G L D N Y Q C D C T R T G Y S G P N C T I P IPC- - C Y Y P - C Q N Q G V C V R F - G L D H Y Q C D C T R T G Y S G P N C T I P NPC- - C Y Y P - C Q H Q G I C V R F - G L D R Y Q C D C T R T G Y S G P N C T I P NPC- - C S L P - C Q N R G V C M T T - G F D R Y E C D C T R T G Y Y G E N C T T P NPC- - C S H P - C Q N R G V C M S V - G F D Q Y K C D C T R T G F Y G E N C S T P NPC- - C S N P - C Q N R G E C M S T - G F D Q Y K C D C T R T G F Y G E N C T T P NPC- - C S N P - C Q N R G E C M S I - G F D Q Y K C D C T R T G F Y G E N C T T P TGCPPSYDGYCLNGGVCMYVESVDRYVCNCV-IGYIGERCQHR SECPPSHDGYCLHGGVCMYIEAVDSYACNCV-FGYVGERCQHR SECPLSHDGYCLHDGVCMYIEALDKYACNCV-VGYIGERCQYR

(-) Indicates gap; o and •

indicate the sites occupied by physicochemicaly similar amino acids and

invariant sites, respectively.The abbreviated names and the references for COXs are the same as those in Fig. 3. EGF_RAT [56], EGF_PIG [57], and EGF_HUMAN [58] indicate the EGFs from rats, pigs and humans, respectively.

intracellular localization of the e n z y m e . X - r a y crystallographic studies

have

revealed that this region constitutes a novel m o t i f f o r insertion o f the e n z y m e into the m e m b r a n e [48]. T h e domain includes four amphipathic a-helices, A , B , C and D ( F i g . 7 ) , w h i c h are a r r a n g e d w i t h t h e i r h y d r o p h o b i c s u r f a c e s f a c i n g o u t w a r d . I n t h e h o m o d i m e r o f C O X , t h e h y d r o p h o b i c s u r f a c e o f a m o n o m e r is p l a c e d t o f a c e t h e s a m e d i r e c t i o n as t h a t o f a n a d j a c e n t m o n o m e r , f o r m i n g a l a r g e h y d r o p h o b i c p a t c h . T h e h y d r o p h o b i c patch does n o t span the lipid bilayer of the m e m b r a n e , b u t i n t e g r a t e s i n t o o n l y o n e l e a f l e t o f t h e b i l a y e r . P i c o t et al. [ 4 8 ] s u g g e s t e d t h a t t h e f o u r helices f o r m the entrance into the catalytic domain of the enzyme, and that the

Fig. 6

A l i g n m e n t of m e m b r a n e - b i n d i n g domains of C O X

heiix A

COXl_HUMAN COXl_MOUSE COXl_RAT COX1 SHEEP COX2_CHICK COX2_HUMAN COX2 MOUSE COX2_RAT

helix B

helix C

helix D

GLWTWLRNSLRPSPSFTHFLLTHGRWFWEFVNA-TFIREMLMLLVLTVRSN EIWTWLRNSLRPSPSFTHFLLTHGYWLWEFVNA-TFIREVLMRLVLTVRSN EIWTWLRSSLRPSPSFTHFLLTHGYWIWEFVNA-TFIREVLMGWVLTVRSN EIWTWLRTTLRPSPSFIHFLLTHGRWLWDFVNA-TFIRDTLMRLVLTVRSN EFFTWLKLILKPTPNTVHYILTHFKGVWNIINNISFLRDTIMRYVLTSRSH EFLTRIKLFLKPTPNTVHYILTHFKGFWNWNNIPFLRNAIMSYVLTSRSH EFLTRIKLLLKPTPNTVHYILTHFKGVWNIVNNIPFLRSLTMKYVLTSRSY EILTRIKLLLKPTPNTVHYILTHFKGVWNIVNNIPFLRNSIMRYVLTSRSH

(-) Indicates gap; O and •

indicate the sites occupied by physicochemically similar amino acids and

invariant sites.The abbreviated names and the references for COXs are the same as those in Fig. 3.The helical regions of ovine COX-1 are underlined.

Cydooxygenases: prostaglandin H synthases

substrate of the enzyme, arachidonic acid released from the membrane, can directly access the catalytic domain through the hydrophobic entrance. M ost parts of the dom ain are encoded by the fourth exon of C O X -I gene o r the th ird exon of C O X -2 gene (Fig. I). T he C -term inal half of helix D is, however, encoded by the following exon in each gene, which does not belong to the m em brane-binding dom ain, but is a p art of the catalytic domain. The N -and the Cterm inal halves of helix D m ay have been encoded by different genes, w hich have been integrated into an ancestral gene fo r C O X by exon-shuffling, although they now function to form a single helix, D.

The catalytic domain T he th ird folding u n it is the catalytic dom ain of about 450 am ino acid residues in length. T he catalytic dom ain is encoded by the fifth to eleventh exons of the C O X -I gene o r by the fo u rth to the ten th exons of the C O X -2 gene. As described above, C O X has tw o catalytic activities: cyclooxygenase and peroxidase activity. The dom ain carries the active sites fo r the tw o functions. Fig. 8 shows a proposed schem e fo r the catalytic m echanism to explain the relationship betw een the two activities [59,60]. The secondary structure of the catalytic domain is predom inantly α -helical w ith very little β -sheet [48], The tertiary structure comprises two distinct lobes th a t are intertw ined w ith each other (Fig. 9). The larger lobe is com posed of seven helices (H 2, H 3, H 5, H 6, H lO , H 18 and H 19), w hile the smaller dom ain is Fig. 7

T ertiary str u c tu r e o f t h e m em b r a n e -b in d in g d o m a in o f ovine COX-1

The figure was produced by MOLSCRIPT [49] with the coordinates o f IPRH [48] in the Protein D ata Bank.

33

H.Toh andT.Tanabe

34

Fig. 8________ Model for th e catalytic m echanism of COX AA

F e (IV)

F e (IV) AA* Tyr

F e (IV) Tyr* (I n te rm e d ia te II)

F e (IV) P P * (I n te rm e d ia te I)

PGHF e (III)

F e (IV) O 2AA* O O Tyr PGG-

C y c lo o x y g e n a se

P e ro x id a s e

AA = arachidonic a d d.T he oxidation state o f iron is shown by Rom an numerals. Fe(III) indicates the resting enzym e. A two-electron oxidation o f the h a em group by a hydroperoxide forms an interm ediate I, indicated by Fe(IV) PP*.The interm ediate I abstracts a hydrogen from the side-chain o f a neighbouringTyr residue, yielding an interm ediate Il [Fe(IV) Tyr*], which is associated with a Tyr radical.The interm ediate Il or th e Tyr radical abstracts th e 13-pro-S hydrogen from AA to initiate the cyclooxygenase reaction.This sta te is shown as Fe(IV) AA* Tyr. Figure based on W ei e t al. [60] and Hsi e t al. [61],

built w ith six helices ( H I , H 8, H 12, H 14, H 15 and H 16). T he tw o lobes are connected w ith six poly p ep tid e segm ents. T he dom ains, except the N - and C term inal regions, show weak b ut significant similarity in amino acid sequence to the m em bers of the peroxidase family. Figs. 10(a) and 10(c) show the sequence alignm ents of the N - and C -term inal regions of the catalytic dom ains. Fig. 10(b) show s the sequence alignm ent betw een the catalytic dom ains and 23 peroxidases. The sequence identities between the domains and peroxidases are only around 20%. H ow ever, the X -ray crystal structure of canine m yeloperoxidase [76], a m em ber of the peroxidase family, shares a quite sim ilar folding p attern w ith th a t of ovine C O X -1. As show n in Fig. 10(b), m ost of the α -helices of both enzymes are found at the corresponding positions in the alignment. T here is a h y drophobic channel in the catalytic dom ain to constitute the cyclooxygenase active site, w hich is long, n arrow (about 8 X 25 A) and present betw een the large and small lobes. The channel runs from the o u ter surface of the m em brane-binding dom ain to a place near the edge of haem plane. T he form er position is here referred to as the ‘entrance’ of the channel, and the latter as the ‘exit’ o f the channel. As show n in Fig. 10(b), the Ser residue is specifically conserved am ong C O X s at the alignm ent site 493, although a Ser is n o t fo u n d in o th er peroxidases at the site. T he residue corresponds to Ser-530 of ovine C O X -1, w hich is p resent in the h y d ro p h o b ic channel in the crystal stru ctu re. T he residue is n o t required fo r the cyclooxygenase activity. H ow ever, the acetylation of the residue

Cyclooxygenases: prostaglandin H synthases

Fig. 9

T he te rtia ry stru c tu re of th e catalytic domain of ovine COX-I

helix H3

helix H15

helix HlO

Small Lobe The figure was produced by MOLSCRIPT [49] with the coordinates o f IP RH [48] in the Protein Data Bank.The h aem group in the e n zym e is shown in ball-and-stick representadon.

w ith aspirin inhibits the enzym e by blocking the channel [35,77], Interestingly, acetylation o f C O X -2 by aspirin reveals a 15-lipoxygenase activity [78]. A Tyr radical has been detected in ovine C O X -I by electron param agnetic resonance (EPR) spectroscopy [79], w hich is considered to initiate the cyclooxygenase reaction by abstraction of 13-pro-5 hydrogen from arachidonate, as shown in Fig. 8. Spectral and biochem ical studies suggest that a Tyr group(s) is required for cyclooxygenase activity of the enzym e [80-83], T he m utation of Tyr-385 of ovine C O X -I to Phe abolishes the cylooxygenase activity [84]. Tyr-385 is found at the alignm ent site 313 in Fig. 10(b), where this Tyr residue is conserved among C O X s, but not conserved in peroxidases. In the crystal structure of ovine CO X -1, Tyr-385 is p resent at the exit of the h y d ro p h o b ic channel surrounded by six arom atic residues, and is in close proxim ity to the haem. T herefore, Tyr-385, or the Tyr residue at the alignm ent site 385, has been considered to be a candidate for involvem ent in radical form ation, although a recent m utation study is n ot in agreement w ith this hypothesis [61]. The Arg-120 and Glu-524 of ovine C O X -I are the only p olar residues in the hydrophobic channel, w hich are closely located to each other. The form er is present on the C-term inal half of Helix D and corresponds to A rg at the alignm ent site 49 o f Fig. 6, w hile the latter corresponds to Glu at the alignm ent site 486 of Fig. 10(b). Both residues are specifically conserved among

H.Toh andT.Tanabe

36

Fig. 10

Sequence alignments for COX-1 and COX-2, and for C O X with peroxidases 1

(a) COXl_HUMAM COXl_MOUSE COX1_RAT COXl__SHEEP COX2_CHICK COX2_HUMAN COX2_HOUSE COX2_RAT

(b)

l

•

+

•

+

LIPSPPTYNSAHDYISWESFSNVSYYTRILPSVPXDCPTPMGTKGKKQL LIPSPPTYNSAHDYISWESPSNVSYYTRILPSVPKDCPTPMGTXGXKQL LIPSPPTYNTAHDYISWESFSNVSYYTRILPSVPKDCPTPMGTKGKKQL I.TPSPPTYWIAHDVTSHPSFSHVSYYTRILPSVPROCPTPMDTKGKKOL LIDSPPTYNSDYSYKSWEAYSNLSYYTRSLPPVGHDCPTPMGVKGKKEL LIDSPPTYKADYGYXSWEAFSNLSYYTRALPPVPDDCPTPLGVXGKKQL LIDSPPTYNVHYGYXSWEAFSNLSYYTRALPPVADDCPTPMGVXOHKEL LIDSPPTYKVHYGYXSWEAFSHLSYYTRALPPVADDCPTPKCVXGNKEL •• •••••• • »••oo«*o****« ••o«o •••••O * • oo*

•

h e l i x 112

•

•

•

•

•

*

PDAQLLARRPZ/ - LRRXFIPDPQGTNLMFAPPAQHFTUQPPX-TSGX -MGPG -FT

COX J »OMAN COXI"MOUSE COXL_SHEEP COX2~CHICK COXJFHUMAN COX2JMOUSB COX2"RAT PER>THUAMN PERM~MOUSE PERE"HUMAN PERK_MOUSE PERI."BOVIN PERT_HUMAN PERT"PIG PERTJ40USB PERT~RAT PERL~DROMS PBR2~DROME PER)~EUPSC PER2"EUPSC PERN~PLEHT PBR1~CABLE PER7-~CAEI.8 P8R3~CAELB

