Lysozymes: Model Enzymes in Biochemistry and Biology 978-3-0348-9952-9, 978-3-0348-9225-4

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EXS 75

Lysozymes: Model Enzymes in Biochemistry and Biology Edited by P. JoBes

Birkhiiuser Verlag Basel· Boston . Berlin

Editor Prof. Dr. P. Jolles Laboratoire de Chimie des Substances Naturelles URA C.N.R.S. No. 401 Museum National d'Historie Naturelle 63, rue Buffon F-75005 Paris France

Library of Congress Cataloging-in-Publication Data Lysozymes: model enzymes in biochemistry and biology / ed. by P. Jolles. p. cm. - (EXS; 75) Includes bibliographical references and index. \. Lysozyme. I. Jolles, Pierre, 1927 QP609.L9L96 1996 574.19'25 - dc20

. II. Series.

Deutscbe Bibliotbek Cataloging-in-Publication Data EXS. - Basel; Boston; Berlin: Birkhiiuser. Friiher Schriftenreihe Fortlaufende Bei\. zu: Experientia 75. Lysozymes: model enzymes in biochemistry and biology. 1996 Lysozymes: model enzymes in biochemistry and biology / ed. by P. 10Iles.-Basel; Boston; Berlin: Birkhiiuser, 1996 (EXS; 75) ISBN-13: 978-3-0348-9952-9 001: 10.1007/978-3-0348-9225-4

e-ISBN-13: 978-3-0348-9225-4

NE: Jolles, Pierre [Hrsg.] The publisher and editor can give no guarantee for the information on drug dosage and administration contained in this publication. The respective user must check its accuracy by consulting other sources of reference in each individual case. The use of registered names, trademarks etc. in this publication, even if not identified as such, does not imply that they are exempt from the relevant protective laws and regulations or free for general use. This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, re-use of illustrations, recitation, broadcasting, reproduction on microfilms or in other ways, and storage in data banks. For any kind of use permission of the copyright owner must be obtained.

© 1996 Birkhiiuser Verlag, PO Box 133, CH-4010 Basel, Switzerland Printed on acid-free paper produced from chlorine-free pulp. TCF OC! Softcover reprint of the hardcover Ist edition 1996 987654321

Contents Contributors

v

Introduction

P. Jol/es From the discovery of lysozyme to the characterization of several lysozyme families . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3

Lysozyme: A model enzyme in protein chemistry

E.M. Prager and P. Jol/es Animallysozymes c and g: An overview. . . . . . . . . . . . . . . . . . . . .

9

Discovery of several families of lysozymes

J. Fastrez Phage lysozymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

35

J.- V. Hjjltje Bacteriallysozymes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

65

J.J. Beintema and A.C. Terwisscha van Scheltinga Plant lysozymes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

75

D. Hultmark Insect lysozymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

87

Lysozyme: A model enzyme in enzymology

J.- V. Hjjltje Lysozyme substrates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..

105

M. Karplus and C.B. Post Simulations of lysozyme: Internal motions and the reaction mechanism ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 111 B. Fischer Folding of lysozyme. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..

143

T. [moto Engineering of lysozyme. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..

163

Lysozyme: A model enzyme in protein crystallography

N.C.J. Strynadka and M.N.G. James Lysozyme: A model enzyme in protein crystallography. . . . . . . ..

185

vi

Contents

Lysozyme: A model enzyme in molecular biology and genetics

D.M.lrwin, M. Yu and Y. Wen Isolation and characterization of vertebrate lysozyme genes ... "

225

M.L. Short, J. Nickel, A. Schmitz and R. Renkawitz Lysozyme gene expression and regulation .................. "

243

Lysozyme: A model enzyme in immunology

E.M. Prager Polyclonal antisera elicited by lysozymes: Insights into antigenic structure and evolution. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 261 SJ. Smith -Gill Molecular recognition of lysozyme by monoclonal antibodies. ..