PDVQLLAQQ L t» - LRREPI PAPQGTNILFAFPAQHFTHQFFK - TSGK - MGPG - FT PDAEFLSRRPL-LRRKFIPDPOCTNLMPAPPAOH FTHOF F K-TSGK KGPG- FT PDSKLIVBXPL-LRRKFIPDPQGTNVMPTFFAQHFTHQFFK-TDHX KGPG- FT PDSNBIVEKLL-LRRKFIPDPQGSNMMFAPFAQMFTHQFFX-TDHX-RGPA-FT PDSXEVLBXVL-LRREFIPDPQGSNBMFAPFAQHPTHQPFX-TDHK-RGPG-PT PDSKEVLEKVt, •LRREFIPDPQGTNMMFAFFAQHFTHQFFK-TDQK-RGPG-FT P LA RQV SNAIVRFPNDQ LTKDQE R A LM FMQWGQ PLD H DITL • TPEPATRPS - FPTGLNCETSCLQQPP CPPLX PLVRAVSNQIVRPPNERLTSDRGRALMFMQWGQFIDHDLDF-SPESPARVA-FTAGVDCERTCAQLPP CFPIK PLVRDVSNQIVRFPSKKLTSDRGRALMFMQWGQFIDHDLDF-SPESPARVA-FSKGVDCEXTCAQLPP CFPIK PLARBVSNKIVGYLDBEGVLDQNRSLLFMQWGQIVDHDLDF-APETELGSN-EHSKTQCEEYCIQGON CFPIM PPVREVTRHVIQVSNEVVTDDDRYSDLLMAWGQYIDHDIAF-TPQSTSKAA•FGGGADCQMTCENQNP CFPIQ PPVREVTROVIHVSNEAVTEDGQYSDLLMAWGQYIDHDIAP-TPQSTSKAA-PAGGADCQLTCEHRSP CFPIQ PPVREVTRHLIQVSNEAVTBDDQYSDFLPVWGQYIDHDIAL-TPQSTSTAA-FMiMVDCQLTCENQNP CPPXQ PPVREVTRHLIQVSNEAVTBDDQYSDFLPWfGGYXDHDIAL-TPQSTSTAA • FWGGVOCQLTCENQNP CFPIQ PSARLVS• •LVAPGEQDV-PDPEFTLHNMQWGQIMTHDHSM-QAGGTQSK•- -KHPTRCCTDDGRLICLDTAHKTCFAII PSAR LVSTSLVA•-TKBITPDARITHMVMQWGQPLDHDLDHAIPSVSSES- •-WDGIDCXKSCEMAPP CYPIB PGARLISTNPHGFSTPA•D RDDQLTHLTTLFG V F LNH DLQI•YPSMPTSGGDLEESIDCCNSDNTAV CYPID PGARLISTHFHGFSTPA-DRDOQLTHLTTLFGVFLNHDLQI-YPSMl'TSGGDLEEStDCCNSDNTAV CYPID PPPRPISNNVL-LDV-H-QPDELFTSSVHQWAQFIDHBFAH-VPFPTLEHGD- - -GXBCCPNGTQASGTLSHPR-CFPID PSVRSLSLTIF-- -TPRGEVHSDVTTMMGLWMQLIASDMVNIVPFQAVNEG-TSSALPCCKRGFNHSE CDAID PHPRRVSNLVC-- -EDKDVSHVXFTHXVMQFGQLL,DBELTH- SPVARGPND- • •EILNCT•KCDSP2XISVH••• CMP1R PNPREVSVFLL- • SSERSLP-GHVHSLLMLFGQPVSKDITS•HAAQNP CO - CQNSGPM CASIF PNARKVSRVLI - - GTDETTPHSHLSAMTMQKGQPIDHDLTLTAPALTRHSYK- - EGAFCNRTCENADP CFNIQ PSAREAMRVML- •SSAQSVVHDKFNNMMHOWGQPMSHDMSX-TTLOJ'SNA CK -TCDPVPSK CMPIP PSPREITRRLT • • SSQASVESPDYNALIMQPGQFISHDMAK-TTLVPSSX CN-VCQNITSR CMSVP O o o

PBR4~CAELe

PERB"CAELE P£R6~CABLB 81 C0X1__HUKAN COXI"WOUSB COXL~SHEEP COX2 CHICK COX2~HUMAN COXSJiOVSB COX2 RAT PBRM~HUAMN PERM_MOUSE PERE__HUMAN PBRB_MOUSE PERL BOVIN PERT~HOMAN PERT PIG P£RT~MOUSE PERTJRAT PERL__DROME PBR2 DROME PER1~BUPSC PER2 BUPSC PERN~PLENI PER1_CAELB PER2 CABLE PER3~CAELE PERL""CAELE PER5~CAELB PER6_CABLE

161 COX1 HUMAN COXL~MOUSH COXL~SHBBP COX2~CHICK COX2~HUMAN COX2^MOUSE COX2"RAT PBRM'HUAMN PERM~"MOUSR PERB"*HUMAN PBRB~MOUSE PERL_BOVIN PERT HUMAN PERT~PIG PBRT~MOUSE PERT RAT

h e l i x HI

helix E

•

•

*

•

•

*

h e l i x H3

•

•

•

KALGHGVDLGHIYGDNLERQYQLRLFKDKALGHGVDLGHIYGDNLERQYHLRLFKDKALGHGVDLGHIYGDNI.EROYOLRLFKDKAYGHGVDLMHIYGETUERQLKLRLRXDNGLGMGVDtKHIYGETLARQRKLRLFKD•

RGLGXGVQLNXIYGSTWRQHKLithPXDRGLGHGVDLNHVYGETLDRQHKLRLFQDIPPNDPR IKNQA • DCIPFFRSCPAC- PGSN- ITI RNOINALTS FVDASMVYGSB EPLARHLRHMSHO IPPNDPR IKNQK-DCIPFFRSCPAC-TRNN- ITI RNQINALTSFVDASGVYGSEDPLARXLRNLTNQ IPPNDPR- - - - IKNQR - DCIPFFRSAPSC- PQNK-NRV RNQINALTS FVDASMVYGSEVSLSLRLRNRTHY IPRNDPR IKHQR-DCIPPFRSAPAC- PQNR-NKV RNQIHALTSFVDASMVYGS BVT LALR LR NRTHF FPKNDPK.- - - - LKTQG - KCMPFFRAGFVC • PTPPYQSL -AREQIWAVTSFU5ASLVYGSEPSLASRLRHLSSP LP • BEAR PAAGT • A C LPFY RSS AACGTGDQ - GALFGN LST AN PRQQKNGLTS F LOASTVY GS S P ALERQLRNW?S A LP-TKAS-• - -GAAGA-TCLPFYRSSAACCSCRQ-GALVGHLSWAAPRQQMNGLTSFLOASTVYGSSPAQEQRLRffWTSA LP SSSS GTT•ACLPFYRSSAACGTGDQ•GALFGNLSAAN PRQQMNGLTSFLDASTVYGSSPGVBKQLRNWSSS LP-SNSS RTT-ACLPFYRSSAACGTGDQ-GALFGNLSAAHPRQQMiJGLTSFLDASTVYGSSPGVKKQLRHWSSS VPPHDPAY- -•SQVGT-ECLNFVRTLTDRDSNCQ YQGGPAEQLTWTSYLDLSLVYGNSIQQNSDIRBPQ- VPPKDPR VRNR-RCIDVVRSSAICGSGMT--SLF--FDSVQHREQINQLTSYIDASQVYGYSTAPAQLARNLTSQ IPVNDTYF-- -GVYGR•TCHEFVRSLASPALTCG LGPREQLHTATGYIDASQVYGSDIDRQLLLRAMU- • IPVNDTYF- - -GVYGR-TCMBFVRSLASPALTCG LGPREQLNTATGYIDASQVYGSDIDRQLLLRAMB- LT-GDPFY-- -GPLGS-TCHNFVRSHVAVGVGSA CAFGYADBLNQLTHWIDASMVYGSTABBBRELRAGQ-IPAADPAY RTRL-NCIPHARSIIAPRBACR LGPREQANFASSYLDASFIYGSNMEKAKQLRTFR - VEKDDPFFPTNYPNGEPRCLPFARSLLGQLN LGYRNQLNQLTAYVDGSAIYGSTKCEAKNLRLFT • APPSDRSR RCIPFTRSFPICGTGQ FGRV REQLNMNTAAXDASLIYGSEAITARSLR- F • • • LEADDPKLHT-GLYQKHPCMEFERMGAACGSGET-SPIFQRV••-TYRDQLSLLTSYLDASGIYGHSEEQALELRDLYSD ICSKDPNL- - -GFKSK -QCLKVSRSAPIC-RVB PREQLNENTAYIDGSMIYGSSI.XDLHKFRDGR • • ITFDDSNA- - - NFRQA • QCIRVSRSSPICGSGN LX PRQQLN EHTGYI DA S PIYGS S VHDS X K PRDGil - -

O OO

•

( h e l i x H4)

•

•

•

•

O**

•

h e l i x HS

,

•

•GXLKY-QVL--DGEKYPPS-- -VEBAPVLMHY-PRGIPPQSQMAVGQBVPGLLPGLMLYATLWLRBHNRVCOLLKABHP •GXLKY-QVL- DGEVYPPS- - -VEQASVLMRY•PPGVPPERQMAVGQEVPGLLPGLMI.FSTIWLRBHNRVCDLLKEEHP GKLKY -QHL- -NGBVYPPS-- - VEEAPVLKHY - PRGIPPOSOMAVGOBVFni.I.PCLMI.VA.TIWI,RKHHBVf>f>T.T.irf f.HP •GKLXY-QUI -•DGBMYPPT•-•VKDTQAEHIY-PPHVPEHLQPSVGQEVPGLVPOLMKYATIWLREHNRVCDVLKQEHP •GKMXY-QIt - -OGBMYePT- - VKDTQAEMIY • PPQVPEKLRFAVGQEVPGLVPGUiKYAT iWLREHKftVCESVl^KQBHP -GKLKY-QVI•-GGEVYPPT--•VKDTQVEMIY-PPHIPENLOFAVGOEVFGI.VPGLHMYATIWLREHNRVCDILKQEHP -GKLXY-QVI--GGEVYPPT-- -VXDTQVDMIY-PPHVPBHLRFAVGQEVFGI.VPGLMMYATIWLRBHNRVCDILKQEHP LGLLAVHORFODNGRALLPPDM- LHDnPCM.TM - • - R S A RI PC F LA GDTRS S RM PE LT SMHTI.I^ipRHWRl.ATRf.KS pMP LGLLAVHTRFQDNGRALMPFDS-LHDDPCLLTN---RSARIPCFLAGDMRSSEMPELTSMHTLFVREHNRLATQLKRLNP LGL1AINQRFQDNGRALLPFDN-LHDDPCLLTN--•RSARIPCFLAGDTRSTETPKLAAMHTLFMREHNRLATELRRLHP LGLLATNQRFQDNGRALLPFDN-LHEDPCLLTN-•-RSARIPCFLAGDTRSSETPKLTALHTLFVREHNRLAAELRRLNP LGLMAVNQEAWDHGLAYLPFNN-KKPSPCBFIN-•-TTARVPCFLAODFRASEQILLATAHTLLLREHNRLARBLKKLKP BGLLRVHARLRDSGRAYLPFVPPRAPAACAPEPGIPGBTRGPCFLAGDGRASKVPSLTALHTLWLREHNRLAAALKALNA BGLl.RVNTRHRDAGRAFLPFAPPPAPPACAPEPGTPA-ARAPCFLAGDSRASEVPGLTALHTliWLRBHNRLAAAFKAIiNA AG LLRVNT LHLOAGRAYLPFA TAACA PBPGTPRTNRTPCFIIAGDGRAS EVPALAAVHT LHLREHNRLASAFKAINK KAT . _ . .CliriiDO Q^i QU AUD7*QrPI hrnnOlLet>lin>f ft klllwi Uf nDff»nt %m

pER2^DROME PERI^EUPSC PER2__eüPSC PERN__PLENI PERl^CAELE PER2^CAELE PER3_CAELE PER4~CABLE PER5_CAELE

ASEVCYRSCDVRVNQNPGLAILQr rLLRBfíNR rAOALSALNP

EGLLRVG-VHFPRQKDMLPPAAPQDGMDCRRNL-.-DEllTMSCFVSGOIRVNBOVG^f.MH'riWMREHSRlASRLKOINS GGLMRTT- - - PTDDLDLMP-QDN - - STFCRATE GNLCFlGGDGRVNVQPMMMSLHHLFVREHNRLANlISTANP GGLMRTT- - -PTODLDLtoP-Q0N- -STFCRAAE gnlcfiggdgrvnvqpkhmslhhi.fvrehhrlanxissanp NGLI-KV • • SANNLLPINPNQ- GGSCEARV RGAKCFMAGDSRVNEQPGLTALHTLLVRQHNLVARDl.KALSP NGQI'RT AGSIGELP • ATDG • TLQCQATH SRCALSGTDEVNILPSVAALHTVFXRHHNRX ADNLRSINR RGLLNFTDF - • GHGQMMLP - QGNQ - EKDCRSTI, • • • BKRHMPCFVAGDERNSHQPGLTIMHTPFVREHNRIfcMQLSALNP AAMI.RTSMl GGRMfp PNTNPGSLTAGDGRAILFVGLAALHTSFLRLHNNVAARLQNMNR HGLLRFDIV-SGANKPYMPFEKDS-DMDCRRNP- •SRENPIKCFIAGDVRANF.QLGLMSMHTIFI.RBHNRTASRLLEVHB TGFLRVTRp. - • NHQNVLPF-DQS• • -KCANKD KCTASFTAGDIRANLFIGLSSLHIMPAREHNRIAQKLTELNP SGFLKLPMF- - • WGKAFLPF- DQN- - - KCRNRG QCSVIFTAGDSRVNLFVGLSAWHTIFTEEHNRLVTAFKRLNP

PERSICAELE

0 0

241

COXl_HUKAN

• *

•

h e l i x HS

( h e l i x H7) *

TWGDEQLPQTTRlilLIGETlRlVIEEYVQQ-LSGYPLQLKF TWDDEQLPQTTRLILIGETIKIVIEEYVQH - LSGYPLQLKF

COXl_MOUSB COXi_SHEEP COX2_CHICX COX2_HUMAN COX2JXOÜSE COX2_RAT PERM_HUAMN

O •

•• O „

h e l i x H8 +

,

DPELLFGVQPQYRNRXAMEFN-HI,-YHW-HPLM DPELLFRAQPQYRNR1AKEFN • HL-YHW-HPLM

EWDDEQLPQTTRLILIGETIKIVIEDYVQH- LSGYliFKLKF EWGDBQLFQTSRLILXGETIKIVTEDYVQH - LSGYHFKLKF EWGOEQLFQTSRLILIQETIKIVIEOYVQH-LSGYHPKLXP EWDDERLFQTSRLILIGETIKIVTEDYVQH-LSGYHFKLKP

PERM_MOUSK

DPBLLFNQRFQYQNRIAAEFN-TL-YHW•HPLL nPELLFNKQFQYQNRIAAEFN-TL • YHW-HPLL I>PELLFNQQPQYQNR2ASKPÍÍ-TL- YHW- HPLL DPELLFNQQFQYQNRIASEFN-TL-YHW-HPLL

RWNGBKLYQEARKIVGAMVQIITYRDYLPLVLGPAAMXKYL PQYRS YNDSVDPRIANVFT - NA - FRYGHTLI RWNGDK LYNEARKIMGAMVQIITYRDPLPLVLGKARARRTL GHYRG-YCSNVDPRVANVFT- LA- FRFGHTHL HWSGDKLYN8ARKIVGAMVQIITYRDFLPLVLGRARIRRTL GPYRG YCSHVDPRVAHVFT - LA • FRPGHTML HWNGEKLYQEARKILGAFIQIITFRDYLPIVLG-SEMQKWI PPYQG-YNNSVDPRISNVFT-PAFRFGHMBV HWSADAVYQEARKVVGALHQIITLRDYIPRILGPEAPQQYV GPYEG-YDSTANPTVSNVFS-TAAFRPGHATI HWSADTVyQEARKWGALHQXVTLRDYVPKILGAEAFGQHV GPYQG-YDPAVDPTVSNVFS-TAAFRPGHATI H WS ANTA YQEARKWGALHQIITMRDYIPKILGPDAFRQYV GPYEG-YNPTVNPTVSNIFS • TAAPRFGHATV HWSANTAYQEARKWGALHQIITMRDYIPKILGPDAPRQYV GPYEG - YNPTVNPTVSNVFS• TAAPRFGHATV H YDDR TL FQEA R KINIAQYQQISY YE VLPIFLGGENMLKNRLIYKAPSGS¥INP PD PNIDPS VI»NEH A • TAA PR Y FH S 01 HWDGDTLYQEARKIVGAQMQHITPKQWLPLIIGESGME-MM SEYQA TSP•TESSIANEFA•TAALRFGHTII DWTDEVIFQETRKLVIAEMQHVTYNEYLPKXVGPTMMETYSLNTLTQ-G-YSH-YLANINPSIRNGFA-SAGItYSHSGI,

PERB_HUMAN PERE_MOUSE PERL_B0VIN

PERT_HUMAN PERT_PIG PERT_MOUSE PERT_RAT PBRl_DRO«E PER2_DROME PER1_EUPSC PBR2_BCTPSC PERN_PLENI

onrDZVIFQETRKLVIAeWKVTYHE'Ci.PKIVGPTttMETYSLNTLTQ-G-YSM-YLANrNPSIRNGPA-SAGIIYSHSGL

QWSDNALFQBTRRIIIAQTQHIIFNEWLPXILGKDFMXSFGLTVLRS-G-PSADYNPNINPNMNSKPSTAAFRFGHTLV HWTDDKLYEEARKIVAAQVQHITYNEPLPVLLGRENMRNYGLN-LHSAGFDSN-Y8MNL.KGTTPNEFAVTITYYP-WAliL QWNDDTVFEEARRIVTAEMQHITFAEPLPKIIGLDliLNAQNIiV-PKKNGYFGG YDNTCDASXSQPFA-TAAFRFGHTLr HWNADRIFQESRKIVGGIVQVITYQEPVPELIG-DASKTXL GAYNG-YNPNVBlGVLNEFA-AGAYRL-HGMI NWDGETIFQETRXLIGAMLQHITYNAWLPKlLGKATYNTII GEYJCG - YWPDVNPTIANRFA • TAALRPAHTLI TWSGDRVFQEARKIVGAQIQNVLYKEYLPKLLG-VSPDKVI GPYKG-YOTNVDATIANKFT-TSAFRFGHGMI HWDGERLYQEARKHIGAQVQAíVYREWl/PKVLG - ASPATVV . -GPfRC-füSDVDSrJANKPT • SAAfRFGHCMl

PER1_CAELE

PER2_CAELE PER3_CAELE PER4_CAELE PER5_CABLE PER6_CAELE

O

321 COXl_HOMAN COXl_MOUSE COXl_SHEEP

COX2 CIIICX COX2_H UMAN COX2J40VSE

COX2_RAT PERM_HUAMN PERM_MOUSB PBRE_HUMAN

Oo O» O o

* -P -P •P •P -P -P -p. - . -

O

*

OO

OO

h e l i x H9 •

O

h e l i x H10 « E PER5_CAELE PER6_CAELE

401

h e l i x Hi2

•

h e l i x H13

•

•

h e l i x H14

•

h e l i x H15

*

h e l i x H16

* — •—

+

COXl_HUMAN COXl_MOUSB COXl_SHEEP COX2_CHICK COX2_HUHAN COX2_MOUSB COX2_RAT PERM_HUAMN

LHV AVDVIRESREMRLQPFNEYRKRP-GHKPYTSFQBLVGB--XB--MAAELBELYGDIDALEFYPGLLLEKCHPNSIFG LHVAVDVIKESREMRLQPFNEYRKRF-GLKPYTSFQELTGB•-KE-•MAAELEELYGDIDALEFYPGLLLEKCQPNSIFG T.HVAVPVIKRSRVLRLOPFW^YRKRF• GMXPYTSEQELTGB- -K£; -^l¿,flELBELYGDlPALEEl£aLIíLgKCHPNSlFG QKVA KASIDQSRQMRYQSLNEYRKRF-MLKPFKSFBB LTGB • • - HMBíjBEL YGDIPAMBL YPGLLVBXPRPGAIPG QKVSQASTOOSRQMKYQSPNEYRKRP-KLKPYBSFEELTGK--KE--HSAELEALYQOIDAVELYPALLVBKPRPDAXFG QAVAKASIDQSREMKYQSLNEYRKRF-SLKPYTSFEELTGE•-KE--MAAELKALYSDXDVMELYPALLVEKPRPDAIFG QAVAKASIDQSREMKYQSLNEYRKRF-S LKPYTS FEELTGE- -KE-•MAABLKALYHDX DAMBL YPALLVBKPRPDAIFG

PERB_HOMAN PERE_MOUSE

D- I.AALNMQRSRDHGLPGYNAWR-RPCGLSQPRNLAQL-SRVLKNQDLARKFLNLYGTPDNIDIWIGAIAEPLLPGARVG D-LAALNMQRSRDHGLPGYNAWR-RPCGLSQPRNLAQL-SRVLKNQDLARKPLRLYKTPDNIDIWVGATAEPLLPGARVG

PERM^MOUSE D- LP&UtMQRSRDHGLPGYNAWR•RPCÚLPQPSTVGEL-GTVLKNLBLARKLMAQYOTPHNXDIWMGGVSEP

contd.

9 H.Toh andT.Tanabe

38

Fig. 10 (Contd.) PFRL_BOVTN PERT_HUMAN PERT_PIG PERT_MOUSE PERT_RAT PERl_DROMK PER2_DROMB PRK1_EUPSC PER2_BUPSC PERM_PLEN1 PER1_CABLB PER2_CAEI.R PER3_CAELB PBR4_CABLE PER5__CAELE PKR6_CABLB

D-LAAINLQRCRDHGMPGYNSWR-GPCGLSQPKTLKGL-QTVLKHKILAKKLMDLYKTPDNIDIWIGGNAEPMVERGRVG D-LASINLQRGRDHGLPGYNEWR-EFCGLPRLETPADL-STAIASRSVADKILDLYKHPDNIDVWLGGLAENPLPRARTG D I.ASINLORGRDHGLPGYNEWR - EPCGLSRLETWADL-SAATANGRVADRILGLYQHPDNIDVWLGGLAESFLPCARTG D•I.ASLNLQRGRDHGLPDYNBWR-EPCGLSRI.ETPAEL-NKAIANRSMVNKIMDLYKHADNIDVWLGGLAEKPLPGARTG D-LASLNLQRGRDHGLPGYNBWR-EFCGLSRLDTGAEL.NKAIANRSMVNKIHELYKHADNIDVWLGGLAEKKLPGARTG D - IiRSLDIQRNRDHGLASYNDMR- EFCGLRRAHSWBGY -GDLISPP- ILEKLKSLYPSHEDVDLTVGASLliAHVAGTLAG D-LAAINIQRGRDHGMPGYNVYR-KLCNLTVAQDFEDL-AGEISSABXRQKMKELYGHPDNVDVWLGGILBDQVEGGKVG D -1.AAIWVQAGRDÏGLPTYNAWR - QWCGLDVATNFTTL-ADH • SEDD • AN LLAS LY T SV EDIDVWTGGVS EIPIEGGSVG D-LAAIKLQAGRDIGLPTYNAWA-QWCGLDVATNFTTL-ADH-SEDD-ANLLATVYTSVBDIDVWTCGVSEIPIBGGSVG D.LMSLNIQRGRDHGIATYNSMR-OVCGLPRARTFNDL-TDQISPEN-VQKLARIYXNVDDIDLFVGGITENSVRGGLLG D-LISIALKQGRDHGIPGYTALR-ASCGLGRIASFNDLREIFLPEVKF-EQVSSAYTRVEDVDLLVGVLAEKPLKGSLVG D- MVLNILRARDHGVQPYNDLR-BFCGLRRAVKWDDLKGEM-DQDNI-NILQSLYESVDDVDLFPGLVSERPLRGALLG D-MAAVNIQRGRDHGLRSYNDYR-RFCNLHPITSFNDW-PKV-PDENVRQRIGQLYRTPDDLDFYVGGILEQPAAGSLLG D • LAALNIQRGRDHGLPSWTEYR - KFCNLTVPKTWSDM- KNIVQNDTVISKLQSLYGVTENIDLWVGGVTEKRTADALMG D-LGSLNIQRGRDHGIPSYNKMR-QFCGLXSANTFDDFADMIL-DRNI.RAGLARNYNTTNDVDFYVGSMLEDPVIGGLVG D-LSTINIQRGRDHCHPAVVKYR-ELCGMGTAFNFEHLSRBIL-NTGTRNKLQEIYGSVDKIDLWVGALLEDPIIRGLVG o « O • O OO O • O •

481 C0X3__HUMAN C0X1_M0USE C0X1_SHEEP COX2_CHICK COX2_HUMAN COX2_MOUSB C0X2_RAT PERM_HUAMN PERM_MOUSB PERE_HUMAN PERE_MOUSB PERI._BOVIN PEKT_HUMAN PERT_PIG PERT_MOUSB PERT_RAT PERl_DROME PER2_DR0ME PER1_BUPSC PER2_EUPSC PERN_PLENI PER1_CAELB PBR2_CAELE PER3_CAELE PER4_CAELB PER5_CAELE PER6_CAELE

helix H17 »

*

helix H19 *

»

•

ESMI-KIGAPP-SLKGLLGNPICSPEYWKPSTFGGBVGFNIVKTATLKKLVCLNTKT-••-CPYV-SFRV BSM1-EMGAPP-SLKGLLGNPICSPEYWKPSTFGGDVGFNLVNTASLKKLVCLNTKT-- --CPYV-SFRV ETMV-EIGAPF SLKGLMGNTICSPBYWXPSTFGGKVGFBIINTASLQKLICNNVKG-•--CPFT-AFHV ETMV BVGAPF SLKGI.MGNVICSPAYWKPSTFGGEVGFQIINTASIQSLICNNVKG CPFT-SFSV ETMV-ELGAPF SLKGLMGNPICSPQYWKPSTFGGEVGFKIINTASIQSLICNNVKG CPFT-SFNV ETMV-ELGAPF-SLKGLMGNPICSPQYWKPSTFGGEVGFRIINTASIQSLICNNVKG- - --CPFA-SFNV PL·LACTTGTOFHKT.RD- - GHRFWWRN- - - EGVFSMO - ORpALAOISI.PRI ICDNT - O - ITTVSKNN1FMS QLLACLIGTQFRKLRD--GDRFWWEN--•PGVFSXQ-QRQALASISLPRLICDNT-G -ITTVSKNNÏPMS PLLACLFENQFRR-AE•-TETGSGGR-- -TRCFHQR-QRKALSRISLSRIICDNT-G•ITTVSRD-1 FRA PLLACLPBNQFRRARD- -GDRPWWQK•--WGVFTKR-QRKALRRISLSRIVCDNT-G-ITTVSRD-I FRA PLLACLLGRQFOQIRD--GDRFWWEN-- -PGVFTEK-QRDSLQKVSFSRLICDNT-H -ITKVPLH•AFQA PLFACLIGKQMKALRD-•GDWFWWEN- -•SHVPTDA-QRRELEKHSLSRVICDNT-G•LTRVPMD•AFQV PLFACIIGKQMRALRD--GDRFWWEN-- -PGVFTEA-QRRELSRHSMSRVICDKS-G-LSHVPLD-AFRV PLFACIIGKQMKALRD--GDRFWWEN--•TNVFTDA-QRQELEKHSLPRVICDNT- G -LTRVPVD-AFRI PLFACUGKQMKALRD--GDRFWWEN--•SHVFTDA-QRQELEKHSLPRVICDNT-G-LTRVPVD-AFRI PT PLCILTEQFYRTRV- •GDRFFFENGDKLTGFTPD-QLEELRKASMARLLCDNG-NHISSMQPE-AFRT PbFQCLLVEQFRRLRD--GHRLYYEN PGVFSPE-QWQIKQANFGRVLCDVG-DNFDQVTEH • VF1L PLFACIAARQFQALKM-•GDRPWYENAG-PNQLSVD-TVNAIRNVTMSRLICDNT--NIQQIQGD- AFIA PLFACIAARQFQALKM- -GftRFWYBNAG -PNQLSVXi -TVNAIRNVTMSRLTCDNT- -NIQQIQGD - AFIA WTFLCIVGDQFARLKK--GDRYPYDLGGQAGSFTEP-QLQQIRASSWARIICDTA--NVPAVQPL•AFRQ PTMACIIGKQMQRTRR--ADRFWYENYFAQSGFNEA-QLSEIRNTKLAEÏICSNI-D-IRRIQRN-VFFR TTMSCIIAEQFGRLKK--CDRFYYBNDNSAAKFTPG-QLNEIRKVKLASIFCSNS-KYLKTIQPN•VFDV ATFACVIGKQPHRLRD--GDRFYYEN- - -PGVFTS P-QLAELKRTTLSWVLCQTG-DNMV RVGRR- A FDI PTLACIIADQFKRLRD--GDRFWYEN-•-EEMFSKA-QI.RQIKKVTLSKIXCTNG-DD1DRIQRD-IFVY TTLSCAIGEQFKRARD--GDRFYFEN-- -PGIFTRS-QMEEIKKSSLSRIICDNA-DNFELVSQD-AFLL PTVACIIGPQFKRTRD--GDRFYYEN--•PGVFSRR-QLVEIRKSSLSR1ICDNT-NTIS•VRNI•KFSL o o o • •