277

G.A. Bentley The crystal structures of complexes formed between lysozyme and antibody fragments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 301 Lysozyme: A model enzyme in evolution

E.M. Prager Adaptive evolution of lysozyme: Changes in amino acid sequence, regulation of expression and gene number .................. " 323 D.M. Irwin Molecular evolution of ruminant lysozymes ................ "

347

Proteins and enzymes related to the lysozyme family

B.A. McKenzie ｾＭｌ｡｣ｴｬ｢ｵｭｩｮｳ＠

and lysozymes .......... , ................. "

365

C. Dupont, D. Kluepfel and R. Morosoli Evidence for lysozyme-type mechanism of hydrolysis in xy1anases..................................................... 411

f.-V. BOltje Lytic transglycosylases ................................... "

425

Pharmacological aspects and therapeutic applications of lysozymes

G. Sava Pharmacological aspects and therapeutic applications of lysozymes ................................................... 433

Contributors Beintema J.1., Department of Biochemistry, Rijksuniversiteit Groningen, Nijenborgh 4, NL-9747 AG Groningen, The Netherlands Bentley G.A., Unite d'Immunologie Structurale, C.N.R.S. URA 1961, Departement d'Immunologie, Institut Pasteur, 25 rue du Dr. Roux, F-75724 Paris, France Dupont c., Centre de recherche en microbiologie appliquee, Institut Armand-Frappier, Universite du Quebec, 531 boul. des Prairies, Laval Quebec, H7N 4Z3 Canada Fastrez J., Laboratoire de Biochimie Physique et des Biopolymeres, Universite Catholique de Louvain, Place L. Pasteur, 1, Bte 1B, B-1348 Louvain-la-Neuve, Belgium Fischer B., IMMUNO AG, Biomedical Research Center, Uferstr. 15, A-2304 Orth an der Donau, Austria H6ltje J.-V., Max-Planck-Institut fUr Entwicklungsbiologie, Abteilung Biochemie, SpemannstraBe 35, D-72076 Tiibingen, Germany Hultmark D., Department of Developmental Biology, Wenner-Gren Institute, Stockholm University, S-106 91 Stockholm, Sweden Imoto T., Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-82, Japan Irwin D.M., Department of Clinical Biochemistry, and Banting and Best Diabetes Centre, University of Toronto, 100 College St., Toronto, Ontario, M5G lL5, Canada James M.N.G., MRC Group in Protein Structure and Function, Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada Jolles P., Laboratoire de Chimie des Substances Naturelles, URA C.N.R.S. No. 401, Museum National d'Historie Naturelle, 63, rue Buifon, F-75005 Paris, France Karplus M., Department of Chemistry, Harvard University, Cambridge, MA 02138, USA, Laboratoire de Chimie Biophysique, Institut Ie Bel, Universite Louis Pasteur, F-67000 Strasbourg, France

Vlll

Contributors

Kluepfel D., Centre de recherche en microbiologie appliquee, Institut Armand-Frappier, Universite du Quebec, 531 boul. des Prairies, Laval Quebec, H7N 4Z3 Canada McKenzie H.A., School of Chemistry, University College, University of New South Wales, Australian Defence Force Academy, Canberra, ACT 2600, Australia, and lohn Curtin School of Medical Research, Institute of Advanced Studies, Australian National University, Canberra, ACT 2601, Australia Morosoli R., Centre de recherche en microbiologie appliquee, Institut Armand-Frappier, Universite du Quebec, 531 boul. des Prairies, Laval Quebec, H7N 4Z3 Canada Nickell., Institut fur Genetik, lustus-Liebig-Universitiit, Heinrich-BuffRing 58-62, D-35392 Giessen, Germany Post C.B., Department of Medicinal Chemistry, Lilly Hall, Purdue University, West Lafayette, IN 47907, USA Prager E.M., Division of Biochemistry and Molecular Biology, Barker Hall, University of California, Berkeley, CA 94720-3202, USA Renkawitz R., Institut fUr Genetik, lustus-Liebig-Universitiit, HeinrichBuff-Ring 58-62, D-35392 Giessen, Germany Sava G., Fondazione Callerio, Institutes of Biological Research, via A. Fleming 22-31, 4127 Trieste, Italy Schmitz A., Institut fur Genetik, lustus-Liebig-Universitat, HeinrichBuff-Ring 58-62, D-35392 Giessen, Germany Short M.L., Institut fur Genetik, lustus-Liebig-Universitiit, HeinrichBuff-Ring 58-62, D-35392 Giessen, Germany Smith-Gill S.l., Division of Basic Sciences, National Cancer Institute, Building 37, Room 2BI0, Bethesda, MA 20892, USA Strynadka N.C.l., MRC Group in Protein Structure and Function, Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada Terwisscha van Scheltinga A.C., Department of Biophysical Chemistry, Rijksuniversiteit Groningen, Nijenborgh 4, NL-9747 AG Groningen, The Netherlands