1 (C)

helix H18 »

COXl_HUMAN COXl_MOUSE COXl_RAT COXl__SHEEP COX2_CHICK COX2_HUMAN COX2_MOÜSE COX2_RAT

•

f

• -PUASQ DD-GPAVE- • RPSTEL • PDYPG DD-GSVLV- -RRSTEL - -PDYPG DD-GSVRV- RPSTEL • - PDPRQ ED-RPGVE- -RPPTEL I.NPEPTE-ATINVSTSNTAMEDINPTLLLKEQSABL PDPELIXTVTINASSSRSGLDDIHPTVLUKERSTEL QDPQPTKTATINASASHSRLDDINPTVLIKRRSTEL QDPQPTKTATINASASHSRLDDINPTVLIKRRSTEL •O O* o oo**

(a) Alignment of the N-terminal regions of COXs. (-) indicates gap; O and •

indicate the sites

occupied by physicochemically similar amino acids and invariant sites.The abbreviated names and the references for COXs are the same as those in Fig. 3.The regions constituting helices in ovine COX-1 are underlined, (b) Alignment among COXs and peroxidases: (-) indicates gap; O and •

indicate the sites

occupied by physicochemically similar amino acids and invariant sites respectively. Asterisks indicate the invariant sites among COXs.The regions constituting helices in ovine COX-I are underlined.The abbreviated names and the references for COXs are the same as those in Fig. 3.The abbreviated names for peroxidases are as follows: PERM_HUMAN and PERM_MOilSE, myeloperoxidases from humans [62] and mice [63] ;PERE_HUMAN and PERE^MOUSE, eosinophil peroxidases from humans [64] and mice [65]; PERL_BOVIN, bovine lactoperoxidase [66]; PERT_HUMAN, PERT_PIG, PERT_MOUSE and PERT_RAT, thyroid peroxidases from humans [67], pigs [68], mice [69] and rats [70]; PER I_DROM£, putative peroxidase from Drosophila melanogaster [71]; PER2_DROME, peroxidasin from Drosophila Kc 7E10 cell line [72]; PERLEUPSC and PER2_EUPSC, peroxidase-homotogues from the bacterial light

contd.

C O X s at the Sitesj b u t peroxidases do n o t have the residue at the corresponding sites. T he tw o residues are considered to form a salt bridge in the channel. It is proposed that the A rg residue serves as anchoring counter-ion for the carboxylate group of the fatty acid substrates [14,31,48], T he peroxidase active site is present at the interface between the large and small lobes o f the catalytic dom ain, w here the iro n (III)-p ro to p o rp h y rin IX is placed. T he m ajor helices involved in the haem binding are H 2, H 5, H 6, H 8, and H 12 in both ovine C O X and canine myeloperoxidase, whose C a backbones can be superim posed on each oth er w ith a ro o t mean square difference of 1.4 A [48], H is388 and His-207 of ovine C O X -I act as the proximal and distal H is residues respec­ tively, w hich correspond to the H is residues at the alignment sites 317 and 37 of Fig. 10(b) [48,85], O n ly a peroxidase-like sequence from Caenorhabditis elegans lacks the H is residues at bo th sites. T he form er resides on a loop follow ing a helix H 8, w hile the latter is o n a helix, H 2. T he C O X s lack a long polypeptide segment connecting H 2 w ith H 3 of about 60 residues in length (alignm ent sites 55-131 in Fig. 10b). T he polypeptide form s six strands of extended chain and a loop consti­ tuting a calcium ion binding site in the canine myeloperoxidase [76]. It was reported that som e E G F-Iike dom ains are involved in calcium ion binding [86], Therefore, the lack of the p olypeptide in C O X s may be com pensated by the im p o rt of E G FIike domain. T he alignm ent site 228 in Fig. 10(b) is occupied by a H is residue that was previously considered to act as a ligand fo r the haem iron (for example, see review [87]). H ow ever, the determ ined structure of ovine C O X -I revealed that the residue H is-309 in the enym e does n o t bind to the haem group, b ut constitutes an interface betw een helices H 5 and H 6. As show n in Fig. 10(b), helices H5 and H6 are present at a highly conserved region am ong C O X s and peroxidases. In the structures of both ovine C O X -I and canine myeloperoxidase, the two helices form a ‘V ’-shaped core stru ctu re of the large lobe, around w hich helices H 3, H lO , H 18 and H 19 are

Fig. 10 (C ontd.) organ o f the squid, Euprym na scolopes [73];PERN_PLENI, peroxinectin from blood cells o f the crayfish, Pacifostacus Ieniusculus [74]; and PER CC A E L E PER2_CAELE PER3_CAE1£, PER4_CAELE, PER5_CAELE a n d PER6_CAELE peroxidase-like sequences from chrom osom e III o f nem atode, Caenorhabditis elegans [75], whose accession codes in GenBank are Z 4 9 129, Z 4 9 130, Z 6 6 5 20, Z 6 8 0 0 6 , U 4 2 8 3 3 and L120864, respectively.The amino acid sequence o f canine myeloperoxidase, determ ined by X-ray crystallography, contained several unidentified segments.Therefore, the sequence was n o t included In the alignm ent Instead, the regions constituting a-helices in the canine myeloperoxidase are shown in th e corresponding regions o f hum an myeloperoxidase by underlines.The nam es for th e helices not found in ovine COX-I are in parentheses, (c) Alignment o f the C-terminal regions o f COXs; (-) indicates gap; O a n d *

indicate the sites occupied by physicochemically similar

am ino acids and invariant sites respectively.The abbreviated nam es and the references for COXs are the s o m e o s those in Fig. 3.

H.Toh andT.Tanabe

40

packed. In addition, the tw o helices grip helix H 2 in a ‘ch o p stick ’ fashion. T herefore, the sequence conservation at th e helices H 5 and H 6 suggests th a t the region is subjected to high stru ctu ral co n strain t to m aintain the fold of th e large lobe. T he C -term inus of m ature C O X has been determ ined o nly fo r ovine C O X -1. N ucleotide sequencing of cD N A s and genes for C O X s has revealed that the deduced C-term inal region of the catalytic dom ain is diverged between C O X -I and -2 (Fig. 10c). H ow ever, their C -term ini are conserved and end in (Ser o r P ro)(T hr or A la)-G lu-L eu, w hich fits one of the C -term in al E R reten tio n signals. T herefore, th e C -term ini of C O X -I and -2 are considered to lead the enzym e to ER. H ow ever, a recent study w ith am ino acid replacem ent at th e region has suggested that the C -term inal region plays an im portant role for the folding process of the enzyme, but is n ot required for the intracellular targeting of the enzyme [88]. Fig. 11 show s an u n ro o ted phylogenetic tree fo r 28 enzym es, including tw o isoform s of C O X and peroxidases, based on the alignm ent of Fig. 10(b). The tree topology suggests that C O X s form a distinct group in the peroxidase family, and the divergence of the enzym es from the other peroxidases is quite ancient. N o d e X indicates the divergence betw een C O X -I and -2, w hile nodes Y and Z are considered to correspond to m am m alian divergence fo r C O X -I and C O X -2 respectively. As show n in Fig. 11 and Table I, the branch betw een X and Y is about tw o tim es longer th an th a t betw een X and Z. T he b ranch length indicates the num ber of amino acid replacements that occurred between the nodes of the branch. T herefore, the difference of the branch lengths suggests th a t C O X -2 has been subjected to greater selection constraint than C O X -I d u rin g the tim e from the divergence of the tw o isoforms to the period of mammalian divergence. C ontrary to that, the branch length from Z to hum an C O X -2 is greater th an th at from Y to hum an C O X -1. Moreover, the branch from Z to m ouse C O X -2 is greater than that from Y to m ouse C O X -1. This observation seems to suggest that C O X -I has been subjected to greater selection constraint than C O X -2. H ow ever, the branch length from Y to ra t C O X -I is greater than th a t from Y to rat C O X -2 . T hus the evolutionary rate of C O X has fluctuated a great deal. It is know n th at the ph y sio ­ logical roles of PG s are often different from species to species. T his functional diversity of PG s m ay have fluctuated the evolutionary rate of C O X . In any event, it is difficult to assume the constancy of the evolutionary rate of C O X to estimate the divergence tim e betw een the tw o isoform s at present. W hat is know n, how ever, from the studies on avian C O X -2 , is th at gene du plication of the tw o isoform s occurred before th e divergence betw een mam m als and birds, ab o u t 200 m illion years ago.

Cyclooxygenases: prostaglandin H synthases

Fig. 11

Phylogenetic tre e of C O Xs and peroxidases

fL

COX-1 .S H E E P

-C 0 X -1 .M 0 U S E 922 *— COX-1 .R A T C 0X -2.C H IC K C 0 X -2.H U M A N

I------ C'

903 770

M t /

c

COX-2 .M O U S E L O C O X-2.RAT

P ER 2.C LELE PER 1.D R O M E

944

PER N ,PLEN I I PER1.EU PSC

L

PER2_EUPSC P E R 1 .CAELE

PERM .H U M AN I

PER M .M O U SE PERE.HU M AN

L - PER E.M O U SE P E R L . BOVIN P ER T.H U M AN PER T.M O U SE PERT.RAT - PERT.PJG PER 2.D R O M E PER 4.C LELE P ER 3.C LELE PER5_CLELE PER 6.CLELE

The tree was produced with a software package, PHYLIP (version 3,57c) [8 9 ], based on the alignment shown in Fig. 10(b). The genetic distance between each aligned pair was calculated by the maxim um likelihood m ethod with PAMOOI m a trix [9 0 ].T h e sites, including gaps, were excluded from the alignment fo r the calculation o f the genetic distance.The tree was then constructed by the neighbour-joining m ethod [9 1 ], using the genetic distances.The u nit o f the branch length is the num ber o f am ino acid replacements p e r site. Scale b a r indicates the length fo r 0.1 substitution p e r site. Statistical significance o f the obtained tree topology was evaluated by the bootstrap analysis [9 2 ] with I OOO iterations. Each integer associated with a branch indicates the num ber o f occurrences o f the branch during the iterations. A branch with a num ber greater than 9 0 0 is regarded as significantThe figure was drawn byTREETOOL [9 3 ] with the o u tp u t ofPHYUP.

Concluding rem arks T h is ch a p te r has review e d some recent studies on the stru c tu re , fu n c tio n and e v o lu tio n o f C O X , a lth o u g h several in te restin g topics have been o m itte d o w in g to lim ita tio n s o f space. F o r exam ple, cro s s -ta lk betw een C O X and n itr ic oxide p a th w a ys has been re c e n tly d e m on stra te d [94,95], I t was re c e n tly fo u n d th a t te p o xa lin can in h ib it n o t o n ly the cyclooxygenase a ctivity, b u t also the peroxidase a c tiv ity [96 ]. T h u s rece nt progress in the fie ld has been v e ry ra p id . A s described here, the re are s t ill m a n y p ro b le m s w ith C O X such as the fu n c tio n a l ro le o f the E G F -Iik e dom ain, the o rig in o f tyro sin e radical and the m echanism o f in tra c e llu la r

Table 1

The list of genetic distances between tw o nodes in the COX cluster of Fig. 11

The branch length between two nodes in the figure corresponds to the genetic distance between them.The unit of genetic distance is the number of amino acid replacements per site. N odes

G en etic distance

X and Y

0.19534

X and Z

0.09345

Y and hum an C O X -1

0.04506

Z and hum an C O X -2

0.08302

Y and m o u se C O X - 1

0.03488

Z and m o u se C O X -2

0.07405

Y and r a t C O X -1

0.07615

Z and ra t C O X -2

0.05620

targeting to E R a n d /o r N E . H ow ever, rapid progress in the field sh o u ld soon resolve these problem s. F urther study on C O X will n ot only reveal a new aspect of intracellular signal transduction, but will also bring great progress for the therapy for various inflammatory reactions such as pain and fever.

R ecent findings Since this chapter was w ritten, studies on C O X have developed rapidly and several im portant reports have been published. This final section of the chapter provides an update of some of these advances. T he crystal structures of C O X -I and -2 have been extensively investigated. As already described, the structures of C O X -I com plexed w ith flurbiprofen [48] and brom oaspirin [77] have been resolved. In addition, the crystal structures of C O X -I com plexed w ith iodoindom ethacin and io d o su p ro fen have now been determ ined [97]. F u rtherm ore, the crystal structures of C O X -2 complexed w ith various N SA ID s have been independently reported by tw o groups [98,99]. N on-selective N S A ID s do n o t induce conform ational change of b oth C O X -I and -2, while the C-term inal region of helix D of C O X -2 is unfolded by the binding of the selective C O X -2 inhibitors, SC-558 [98] and RS57067 [99]. In addition, a secondary pock et adjacent to the h y d ro p h o b ic channel has been reported to be involved in the C O X -2 selectivity of SC-558 [98]. H ow ever, neither the conform ation change n o r the secondary pocket has been able to explain the C O X -2 selectivity of other N SA ID s such as RS104897 to date. We described in the text (see above) that the C-term inal tetrapeptide may be involved in ER-targeting of the enzymes, and it was recently revealed that the C term inal tetrapeptides of bo th C O X -I and -2 actually target the enzym es to ER

[100],

Cyclooxygenases: prostaglandin H synthases

Finally, w e conclude this note w ith an introduction of recent sequencing w orks on C O X . T he nucleotide sequences of cD N A s for C O X -2s from sheep [101], guinea pigs [102] and rabbits [103] have been determined. Furtherm ore, initial findings on the sequence of C O X -2 in rainbow tro u t leucocytes have been reported [104]. H ow ever, m ore sequence and structure data are required to reveal the evolution of both C O X -I and CO X -2. The authors acknowledge Drs. Suyama, Kanai, and K iku n o fo r the instruction in the use o f M O L SC R IP T , T R E E T O O L a n d P H Y L IP

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.

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9 H.Toh andT.Tanabe

44

26.

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Dinchuk, J . E . , Car, B . D . , Focht, R . J . J o h n s t o n , J . J . , Jaffee, B . D . , Covington, M.B., Contel, N.R., Eng, V.M., Collins, R.J., Czerniak, P.M., Gorry, S.A. and T r z a s k o s J . M . (1995) Nature (London) 3 7 8 , 4 0 6 - 4 0 9 Morham, S.G., Langenbach, R., Loftin, C . D . , Tiano, H.F., Vouloumanos, N., Jennette, J . C . , Mahler, J.F., Kluckman, K.D., Ledford, A., Lee, C.A. and Smithies, O . (1995) Cell 8 3 , 4 7 3 - 4 8 2 Tsuji, M. and DuBois, R.N. (1995) Cell 83,493-501 Yokoyama, C., Takai, T. and Tanabe, T. (1988) F E B S Lett. 2 3 1 , 3 4 7 - 3 5 1 Merlie, J.P., Fagan, D., Mudd, J . and Needleman, P. (1988) J. Biol. Chem. 2 6 3 , 3 5 5 0 - 3 5 5 3 DeWitt, D.L. and Smith, W.L. (1988) Proc. Natl. Acad. Sei. U.S.A. 8 5 , 1 4 1 2 - 1 4 1 6 Funk, C . D . , Funk, L.B., Kennedy, M.E., Pong, A.S. and Fitzgerald, G.A. (1991) F A S E B J . 5, 2304-2312 Diaz, A., Reginato, A.M. and Jimenza, S.A. (1992)]. Biol. Chem. 267,10816-10822 Takahashi, Y., Ueda, N., Yoshimoto, T., Yamamoto, S., Yokoyama, C., Miyata, A., Tanabe, T., Fuse, I., Hattori, A. and Shibata, A. (1992) Biochem. Biophys. Res. Commun. 1 8 2 , 4 3 3 - 4 3 8 DeWitt, D.L., El-Harith, E.A., Kraemer, S.A., Andrews, M.J., Yao, E.F., Armstrong, R . L . and Smith, W.L. (1990) J . Biol. Chem. 265,5192-5198 Feng, L., Sun, W., Xia, Y., Tang, W.W., Chanmugan, P., Soyoola, E., Wilson, C . B . and Hwang, D. (1993) Arch. Biochem. Biophys. 3 0 7 , 3 6 1 - 3 6 8 Xie, W., Chipman, J.G., Robertson, D.L., Erikson, R.L. and Simmons, D.L. (1991) Proc. Natl. Acad. Sei. U.S.A. 88,2692-2696 Hla, T. and Neilson, K. (1992) Proc. Natl. Acad. Sei. U.S.A. 89, 7384-7388 Jones, D . A . , Carlton, D.P., Mclntyre, T.M., Zimmerman, G . A . and Prescott, S.M. (1993) J . Biol. Chem. 2 6 8 , 9 0 4 9 - 9 0 5 4 Kujubu, D.A., Fletcher, B.S., Varnum, B . C . , Lim, R.W. and Herschman, H . R . (1991) J. Biol. Chem. 266, 12866-12872 O ' B a n i o n , M.K., Sadowski, H . B . , Winn, V. and Young, D . A . (1991) J . Biol. Chem. 266, 23261-23267 Ryseck, R.P., Raynoschek, C., Macdonald-Bravo, H., Dorfman, K., Mattel, M . G . and Bravo, R. (1992) Cell Growth Differ. 3,443^150 Kennedy, B.P., Chan, C . C . , Culp, S.A. and Cromlish, W.A. (1993) Biochem. Biophys. Res. Commun. 1 9 7 , 4 9 4 - 5 0 0 Yokoyama, C. and Tanabe, T. (1989) Biochem. Biophys. Res. Commun. 165, 888-894 Kosaka, T., Miyata, A., Ihara, H., Hara, S., Sugimoto, T., Takeda, O., Takahashi, E. and Tanabe, T. (1994) Eur. J . Biochem. 221, 889-897 Appleby, S.B., Ristimaki, A., Neilson, K., Narko, K. and Hla, T. (1994) Biochem. J . 302, 723-727 Fletcher, B.S., Kujubu, D.A., Perrin, D . M . and Herschman, H.R. (1992) Proc. Natl. Acad. Sei. U.S.A. 2 6 7 , 4 3 3 8 - 4 3 4 4 Picot, D., Loll, P.J. and Garavito, M. (1994) Nature (London) 3 6 7 , 2 4 3 - 2 4 9 Kraulis, P.J. (1991) J. Appl. Crystallogr. 2 4 , 9 4 6 - 9 5 0 Doolittle, R.F., Feng, D.F., J o h n s o n , M.S. and McClure, M . A . (1986) Cold Spring Harbor Symp. Quant. Biol. 51,447^155 Toh, H . (1989) F E B S Lett. 2 5 8 , 3 1 7 - 3 1 9 Montelione, G.T., Wiithrich, K., Burgess, A.W., Nice, E . C . , Wagner, G., Gibson, K . D . and Scheraga, H.A. (1992) Biochemistry 3 1 , 2 3 6 - 2 4 9 Padmanabhan, K., Padmanabhan, K.P., Tulinski, A., Park, C . H . , Bode, W., Huber, R., Blankenship, D.T., Cardin, A.D. and Kisiel, W. (1993) J . Mol. Biol. 2 3 2 , 9 4 7 - 9 6 6 Li, W.-H. and Graur, D . (1991) Fundamentals of Molecular Evolution, Sinauer Associates, Sunderland, M A Appella, E., Weber, I.T. and Blasi, F. (1988) F E B S Lett. 231, Nexo, E., Jorgensen, P.E.,Thim, L. and Roepstorff, P. (1990) Biochim. Biophys. Acta 1037, 388-393 Pascall, J . C . J o n e s , D.S.C., Doel, S.M., Clements, J.M., Hunter, M., Fallon, T., Edwards, M. and Brown, K.D. (1991) J. Mol. Endocrinol. 6 , 6 3 - 7 0 Svoboda, M., Bauhofer, A., Schwind, P., Bade, E., Rasched, I. and Przybylski, M. (1994) Biochim. Biophys. Acta 1206,35-41 Dietz, R., Nastainczyk, W. and Ruf, H . H . (1988) Eur.J. Biochem. 171,321-328

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3

Mammalian lipoxygenases: structure, function and evolutionary aspects N atsuo Ueda, Hiroshi Suzuki and Shozo Yamamoto* D ep artm en t o f Biochemistry,Tokushima University School o f Medicine, Kuram oto-cho,Tokushim a 770-8503, Japan

Introduction M ammalian lipoxygenases are generally classified on the basis of their oxygenation sites of arachidonic acid (AA) as substrate, namely, 5-, 8-, 12- and 15-lipoxygenases (Scheme I). T he 12-lipoxygenase of platelets was the first lipoxygenase found in mammalian tissues [1,2]. Later, tw o 12-lipoxygenase isozymes (‘leucocyte type’ and ‘p latelet ty p e ') w ere distinguished by their prim ary structures and catalytic p rop erties [3]. A n o th er lipoxygenase was isolated from rabbit reticulocytes [4] w hich was later characterized as a 15-lipoxygenase [5], 5-Lipoxygenase was found in rabbit leucocytes [6], in connection w ith the discovery of leukotrienes (LT) [7]. 8L ipoxygenase was found recently in m urine epidermis treated w ith ph o rb o l ester [8], but its characterization requires further investigation. This chapter deals w ith the functional and structural properties of these mammalian lipoxygenases.

Regio- and stereo-specificity of oxygenation M am m alian lipoxygenases are generally considered to be regio- and stereospecific in term s of the oxygenation site of substrate as illustrated in Scheme I. T heir oxygenation products from AA were identified as (6£,8Z,1 lZ,14Z)-(5S)-hydroperoxyeicosa-6,8,ll,14-tetraenoic acid (5S-HpETE), (5Z,9£,llZ,14Z)-(85')-hydropero x yeicosa-5,9,ll,14-tetraenoic acid (8S-H pETE), (5Z,8Z,10£,14Z)-(12S)-hydroperoxyeicosa-5,8,10,14-tetraenoic acid (12S-HpETE), and (5Z,8Z,llZ,13£)-(155’)hydroperoxyeicosa-5,8,ll,13-tetraenoic acid (15S-HpETE) respectively. However, the regiospecificity is n o t so strict. F or example, 15-lipoxygenase produces a small am o u n t of 1 2-H pE T E as w ell as 15-H pE T E as a pred o m in an t p ro d u ct [5,9], and 12-lipoxygenase produces 15-H pE T E as a m inor p ro d u ct in addition to the major 12-H pE T E [10], M oreover, the positional specificity varies depending on the *

To w h o m correspondence sh o u ld be addressed.

N. Ueda et al.

48

Schem e 1

Reactions catalysed by mammalian lipoxygenases. LOX, lipoxygenase_____________________________ /= V = V

5-LOX

\

Arachidonic acid 8-LOX

/

\

15-LOX

12-LOX •C 00H

,COOH

,OOH COOH

5S-HPETE

v cooh

8S-HPETE

12S-HPETE 12-LOX 15-LOX

,COOH H 00,

'COOH

'COOH OOH HOOs

8S,15S-diHPETE

,COOH

ΌΟΗ

14R,15S-diHPETE

14,15-LTA4

polyenoic structure. R abbit reticulocyte 15-lipoxygenase oxygenates A A to 15SH pE T E and 12S-H pETE in a ratio of 80:15 while 6,9,12,15-eicosatetraenoic acid is oxygenated at the (135]- and (165)-positions in a ratio of 54:46 [11]. 5-Lipoxygenase [6], 8-lipoxygenase [12], 12-lipoxygenase [I] and 15lipoxygenase [8] synthesize predom inantly the (5)-isomer of H pE T E . Concom itant p ro d u ctio n of a small am ount of its (R )-isom er has been rep o rted fo r 12-lipoxy­ genase [13] and 15-lipoxygenase [11]. As listed in Table I, an antarafacial relationship between stereoselective hydrogen abstraction and oxygen insertion has been reported w ith 5-lipoxygenase [14], 8-lipoxygenase [12] and 12-lipoxygenase [15-18] as well as soybean lipoxygenase-1 [20],

Further metabolism of hydroperoxy products T he prim ary hyd ro p ero x y p ro d u cts can be fu rth e r oxygenated at different positions, form ing d ihy d ro p ero x y acids (diH pE T E ). N am ely, p o rcin e leucocyte 12-lipoxygenase converts 5S-H pETE into 5S,12S-diHpETE and 5S,15S-diHpETE, and 15S-HpETE into 14R,15S-diHpETE and 8S,15S-diHpETE [21], Lipoxygenases also metabolize their hydroperoxy products to epoxides of LTA4 type by an apparent dehydration reaction. In addition to the arachidonate 5oxygenase activity, 5-lipoxygenase possesses LTA4 synthase activity, converting 5-H pE T E into LTA4 [22-25]. Such a dual activity of 5-lipoxygenase was confirm ed w ith recom binant enzym es expressed in osteosarcom a cells [26], Escherichia coli [27], and Sf9 insect cells [28,29]. As show n in Table I, the fo rm atio n of LTA4 is

Mammalian lipoxygenases

Table 1

49

S te r e o se le c tiv e hydrogen abstraction in m am m alian lipoxygenase reactions A bstracted

5-Lipoxygenase

S u b strate

P roduct*

hydrogen

R eferences

A rachidonic acid

SS-HPETE

7-D

[14]

5S-HPETE

LTA4

IO-D

[16,17,24, 30,31]

8-Lipoxygenase

A rachidonic acid

12-Lipoxygenase

8S-HPETE

IO-D

[12]

A rachidonic acid

12S-HPETE

IO-L

[15-18]

5S-HPETE

5S,l2S-diHPETE

IO-L

[16,17]

I5S-HPETE

I4,I5-LTA4

IO-L

[18,19]

I5S-HPETE

I4R, l5S-diHPETE

IO-L

[18,19]

I5S-HPETE

8S,l5S-diHPETE

V ariablef

[19]

*HPETE, diHPETE a n d LTA4-type epoxide w ere analysed as sta b le m etabolites such as hydroxy a d d s (HETE) a n d dihydroxy a d d s. f A consistent perc en ta g e (90% ) o f I OD-H w as retained, b u t a co n sta n t result w as n o t obtained for abstraction o f I OL-H.

initiated by stereoselective lO D -hydrogen abstraction [16,17,24,30,31]. Similarly, 12-lipoxygenase and 15-lipoxygenase convert 15S-H pE T E into 14,15-LTA4 (actually detected as its hydrolytic products, 8,15-dihydroxy acids) by their LTA4 synthase activities [21,32,33]. T hus, 15S-H pETE is converted by 12-lipoxygenase into 14,15-LTA4 in addition to the double oxygenation products 14R,15Sd iH p E T E and 8S,15S-diH pETE m entioned above. N o t only arachidonate 12oxygenation, b ut also the form ation of 14,15-LTA4 by 12-lipoxygenase, are initiated by lO L-hydrogen abstraction [18,19].