Introduction

lysozymes: Model Enzymes in Biochemistry and Biology

00. by P Jolla,

© 1996 B"khauser Verlag Basel/Switzerland

From the discovery of lysozyme to the characterization of several lysozyme families P. Jolles Laboratoire de Chimie des Substances Naturelles, URA C.N.R.S. No. 401, Museum National d' Historie Naturelle, 63, rue Buffon, F-75005 Paris, France

Sir Alexander Fleming is especially known for his discovery, in 1928, of penicillin: he was not looking for an antibacterial substance but for Staphylococcal variants because he had been asked to write an article on Staphylococcus. A seeded plate he had put aside was later found to be contaminated with a Penicillium, and around the fungus bacterial colonies were apparently undergoing lysis. Fleming deserved praise and gratitude for his acute observation and for the curiosity which prevented him from discarding an unexpected contaminant, as others might have done. Indeed the circumstances of the discovery of lysozyme were very similar: in fact it was Fleming's first major discovery, in 1921. While suffering from a cold, a drop from his nose fell onto an agar plate where large colonies of a contaminant had grown ... and lysozyme was discovered. He made this important discovery because when he saw that the colonies of the contaminants were fading, his mind went straight to the cause of the phenomenon he was observing: that the drop from the nose contained a lytic substance. In his first paper devoted to this "remarkable bacteriolytic element", Fleming ( 1922) noted: "it was found that nasal mucus contained a large amount of lysozyme, and it was later found that tears and sputum were very potent in their lytic action. It was also found that this property was possessed by a very large number of the tissues and organs of the body". And Fleming found that this was so - the substance was in tears, saliva, leucocytes, skin, finger nails, human milk - thus very widely distributed in animals and also in plants. He published further papers on lysozyme between 1922 and 1927 (Fleming, 1922, 1929, 1932; Fleming and Allison, 1922, 1923, 1925, 1927). Other scientists before Fleming had noticed the bactericidal power of egg-white (Rettger and Sperry, 1912) and much work has been done on the bactericidal action of leucocytes. Probably all these scientists were investigating the action of lysozyme but "all these authors considered that the antibacterial phenomena they observed were peculiar to the substance with which they were working - leucocytes or egg-white and none of them apparently had any inkling that the lytic element was

4

P. JolIes

widely distributed throughout the animal and vegetable kingdom." (Fleming, 1932.) Lysozyme is thus very widespread in nature. This observation was the starting point for studies of the evolution of lysozymes and for the characterization of different lysozyme families. The expression "lysozymes" was employed for the first time in 1932 by Fleming himself (Fleming, 1932): "It has been shown, however, that the lysozyme of different tissues and secretions has quite varied antibacterial powers toward different microbes, and it seems that there are some differences in the antibacterial ferments (which we may call lysozymes) of different tissues whereby the bacterial affinities may be very different." More than seventy years after Fleming's discovery of lysozyme, this enzyme is constantly subject to up-to-date studies in biochemistry and biology, as was already the case in the 1960s. When the word lysozyme is currently used, hen egg-white lysozyme is generally meant: it is the classic representative of this enzyme family and the related enzymes are called chicken-type or c-type or conventional-type lysozymes. The lysozyme of hen egg-white is remarkable in many ways. It was the first protein which was sequenced and found to contain all the twenty usual amino acids (Jolles and Jolles, 1961; Canfield, 1963; Jolles et aI., 1963). It was the first enzyme which was submitted to complete X-ray crystallographic analysis and for which a detailed mechanism of action was proposed (Blake et aI., 1961; Phillips, 1966). Ten or fifteen years ago most of the lysozyme data were obtained from birds. Since then c-type lysozymes have also been characterized in many other animals, such as mammals, reptiles and invertebrates (JoBes and JoBes, 1984). However with the development of the studies devoted to lysozyme, it rapidly became evident that besides the c-type lysozymes other distinct types of lysozymes exist: they were characterized in birds, phages, bacteria, fungi, invertebrates and plants (Jolles and Jolles, 1984). These distinct types of lysozyme differ on the basis of structural, physicochemical and immunological criteria; only the specificity of all these enzymes is the same: they cleave a ,B-glycosidic bond between the C-I of N-acetylmuramic acid and the C-4 of N-acetylglucosamine of the peptidoglycan. Some ill-informed scientists have claimed that everything has been discovered in the lysozyme research field. As a matter of fact, biochemical fashions come and go: the simple observation of the high number of quoted publications on lysozymes indicates that this research field is active. Personally, I have been involved since 1957 in lysozyme research and found it appropriate to organize a multi-author book on lysozyme as it is no longer possible for anyone scientist to cover all the studies in which lysozyme is and continues to serve as a model system. This is true in protein chemistry, in enzymology, in crystallography, in molecular