Substrate specificity T he catalytic activities of m am m alian lipoxygenases have been exam ined w ith unsaturated fatty acids w ith various carbon numbers and polyenoic structures. The C lg fatty acids, such as linoleic and linolenic acids, are scarcely oxygenated by 5lipoxygenase [34,35] and platelet 12-lipoxygenase [2,13,36]. In contrast, the C 18 u nsaturated fatty acids are as active as substrates as C 20 unsaturated fatty acids for leucocyte 12-lipoxygenase [13,36-38]. Thus, leucocyte and platelet 12-lipoxy­ genases are distinguished by their reactivity w ith linoleic and linolenic acids. Similarly, 15-lipoxygenases of reticulocytes [11] and soybean [39] show broad chain-length specificities. Readers should refer to Table 3 in [3] for the details of substrate specificities of various lipoxygenases. It should be noted th at b o th 12lipoxygenase [21,37] and 15-lipoxygenase [40] produce (13S)-hydroperoxy acid from linoleic acid. Furtherm ore, esterified fatty acids are less active w ith platelet 12lipoxygenase [10], and essentially inactive w ith 5-lipoxygenase [41,42]. In contrast,

so

N. Ueda et al.

n o t only free u nsaturated fatty acids b u t also unsaturated fatty acids esterified in m em brane phospholipids and lipoproteins, are oxygenated w ith considerable activities by porcine leucocyte 12-lipoxygenase [10,43], rab b it reticu lo cy te 15lipoxygenase [4,9,44,45] and soybean lipoxygenase-1 [46,47], O xidative m o d ifi­ cations of membrane phospholipid and low -density lipoprotein by 15-lipoxygenase have been noted in relation to breakdow n of m ito ch o n d ria d u rin g reticulocyte m aturation [48] and development of atherosclerosis [45] respectively. A nandam ide (arachidonylethanolam ide) is an endogenous cannabinoid receptor ligand, and its arachidonate m oiety is also oxygenated by p o rcin e leucocyte and rat pineal gland 12-lipoxygenases, rab b it reticulocyte 15-lipoxy­ genase and soybean lipoxygenase-1 [49,50], H ow ever, the reaction of hum an platelet 12-lipoxygenase w ith anandam ide proceeds at a m uch slow er rate, and porcine leucocyte 5-lipoxygenase is totally inactive [49],

Activators It has been found th at 5-lipoxygenase requires calcium ions [51], and th a t the calcium -dependent activity is stim ulated by ATP [34], M ore recently, the stim ulatory effects of these com pounds w ere show n on b o th the 5-oxygenase and LTA4 synthase activities of the enzym e [22-24], N either calcium ions n o r ATP have been rep o rted to be required fo r purified 12- and 15-lipoxygenases although activation of these enzym es by calcium ions or o th er divalent ions has been reported in crude preparations [52,53], Porcine leucocyte 5-lipoxygenase activity increases up to about 2-fold depending on phosphatidylcholine [54], and porcine leucocyte 12-lipoxygenase activity is also enhanced by a low co n cen tratio n of detergent [21], U sually the lipoxygenase reaction starts w ith a lag phase, w hich is abolished by the addition of h yd ro p ero x y fatty acids [55], T hus, the h y d ro p e r­ oxides apparently stim ulate lipoxygenase activity. It is presum ed, on the basis of an E P R study, th a t the h y d roperoxide oxidizes the resting ferrous fo rm of lip o x y ­ genase to the active ferric form [56],

Self-inactivation W hen lipoxygenases and cyclooxygenase are allowed to react w ith AA, the reaction rate rapidly slow s dow n and ceases w ithin a few m inutes. This p h en o m en o n is referred to as ‘self-catalysed d estru c tio n ’ [55] o r ‘suicide in activ atio n ’ [57,58], Suicide inactivation has been reported for 5-lipoxygenase [54], leucocyte 12-lipoxy­ genase [13,36,58], and 15-lipoxygenase [48], In contrast, platelet 12-lipoxygenase does n o t show such a prom inent inactivation during catalysis [13,36,58], T he self­ inactivation was earlier attributed to radical form ation accompanying catalysis [55], M ore recently, how ever, it has been reported th a t covalent binding of an epoxide

Mammalian lipoxygenases

p ro d u ct (5,6-LTA4 and 14,15-LTA4) is attrib u ted to the suicide inactivation of 5lipoxygenase [57] and leucocyte 12-lipoxygenase [58]. It has been reported that a single m ethionine residue of rabbit reticulocyte 15-Iipoxygenase can be oxidized to its sulphoxide by treatm ent of the enzyme w ith 1 3-hydroperoxy-octadecadienoic acid (a 15-lipoxygenase p ro d u ct from linoleic acid) u n d er anaerobic conditions [59]. Since u n d er this condition the enzym e functioned as ‘lipohydroperoxidase’, splitting the hydroperoxide, and resulted in ‘self-inactivation’, a central role of the m ethionine residue was presum ed for the catalysis of the enzym e. Recently, this particular m ethionine has been identified as M et-590 in hum an 15-lipoxygenase and as M et-591 in rabbit 15-lipoxygenase. W hen M et-590 in the hum an enzym e is replaced by leucine by the site-directed m utagenesis, the m utant enzym e is still inactivated by 13-hydroperoxyoctadecadienoic acid, 15-H pE T E or AA. T he result show s th at the enzym e inactivation is not attributable to m ethionine oxidation [60].

cDNA cloning and prim ary stru ctu re As listed in Table 2, cD N A s of various mammalian lipoxygenases have been cloned and their nucleotide sequences determined. The cD N A s for 5-lipoxygenase have an open reading fram e encoding 670-674 am ino acids, including the initiator m ethionine, and 5-lipoxygenases of hum an, rat and m ouse show m ore than 92% identity of amino acid sequence [61-64]. It was found that a cloning artifact caused a unique C-term inus of rat 5-lipoxygenase [65], and taking this into consideration the am ino acid n um ber should be 673 rath e r than 670 [66] (Fig. I). P latelet-type and leucocyte-type 12-lipoxygenases are com posed of 663 amino acid residues [67-76], and 15-lipoxygenase contains 662-663 residues [77-79], The molecular mass of all the mammalian lipoxygenases is in the 74-78 kD a range as calculated on the basis of deduced am ino acid sequence. T he size of the mammalian lipoxygenases is consid­ erably smaller than that of plant lipoxygenases (90-100 kDa). Recent X -ray crystal­ lography on soybean lipoxygenase-1 (839 residues, 94262 Da) has revealed that the enzym e is com posed of tw o domains: an N -term inal 146-residue β barrel and a C term inal 693-residue helical bundle [80]. M ammalian lipoxygenases lack a domain corresponding to the N -term inal domain of the soybean enzyme. In term s of the prim ary structure, as show n in Table 2, leucocyte 12lipoxygenase has a higher hom ology w ith 15-lipoxygenase th an w ith platelet 12-lipoxygenase. A phylogenetic study of lipoxygenases has previously been reported [81]. In agreem ent w ith such a high hom ology, the leucocyte 12- and 15lipoxygenases show similar catalytic properties, such as a high reactivity w ith octadecapolyenoic acids and esterified unsaturated fatty acids, and a rapid self-inactivation, as mentioned above. Since leucocyte-type 12-lipoxygenase and 15-lipoxygenase have n o t to date been found together in one animal species, as discussed later, the leucocyte-type 12-lipoxygenase seems to be a species-equivalent of 15-lipoxygenase.

cD N A s of m am m alian lipoxygenases

Mouse

Rabbit

(Genome)

Reticulocyte

Bronchus

fA n amino acid sequence deduced from the Aloxe genomic sequence.

662

663

662

75

75

-

74.6

75.4

Skin papilloma

Reticulocyte

75

-

75.3

75.3

75

74.9

75.4

75.3

Macrophage

Spleen/leucocyte

663

663

Pineal gland

663

663

Brain

Leucocyte

Skin papilloma

Platelet

Tracheal epithelium

Human

75

78

77.6

75.6

Rat

Mouse

78 77.9

75.5 663

Molecular mass (kDa)

Platelet

Rg Cow

Mouse

663

674

670

674

acids*

Amino

HEL cell

HEL cell

Macrophage

Mouse

Human

*The initiator methionine is included.

Aloxef

15-Lipoxygenase

(leucocyte-type)

12-Lipoxygenase

(platelet-type)

12-Lipoxygenase

HL60 cell

Lung/placenta

RBL cell

Human

5-Lipoxygenase

Origin

Rat

Species

Lipoxygenase

LO X , lipoxygenase; P, platelet-type; L, leucocyte-type; h, human; p, porcine.

Table 2

mRNA

-

-

-

-

2.5,3.5

2.5

-

-

2.7

3.5

3.4

2.5

-

3.0

3.1

2.4

-

2.6

2.7

2.7

length (kb)

39

39

39

40

40

40

41

41

41

93

92

100

h5-LOX

60

61

65

57

59

64

65

84

100

40

39

42

66

79

86

70

71

89

100

64

65

39

39

41

pLI2-LO X

Amino acid identity (%) hP 12-LOX

66

81

100

73

75

86

86

63

65

39

38

39

h 15-LOX

[83]

[79]

[78]

[77]

[71]

[76]

[70]

P 5]

[74]

[73]

[71] [72]

[70]

[69]

[68]

[67]

[64]

[63]

[62]

[61]

Refs.

Mammalian lipoxygenases

Soybean lipoxygenase-1 also acts as a 15-lipoxygenase for AA, b u t the am ino acid id en tity betw een hum an 15-lipoxygenase and the soybean enzym e is only 22% [77], cD N A encoding 15-lipoxygenase cloned from hum an bronchus c D N A library [78] is identical w ith th a t from hum an reticulocyte cD N A library [77]. T hus the presence of tw o form s of 15-lipoxygenase in a hum an leucocyte preparation [82] awaits further investigation. Recently a novel lipoxygenase-like gene has been isolated from a m urine genom ic lib rary by hom ology screening w ith a hum an 15-lipoxygenase cD N A p ro b e [83]. T he id en tity of its deduced am ino acid sequence w ith those of m ouse platelet-type and leucocyte-type 12-lipoxygenases was 60% in b oth cases. A lth o u g h the m R N A was detected predom inantly in epiderm is, its enzym ic product from A A was not identified.

Functional am ino acid residues as iron ligands A n alm ost equim olar am ount of non-haem iron has been found in 5-lipoxygenase [84-87], leucocyte-type 12-lipoxygenase [88], 15-lipoxygenase [89] and soybean lipoxygenase [90-92]. A linear correlation betw een specific enzym e activity and iron content was also observed w ith the purified recom binant 5-lipoxygenase [84], In view of the central role of the iron in lipoxygenase catalysis, amino acid residues as possible ligands w ere extensively investigated by site-directed mutagenesis. A sequence of H is-(X )4-H is-(X )4-H is-(X )17-H is-(X )8-H is in soybean lipoxygenases was suggested as an iron-binding region [93]. The same histidine cluster (His-363, 368, -373, -391 and -400) is present in hum an 5-lipoxygenase. This cluster, together w ith som e o th e r histidine residues (H is-131, -433 and -551 in hum an 5-lipoxy­ genase) is highly conserved in various other mammalian lipoxygenases (Fig. I). The results of m utagenesis of these histidine residues show ed that H is-368, -373 and 551 are im p o rta n t fo r 5-lipoxygenase activity [94-96]. M utation of the corresponding histidine residues also resulted in a total loss of activity of hum an p latelet 12-lipoxygenase [97] and porcine leucocyte 12-lipoxygenase [98]. Iron content was very low in His-373 and -551 m utants of 5-lipoxygenase, and a partial loss of iron was observed in His-368 m utant [86,87]. A very low content of iron was also show n w ith H is-361, -366 and -541 m utants of porcine leucocyte 12-lipoxy­ genase [98]. A n electron density map of the crystallized soybean lipoxygenase-1 confirm ed th a t these three conserved histidine residues function as iron ligands [65,80]. Recent cD N A cloning of m urine platelet 12-lipoxygenase revealed that the enzym e does n o t contain histidine at positions corresponding to His-131 and H is363 of hum an 5-lipoxygenase [70,71]. Earlier, carboxylate oxygens were proposed to be ligands in soybean lipoxygenase-1 that was analysed by extended X -ray absorption fine structure [99]. Later, the crystallographic data show ed th at C O O - of the C -term inal isoleucine was another ligand [65,80]. T he C-term inal isoleucine residue is well-conserved in

N. Ueda et al.

54

Fig. 1

Human 5 -LOX 5 -LOX Rat 5 -LOX Mouse Human P12 -LOX Mouse P12 -LOX L12 -LOX Pig Cow L12 -LOX L12 -LOX Rat Mouse L12 -LOX Human 15 -LOX Rabbit 15 -LOX Aloxe Mouse

Partial amino acid sequences of m a m m a l i a n lipoxygenases

126 125 126 122 122 123 123 123 123 122 123 122

131 HILKQHRRKEL— HILKQHRRKEL— HILKQHRRKEL— DMFQKHRBKEL— DOTQKYREKEL— GLFKKHREEEL-GLFKKHREEEL— GLFRKHREEEL— GLFRNHREEEL— GLFQKHREEEL— GLFQKHREQEL— NLFRKÏREQEL—

426 433 AINTKAREQLICECGLFDKA N ATGGGGH— 1 AINTKAREQLNCEYGLFDKA N ATGGGGH— AINTKAREQLNCEYGLFDKA N ATGGGGH— EINTRARTQLISDGGIFDKA V STGGGGH— EINTRTRTDLISDGGIFDQV V STGGGGH— EINVRARNGLVSDLGIFDQV V STGGGGH— EINIRARTGLVSDSGVFDQV V STGGGGH— EINVRARSDLISERGFFDKA M STGGGGH— EINVRARSDLISERGFFDKV M STGGGGH— EINVRARTGLVSDMGIFDQI M STGGGGH— EINVRARNGLVSDFGIFDQI M STGGGGH— EINTLARNNLVSEWGIFDLV STGSGGH—

©

363 368 373 391 400 363 HQTIT H LLRT H LVSEVFGIAMYRQLPAVHPIFKLLVAHVRFTÏ 362 HQTIT a LLRT H LVSEVFGIAMYRQLPAVHPLFKLLVAHVRFTI 363 HQTIT H LLRT H LVSEVFG lAMYRQLPAVHPLFKLLVAHVRFTI 355 HEIQY H LLNT H LVAEVIAVATMRCLPGLHPIFKFLIPHIRYTM 355 QELQF H LLNT H LVAEVIAVATMRCLPGLHPIFKLLVPHIRYTM 356 HELHS H LLRG H LMAEVIAVATMRCLPSIHPIFKLLIPHFRTIM 356 HELHS H LLRG H LVAEVIAVATMRCLPSIHFMFKLLIPHLRYTM 356 HELQA H LLRG H LMAEVFAVATMRCLPSVHPVFKLLVPHLLYTM 356 HELQA H LIRG H LVAEVFAVATMRCLPSVHPVFKLLVPHLLYTM 355 HELQS H LLRG H LMAEVIWATHRCLPSIHPIFKLIIPHLRYTL 356 HELNS H LLRG H LMAEVFTVATMRCLPSïHPVFKLIVPHLRYTL 355 HQLQS H LLRG H LMAEVISVATMRSLPSLHPIYKLLAPHFRYTM

546 545 545 535 535 536 536 536 536 535 536 535

55 i TASAQ H AAVNF— TASAQ H AAVNF— TASAQ H AÂVNF— TCTAQ H AAINQ— TCTAQ H AAINQ— TCTGQ H SSNHL— TCTGQ H SSTHL-TCTAQ H SSVHL— TCTAQ H SSIHL— TCTGQ H ASVHL-TCTGQ H SSIHL-TCTGQ M ASTHL—

674 Reference 62 —IPNSVA I 674 63 -«IPNSVA I 673 — IPNSVA I 674 64 —IENSVT I' 663 68 70 — IENSIT I 663 72 —VENSVA X 663 73 —VENSVA I 663 75 —VENSVA Si 663 —VENSVA i 663 70 77 --VENSVA i 662 78 --VENSVA i 663 79 —VENSVT i 662

*A possible frame shift error was considered as proposed in [66], LOX, lipoxygenase; P, platelet-type; L leucocyte-type.

mammalian lipoxygenases (Fig. 1), and its deletion in human 5-lipoxygenase results in a loss of enzyme activity as well as of iron [87,95]. Deletion of C - t e r m i n a l isoleucine residue or its substitution with other amino acids also abolished activity of murine platelet-type and leucocyte-type 12-lipoxygenases [70] and porcine leucocyte-type 12-lipoxygenase [100]. Asn-694 was also proposed to be one of the iron ligands in soybean lipoxygenase-1 [65] or to be part of a network that stabilizes the iron centre by hydrogen bonds [80]. Since mutants of Asn-555 (a corresponding residue in 5-lipoxygenase) showed a low 5-lipoxygenase activity but still retained iron, Asn-555 was presumed not to be a permanent ligand [87],

Determinants of positional specificity In connection with the positional specificity of oxygenation, attempts b y sitedirected mutagenesis have been made for the interconversion between 15-lipoxygenase and 12-lipoxygenase. When Met-418 of human reticulocyte 15-lipoxygenase was replaced by a valine, which was found at the corresponding position of several 12-lipoxygenases, this mutant catalysed both 12- and 15-oxygenation of A A almost to the same extent. Furthermore, when Gln-416, Ile-417 and Met-418 of 15-lipoxy-

M am malian lipoxygenases

genase w ere replaced by the corresponding sequence of hum an platelet 12-lipoxy­ genase (Lys-Ala-Val), 12-hydroxy acid (H ETE) and 15-HETE were produced in a ratio of 15:1. Presum ably, M et-418 defined the binding pocket of the m ethyl term inus of fatty acid substrate, and the m utation of m ethionine to valine caused a shift in substrate binding since this change reduced the side-chain volume by 17% [101]. Subsequently, a double m u tan t (Ile-417 and M et-418 to Val and Val) was expressed, purified and show n to convert AA into 12-H pETE and 15-H pETE in a ratio of 20:1 [102]. In reverse, w hen Val-418 and Val-419 of porcine leucocyte 12-lipoxy­ genase w ere replaced by He and M et respectively, a predom inant 15-lipoxygenase activity w as observed w ith a p ro d u ct ratio 12-H E T E :15-H E T E of 1:5.7 [98]. However, a single m utant (Ala-417 to He) and a triple m utant (Lys-416, Ala-417 and Val-418 to G in, He and M et, repectively) of hum an platelet 12-lipoxygenase caused o n ly a slight increase in 15-lipoxygenase activity. W hen all am ino acids between positions 398 and 429 of hum an platelet 12-lipoxygenase w ere replaced by the corresponding sequence of hum an 15-lipoxygenase, the total enzym e activity was m arkedly reduced and 66% of the p ro d u ct was 15-H pE T E [97]. Similar experi­ ments have also been perform ed w ith a leucocyte-type 12-lipoxygenase of rat brain and pineal gland, but neither single m utations (Lys-417 to Gln or Ala-418 to He) nor double m utation (Lys-417 and Ala-418 to G ln and He) increased 15-lipoxygenase activity [75,103]. Im portantly, leucocyte-type 12-lipoxygenases of rat and mouse have a m ethionine at the p osition corresponding to M et-418 of hum an 15-lipoxy­ genase whereas other 12-lipoxygenases have valine at this position. The presence of m ethionine at this p ositio n m ay be related to the finding th at leucocyte-type 12lipoxygenases of rat and m ouse produce a relatively large am ount of 15-H pETE (a p ro d u ct ratio 12-H E T E : 15-H ET E of 3-6:1) [102]. A trial to convert platelet-type 12-lipoxygenase into 15-lipoxygenase by mutagenesis was unsuccessful [97], Recently, an additional sequence determ inant of the positional specificity of 12/15-lipoxygenases has been described [103a]. W hen, in the rabbit 15-lipoxy­ genase, Phe-353 was substituted fo r a less space-filling am ino acid the m utant enzym e exhibited arachidonate 12-lipoxygenase activity. Sequence alignm ents of the k now n m ammalian 12/15-lipoxygenases indicate that all enzymes containing a small am ino acid at this position are 12-lipoxygenases. W hen there is a less space­ filling am ino acid at po sitio n 353, the bulkiness of am ino acids 417 and 418 determine w hether the enzyme catalyses a 12- or 15-oxygenation of AA.

Genom ic DNA cloning As show n in Table 3, genes of various mammalian lipoxygenases have been isolated. AU the lipoxygenase genes consist of 14 exons and 13 introns, but the total length of exons and in tro n s are considerably different. Genes of 5-lipoxygenase [104], p latelet-type [70,107,108] and leucocyte-type [70,110] 12-lipoxygenases, and 15-

56

N. Ueda e t al.

lipoxygenase [111] span more than 82,13-17, 7-8 and 8 kb respectively. It is notable that, in term s of the intron sizes, the leucocyte-type 12-lipoxygenase gene is closer to the 15-lipoxygenase gene than to the platelet-ty p e 12-lipoxygenase gene. E x o n -in tro n boundaries fo r hum an platelet-type 12-lipoxygenase gene w ere located in the identical corresponding positions to those for hum an 5-lipoxygenase and rabbit 15-lipoxygenase genes [107]. As described above, a novel lipoxygenase-related gene has been isolated from a mouse strain 129 Sv genomic phage library and referred to as A loxe [83], The gene also contains 14 exons and spans 7.3 kb. O n the basis of th e overlap of the restriction map, this gene is identical to a gene segm ent th a t was p reviously presum ed to be a pseudogene derivative of 12-lipoxygenase [70]. In an attem p t to isolate the hum an 12-lipoxygenase gene, a pseudogene m ore like 12-lipoxygenase than 5- and 15-lipoxygenase genes was also found [107], The chrom osom al localization of lipoxygenase genes is show n in Table 3. T he genes of hum an 12- and 15-lipoxygenases are located in the same chrom osom e (num ber 17) [107], and the genes of m urine p latelet-ty p e and leu co cy te-ty p e 12lipoxygenases are also located in the same chrom osom e (n u m b er 11) [70]. T he hum an 5-lipoxygenase gene is present in a different chrom osom e (n um ber 10) [107], Various as-actin g elem ents w ere found in the 5 '-flan k in g regions of various mammalian lipoxygenase genes (Table 3). All the lipoxygenase genes so far exam ined have neither a typical TATA box nor a C C A A T box in the p ro m o to r region, b u t contain m ultiple G C boxes (potential S p l-binding sites) [70,104-111], T hese lipoxygenase genes are, therefore, considered as housekeeping genes. As listed in Table 3, other putative transcriptional reg u lato ry elem ents have been reported: A P-2, N F -κΒ, c-H a-ras, m yb, A P-1, C A C C C m otif (a com m on feature of adult β -globin gene prom otors), a glucocorticoid-responsive element, C /Ε Β Ρ β, GATA box and N F l . Similarities in p rom otor sequences have been found between porcine and m urine leucocyte 12-lipoxygenases and rabbit reticulocyte 15-lipoxygenase [70,109], A region (—179 to —56 from ATG) including five repeated G C boxes of h um an 5-lipoxygenase gene is essential fo r its transcrip tio n in H eL a and hum an prom yelocytic leukaem ia H L 60 cells on the basis of the chloram phenicol acetyltransferase assay. The S pl can bind to this region as exam ined b y gel-shift assay [105]. The luciferase assay indicated that an N F -κΒ m otif was related to the negative control of transcription of hum an platelet 12-lipoxygenase [109]. Binding of N F - kB p50 and p65 o r c-Rel heterodim ers to this m otif was show n by gel-shift assay. A chloram phenicol acetyltransferase assay dem onstrated a p ro m o to r activity of a proxim al 5 '-flanking region ( - 150 to + 20 nucleotide) of rab b it 15-lipoxygenase genes of b o th erythroleukaem ia cells and fibroblasts [111], It was also suggested that the first 900 bp of the 5'-flanking region of the 15-lipoxygenase gene contains a tissue-specific silencer th a t dow n-regulates the transcription by binding of a factor present in non-expression tissues [112].

-

Guinea Pig

Aloxe

15-Lipoxygenase

12-Lipoxygenase (leucocyte-type)

12-Lipoxygenase (platelet-type)

14

>82

Human

5-Lipoxygenase

Chromosomal

14

8.0

-

7.3

Rabbit

Human

Mouse

14

-

14

7.5

Mouse

-

17

11 central

-

14

8

Pig

11 central

14

13

Mouse

I7pl3.l

14

15-17

-

10

localization

Human

-

Exon

Size (kb)

Animal species

Genomic D N A s of m a m m a l i a n lipoxygenases

Lipoxygenase

Table 3 Putative transcriptional

motif, glucocorticoid-responsive

-

-

motif

Spl,TATA-like box, NF1, C A C C C

Spl,TATA-like box,AP-l

Spl,TATA-like box,AP-2

Spl,TATA-like box,AP-2

GATA box

element, NF-KB, C/EBP0,

[83]

[107]

[III]

[70]

[110]

[70]

[107-109]

[106]

Spl,TATA-like box,AP-2, C A C C C

[104,105]

Spl, c-Ha-ros,AP-2, NF-KB

References

Spl, c-Ha-ros, AP-2, NF-KB, myb

regulatory elements

sasEuaSXxodii ubüeuüuue^

IS

Tissue distribution of lipoxygenases As housekeeping enzymes, lipoxygenases are found constitutively and ubiquitously in various cell types such as haematopoietic cells, epithelial cells and endocrine cells. W hen the tissue distribution of lipoxygenases is discussed, we should note th at the enzym e activity may be attrib u ted to contam inant blood cells o r resid en t macrophages and granulocytes [113]. 5-Lipoxygenase is found in haem atopoietic cells such as neu tro p h ils, eosinophils, m onocytes/m acrophages and m ast cells [114]. W hole b lo o d or peritoneal leucocytes of guinea pig [34], hum an [115], pig [23] and rat [116] as well as leukaemia cell lines like rat basophilic leukaemia (RBL-I) [51] and H L 60 [62,117] have been used as enzym e sources. W hen the tissue distribution of 5-lipoxygenase in pig was examined by a peroxidase-linked immunoassay w ith specific m onoclonal antibodies, n o t only peripheral blood leucocytes b u t also lung, pancreas, intestine and lymphatic organs contained considerable am ounts of 5-lipoxygenase [118]. An immunohistochemical study indicated that the im m unoreactivity of 5-lipoxygenase was m ostly attributable to various types of leucocytes included in the tissue [119]. As an exception, the 5-lipoxygenase of porcine pancreas was localized in acinar cells [120], P latelet-type 12-lipoxygenase is found n o t only in platelets of various animal species [1,2,13,36,69,70,121-123], b u t also in hum an [124,125] and m ouse skin [70,71] (Table 4). A n im m unohistochem ical study dem onstrated the presence of the enzyme in imm ature and m ature megakaryocytes as well as platelets in mouse [122], However, porcine platelets had no 12-lipoxygenase activity [126], In addition to leucocytes of pig [21,37,72,126], cow [13,38] and dog [127], leucocyte-type 12-lipoxygenase is found in peritoneal m acrophages of m ouse [70,76], pineal gland of rat [75,128] and mouse [70], anterior p itu itary of pig [129] and tracheal epithelium of cow [130] (Table 4). Enzym e im m unoassay show ed the presence of leucocyte-type 12-lipoxygenase in porcine gastrointestinal tract, lymphatic organs and other organs as well as leucocytes [131], and later an im m uno­ histochemical study revealed that the enzym e is localized in infiltrating leucocytes rather than parenchym al cells of these organs [113]. In the case of porcine anterior p ituitary, 12-lipoxygenase is localized in parenchym al cells w ith in num erous cytoplasm ic granules [129]. Im m unohistochem ical double-staining indicated the co-localization of 12-lipoxygenase and various anterior pituitary horm ones, such as luteinizing horm one and follicle-stim ulating horm one, in these cells [132]. It was reported that m R N A and p rotein of a leucocyte-type 12-lipoxygenase have been detected in human adrenal glomerulosa cells [133]. 15-Lipoxygenase has been extensively studied w ith a purified preparation from rabbit reticulocytes [40,48]. In the course of reticulocyte m atu ratio n , the decline in 15-lipoxygenase appeared concom itantly w ith the b reak d o w n of m itochondria, and m ature erythrocytes have no 15-lipoxygenase [40]. In hum an tissues, eosinophils [134,135] and airw ay epithelial cells [78,136] are m ajor sources

Platelet [121]

Platelet [70,122] Skin [70,71] Platelet [123]

Rat

Mouse

Dog

Leucocyte [13,38]

Leucocyte [127]

Macrophage [76] Brain [123]

Leucocyte [70]

Airway epithelium [73,130] Brain [74] Pineal gland [75,128]

Platelet [2,13]

Cow

Adrenal glomerulosa cells [133]

Leucocyte [21,37,72,126] Anterior pituitary [129]

Platelet-type 12-lipoxygenase Platelet [1,36,69] Skin [124,125]

Pig

Rabbit

Human

Leucocyte-type 12-lipoxygenase

Distribution of 12- and 15-lipoxygenases in various animal species

References are given in square brackets.