From the discovery of lysozyme to the characterization of several lysozyme families

5

biology and genetics, in immunology and in evolution, and the different chapters of this book are devoted to developments in these different areas with special emphasis on results obtained during the last ten years. Despite these extensive studies many aspects concerning the biological role(s) of the various lysozymes are still not precisely known. The peptidoglycan fragments released by the lytic action of this enzyme family might stimulate the synthesis and secretion of immunostimulating (Jolles, 1976) or antibacterial substances: but lysozymes (or their glycosylated forms or some split peptides) might provoke many other, still unexpected, biological reactions in accordance with Fleming's prophecy: "We shall hear more about lysozyme."

References Blake, C.C.F., Koenig, D.F., Mair. G.A., North, A.C.T., Phillips, D.C. and Sarma, V.R. (1961) Structure of hen egg-white lysozyme. A three-dimensional Fourier synthesis at 2 A resolution. Nature 206: 757-761. Canfield, R.E. (1963) The amino acid sequence of egg-white lysozyme. J. Bioi. Chem. 238: 2698-2707. Fleming, A. (1922) On a remarkable bacteriolytic element found in tissues and secretions. Proc. Roy. Soc. London B39: 306-317. Fleming, A. and Allison, V.D. (1922) Observations on a bacteriolytic substance - Iysozymefound in secretions and tissues. British 1. Exp. Path. 3: 252-260. Fleming, A. and Allison, V.D. (1923) Further observations on a bacteriolytic element found in tissues and secretions. Proc. Roy. Soc. London 44: 142-151. Fleming, A. and Allison, V.D. (1925) On the specificity of the protein of human tears. Brit. J. Exp. Path. 6: 87 -90. Fleming, A. and Allison, V.D. (1927) On the development of strains of bacteria resistant to lysozyme action and the relation of lysozyme action to intracellular digestion. 1. Brit. Exp. Path. 8: 214-218. Fleming, A. (1929) A bacteriolytic ferment found normally in tissues and secretions. Lancet 1: 217 -220. Fleming, A. (1932) Lysozyme. Froc. Roy. Soc. London B26: 71-84. Jolles, J. and Jolles, P. (1961) Structure chimique du lysozyme de blanc d'oeuf de poule: la formu1e developpee. c. R. A cad. Sci. 253: 2773-2775. Jolles, J., Jauregui-Adell, J., Bernier, T. and Jolles, P. (1963) La structure chimique du lysozyme du blanc d'oeuf de poule: etude detaillee. Biochim. Biophys. Acta 78: 668-689. Jolles, P. (1976) A possible physiological function of lysozyme. Biomedicine 25: 891-892. 101les, P. and Jolks, 1. (1984) What's new in lysozyme research? Mol. Cell. Biochem. 63: 165-189. Phillips, D.C. (1966) The three dimensional structure of an enzyme molecule. Sci. Am. 215: 78-90. Rettger, L.F. and Sperry, J.A. (1912) The antiseptic and bactericidal properties of egg white. 1. Med. Res. 26: 55.

Lysozyme: A model enzyme in protein chemistry

lysozymes: Model Enzymes In Biochemistry and Biology ad. by P. Jollas © 1996 B"khauser Verlag Basel/Switzerland

Animal Iysozymes c and g: An overview E.M. Prager I and P. Jolles 2 t Division of Biochemistry and Molecular Biology, Barker Hall, University of California, Berkeley, CA 94720-3202, USA 2Laboratoire de Chimie des Substances Naturelles, URA C.N.R.S. No. 401, Museum National d' Historie Naturelle, 63, rue Buffon, F-75005 Paris, France

Summary. Amino acid sequences for 88 distinct Iysozymes c, obtained from members of four vertebrate classes and two orders of insects, are summarized. A model for the relationships and origins of major lineages within the lysozyme c superfamily - which consists of conventional Iysozymes c, calcium-binding Iysozymes c, and !X-lactalbumin - is presented and supported by evolutionary analyses. Pioneering events in the discovery and sequencing of Iysozymes c are traced, and salient contributions to knowledge made by sequences from various kinds of animals highlighted. A summary of the four known amino acid sequences of bird Iysozymes g and an outline of the investigations on this very different kind of vertebrate lysozyme are provided. Areas of future research aimed at further elucidating early events in the evolutionary history of the lysozyme c superfamily and at understanding differences in patterns of lysozyme gene expression are outlined.