Table 4

15-Lipoxygenase

Reticulocyte [4,40,48,79] Leucocyte [52]

Airway epithelium [78,136] Reticulocyte [77]

Eosinophil [134,135]

sasEusSXxodii UE!|euuiuew

6S

N. Ueda et al.

60

of the enzyme. Interestingly, hum an airway epithelium has a 15-lipoxygenase while bovine airway epithelium has a leucocyte-type 12-lipoxygenase [137].

Intracellular localization and translocation of lipoxygenases 5-Lipoxygenases are found in the cytosol fraction of resting leucocytes as examined by differential centrifugation [115,118]. The predom inant localization of 5-lipoxy­ genase is in the cytoplasm of porcine leucocytes as revealed b y im m u n o electro n m icroscopy [119]. In agreem ent w ith these observations, no h y d ro p h o b ic m em brane-spanning domains were found in the prim ary structures of mammalian lipoxygenases [138]. T he subcellular localization of platelet 12-lipoxygenase is a debatable subject. The activity was detected either predom inantly in the cytosol fraction of platelets [2,13,139] o r in the particulate fraction [140]. As observed b y im m u n o ­ electron m icroscopy, the enzym e is p redom inan tly localized in the general cytoplasm of murine platelets [122], In hum an epidermal cells [124,125] and hum an epiderm oid carcinom a A431 cells [141] the p latelet-ty p e enzym e was fo u n d in particulate fractions, and the membrane-associated enzym e could be solubilized by the treatm ent of Tween 20 [124]. L eucocyte-type 12-lipoxygenase exists p red o m i­ nantly in the cytosol of porcine leucocytes [126], and the localization of the enzym e in the cytoplasm was confirm ed by im m unoelectron m icroscopy [113]. Reticulocyte 15-lipoxygenase can be obtained from the supernatant free of strom a (m ainly consisting of plasm a m em brane ghosts and m itochondria) [40]. 15Lipoxygenase activity found in human epidermal cells is localized exclusively in the cytosol fraction [124], C alcium -dependent intracellular translocation of lipoxygenase from the soluble to the particulate fraction was first show n w ith hum an leucocyte 5-lipoxygenase [142]. The translocation of 5-lipoxygenase was associated w ith its activation [143,144], and was caused by various agents such as calcium io n o p h o re A23187 (calcim ycin), im m unoglobulin E, and 7V -(form yl)m ethionylleucylphenylalanine [145]. Membrane association may facilitate 5-lipoxygenase to react w ith AA w hich is released from m em brane phospholipids by phospholipase. T he ro le of 5-lipoxygenase-activating p rotein (FLA P) in the efficient utilizatio n of A A by 5lipoxygenase is discussed in C hapter 5 of this m onograph. Recently, the presence of 5-lipoxygenase in the nucleus and the translocation of the enzym e to the nuclear envelope has been suggested [146,147], 12-Lipoxygenase in hom ogenates from rat platelets [121] and hum an erythroleukaem ia (H EL ) cells [148] is translocated from the cytosol to m em brane in a calcium-dependent manner. The calcium-dependent translocation has also been observed w ith rabbit reticulocyte 15-lipoxygenase [149].

Mammalian lipoxygenases

E xperim ental induction of lipoxygenases There are an increasing num ber of reports on the induction of lipoxygenases during cell d ifferentiation o r incubation w ith cytokines. Both activity and m R N A of 5lipoxygenase can be induced during differentiation of H L60 cells to granulocyte­ like cells by dim ethyl sulphoxide [62,117] o r to m acrophage-like cells by 1,25dihydroxy-vitam in D 3 [150]. A heat stable factor in hum an serum up-regulates 5lipoxygenase activity in dim ethyl-sulphoxide-treated H L 60 cells [151], In these cells the factor was tentatively identified as transform ing grow th factor β w hich increased 5-lipoxygenase activity by about 10-fold b u t the enzym e pro tein only increased by 2-fold [152], E nhancem ent o f 5-lipoxygenase m R N A o r protein level has also been observed w ith various cells, including human neutrophils treated w ith granulocyte-m acrophage colony-stim ulating factor [153,154], m ouse bone m arrow -derived m ast cells treated w ith interleukin-3 (IL-3) [155], hum an m onocytic cell line M ono Mac 6 treated w ith transform ing grow th factor β and 1,25-dihydroxy-vitam in D 3 in com bination [156], and human H aC at keratinocytes in the course of differentiation [157], Lectin-activated lym phocytes can induce 5lipoxygenase in m onocytes primarily by the generation of granulocyte-macrophage colony-stim ulating factor and IL-3 [158]. Platelet-type 12-lipoxygenase in H E L cells can be induced [69] or dow nregulated [159] by treatm en t of the cells w ith ph o rb o l ester. T he platelet-type enzym e in a hum an m egakaryocytic cell line D A M I is also induced by p h o rb o l ester [160], and dow n-regulated by treatm ent w ith fibronectin [161]. In addition to these m egakaryocyte-like cells, A431 cells express the enzym e, and epiderm al grow th factor increases its m R N A level by about 2-fold [162], This induction can be suppressed by pretreatm ent of the cells w ith glucocorticoid [163]. T he 15-lipoxygenase gene appears to be transcribed in ery th ro id cells of bone m arrow , b u t the translation of m R N A is initiated w hen the cells reach the peripheral blood [164]. This finding suggests a certain m echanism by w hich the m R N A was kept inactive. It has been reported that rabbit 15-lipoxygenase m R N A contained 10 tandem repeats of a sequence of C 4(A /G )C 3U C U U C 4A A G starting from about 50 nucleotides of the stop codon in the 3'-u n tran slated region [79]. A cytosolic 48 k D a p ro te in can bind to this m otif, and the interaction betw een this p ro tein and the R N A m otif is necessary and sufficient fo r the repression of 15lipoxygenase at the translational level [165]. M odified form s of this m otif are also contained in the 3 '-untranslated region o f m R N A o f hum an 15-lipoxygenase and leucocyte-type 12-lipoxygenase, b u t n o t for p latelet-type 12-lipoxygenase or 5lipoxygenase [138]. IL -4 also induces 15-lipoxygenase m R N A and p ro tein in hum an m onocytes [166] and hum an alveolar macrophages [167]. This induction is inhibited by γ -interferon, hydrocortisone and phorbol ester. IL-13 also induces the enzyme in hum an blood monocytes [168].

N. Ueda e t al.

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Concluding rem arks In the last decade m olecular b iological approaches have b rou gh t ab ou t great progress in the research on mammalian lipoxygenases as described in this chapter. In addition to 5-, 12- and 15-lip oxygen ases, an 8 -lip o x y gen a se has recen tly been foun d as the fourth mam malian lip oxygen ase, but this en zy m e aw aits further characterization. The general physiological significance o f 12-and 15-lipoxygenases is still unclear although a variety o f biological effects o f their arachidonate metabolites has been reported [3,100,138].

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Plant lipoxygenase: structure and mechanism G.A.Veldink*§, M.P. H ilbersf ,W.F. N ieuw enhuizenf and J.F.G.VIiegenthart* *Utrecht University, Bijvoet Center for Biomolecular Research, Departm ent of Bio-organic Chemistry, Utrecht, NL-3584 CH Utrecht1The Netherlands, fDaresbury Laboratory, Daresbury,Warrington, Cheshire, W A 4 4AD, U.K., and fUnilever Research Laboratory, Olivier van Noortlaan 120, N L -3133 ATVIaardingen1The Netherlands

The presence of a lipid-oxidizing enzyme in plants, then termed lipoxidase, was first described by A ndre and H o u in 1932 [I]. A pigment bleaching property, attributed to a separate enzym e activity described as carotene oxidase, was later found to originate fro m this enzym e as w ell [2]. T he name lipoxygenase (linoleate:oxygen oxidoreductase, E C 1.13.11.12) is now used for this enzyme. Lipoxygenase catalyses the dioxygenation of fatty acids containing one or m ore (lZ ,4 Z )-p en tad ien e system s to conjugated hydroperoxydiene derivatives (Scheme I). In addition to the dioxygenase activity, a hydroperoxide dehydrase activity was found w ith arachidonic acid (5,8,11,14-eicosatetraenoic acid, ETE) as the original substrate th a t yields leukotriene A 4 from 5-hydroperoxyeicosatetraenoic acid (5S-H pE T E ) [3]. T he enzym e is ubiquitous in higher plants [4] and has also been found in low er eukaryotes such as yeast [5], algae [6-8] and fungi [9], Its presence has been reported in several prokaryotes as well [10-12], Lipoxygenase has been found in mammals [13,14] w here it is a key enzyme in the biosyntheses of regulatory molecules such as leukotrienes and lipoxins [15,16]. M ammalian lipoxy­ genases w ere recently reviewed by Yamamoto [17]. In plants, usually several lipoxygenase isoenzym es are present that differ in p ro p erties such as p H -o p tim u m , substrate preference and p ro d u ct specificity [18]. It has long been thought that lipoxygenase is a unique enzym e, being able to catalyse an oxidation reaction w ithout the assistance of a prosthetic group or a metal cofactor. In the early 1970s, however, the presence of a non-haem iron atom in a m olar ratio o f 1:1 was reported [19,20], and iron has also been confirm ed as being present in mammalian lipoxygenases [15,21],

§To w hom correspondence should be addressed.

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Scheme 1

General reaction catalysed by lipoxygenase

Substrates are unsaturated fatty acids such as linoleic, linolenic and arachidonic acids.

Catalytic cycles Although soybean lipoxygenase was crystallized as early as 1947 [22], the lipoxygenase isoenzymes from soybean were first purified to homogeneity in the early 1970s [23]. Subsequently, much work has been directed towards lipoxygenase-1 from soybean and the reaction catalysed by this enzyme with linoleic acid as substrate. On the basis of spectroscopic evidence, a model for the catalytic cycles of lipoxygenase was proposed by De Groot et al. [24] (Scheme 2), which accounts for the role of iron, the kinetics observed upon incubation of linoleic acid with lipoxygenase and dioxygen, including the initial lag phase, and for the secondary products formed under anaerobic conditions [25,26]. Scheme 2

Adapted from [24].

Catalytic cycles of t h e anaerobic and aerobic lipoxygenase reactions

Plant lipoxygenase: stru ctu re and mechanism

Resting, catalytically inactive, lipoxygenase needs lipid hydroperoxide for activation as was shown w ith kinetic experiments investigating lipoxygenase [27-31], and are likely to be converted into alkoxy radicals in this process (Scheme 2, step a [24]) The reaction is either started by traces of hydroperoxides, formed during autoxidation of the substrate, and/or by traces of Fe(III)-Iipoxygenase present in the Fe(II)-Iipoxygenase preparation [32]. The activation by the product hydroperoxide explains the existence of a kinetic lag-phase in the reaction of resting lipoxygenase w ith linoleic acid [28,33,34]. The hydroperoxide products formed at the start of the reaction oxidize Fe(II)-Iipoxygenase to the active Fe(III)-form; more hydroperoxide is formed by the increasing amount of Fe(III)-Iipoxygenase, and eventually all enzyme is activated and no further hydroperoxide is consumed. It has recently been proposed that the hydroperoxide needed to activate the enzyme merely binds to the native enzyme to facilitate activation to the Fe(III)-enzyme [35], The hydroperoxide was reported by Jones et al. [35] not to be converted during the activation step. U pon binding of the substrate, a fatty acid radical is formed after the abstraction of a hydrogen atom from the bisallylic methylene group, and concomi­ tantly Fe(III) is reduced to Fe(II) (step b). In the absence of dioxygen, i.e. in the anaerobic cycle, the radical dissociates from the enzyme (step f) and yields a variety of products, part of which are derived from the alkoxy radical formed in step a, via free radical reactions (Scheme 3) [26,36]. In the presence of dioxygen, i.e. in the aerobic cycle, the enzyme-bound fatty acid radical is transformed into aperoxyl radical (Scheme 2, step c). Iron(II) is subsequently oxidized to Fe(III) and a peroxyl anion is formed (step d). The anion reacts w ith a proton and the resulting hydroperoxide is released from the Fe(III) enzyme, which is then ready for another reaction cycle (step e). Although this reaction scheme is widely accepted and has been confirmed by recent w ork [34], some uncertainties exist as to the identity of particular intermediates. In an alternative model, the reduction of Fe(III) to Fe(II) via a single electron transfer from the ιτ-system of the C-12-C-13 double bond in linoleic acid is proposed. This yields a fatty acid radical cation with a lower p Ka for its C -I l hydrogen substituent, thereby permitting its removal as a proton by a base [37]. In another model, it is proposed that the hydrogen abstraction could be provided by the formation of a carbon—iron σ -bond with C -I or C-5 of the (lZ,4Z)-pentadiene system of the fatty acid substrate [38]. In contrast to the above-mentioned lipoxy­ genase mechanisms, this model does not require the reduction of Fe(III) via a single electron transfer. The form ation of fatty acid radical cations or organometal intermediates is not consistent with the observed formation of carbon-centred radicals and peroxyl radicals [39-43], and the detection of spin-trapped linoleic acid radicals [44] during the anaerobic lipoxygenase reaction. In addition, the changes in the iron redox state during the lipoxygenase reaction, as proposed in the free radical

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Schem e 3

P roduct form ation in th e lipoxygenase-catalysed free radical reaction with u n satu rated fatty acids and fatty acid hydroperoxides______ H-

I

H