Introduction A decade after Sanger's landmark sequencing work on the hormone insulin (Sanger and Tuppy, 1951), Smyth et al. (1963) reported the amino acid sequence of bovine ribonuclease, an enzyme that lacks tryptophan. Chicken egg white lysozyme c (EC 3.2.1.17) served as a model system in protein chemistry as it was the first enzyme containing all 20 of the usual amino acids to be sequenced (Canfield, 1963; Jolles et al., 1963). Development of several new methods permitted determination of this sequence, which, like ribonuclease, has four disulfide bridges (Figs 1 and 2). Only several years later was precise knowledge of lysozyme's substrate obtained. At about the same time, Blake et al. (1965) produced an electron density map of chicken lysozyme, the first X-ray crystallographic structure of an enzyme. Phillips (1974) emphasized that interpreting the image was greatly facilitated, perhaps actually made possible, by the availability of the amino acid sequence. The c (chicken) Iysozymes take their name from this classical representative of the protein superfamily. Two decades later (Jolles and Jolles, 1984) fewer than 20 different complete lysozyme c sequences had been established, most of these from galliform and duck egg whites and one each from human, baboon, rat, and cow. Partial sequences from a tortoise and three moths had

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G•• H. .• Q. K.... G. K.....••.••....•.••... K•. N•. G.... A. LKD •• TQ ...... K..• Q....•..•... HK .. HR. LT. .• K.. GV · T. K•.... K ... N... A.....•.... M•. A. G•• N.•• Q. K.... G. K•.................... K.. N.. G.... A. LKD .. TQ ...... K... Q... . .. . ... NK .. NR.LT ... K.. GV *Cow tracheal Z •. S.. RF .M. NFR. 1. ..•. M.. AR ... H . .. Q..... AGDQ .....••..•. H.•...•... G.• NA .• LP. GA. LQD •. TQ ...... RV .. DP ... R..... R... QHQ.LT .. IQ .. GV *Cow kidney ..••.• RF .M .. FR. 1. ••.. M.. AR ....... Q•...• SGDR ...••...... H......... G.. NA .. IP .• A. LQD .. TQ ...... RV .. DP ... R..... R... QNQ. LT .. IQ .. GV Sheep kidney .K.Q ................ R....... V .. AR . .. H•. Cow milk .K. .. ..... R ...... R.. ..M .. AR .. . H. Goat tear 1 • K........... RF .M .. FR. 1. .... M•. AR .• Goat tear 2a · K. 1. ........ RF .M .. FR. I ..... M.. AR .. Goat tear Zb

Chukar partridge .... V. . • .• . . . . . . •. •• .• . • .• . . •. • . . •• . ..•• • . • . . . . ..•.•..••..•.•...•.•...••.•..•.••.•..••...•.•.•••....••.•.•..•••. H.••.•.•• Japanese quail .. y •.............. K.Q.................... . ......................................................... VH ................. N....... . Turkey .. Y....•.•..•. l....... . .....••••..• H. . ..•.•..•...•..... K.•••.••..•..•.•.......... A.G •.......•..•••••..• H••...... Peafowl .. y ........... L ...•...•.•..•••....••••.• H••••••.•..•.•....•••..•..••••••.•..••.••.•..••.•••••••••••••• R••••••••..•••••••• H•••••••• RN pheasanta . ••..•..•••.•.•.•.••.. K•.. H.••.•..••••.•••.•..••••••..•.••••.. KH ..•••• NV ••••.•• G•. y .•......... M...•...•.••.•.•.•..•..•.. G...... Golden pheasant b .. y ...•••.•••• L ••••..............•••..•. H.... . •••..••.•.....•••....•.•• H •.....•.•.................•.....••••.••.... N •• T •.... Kalij pheasant .. y ......... _ .L.. . .y ...... H.... _.. . .•.... K ........ H.. . ........................•......... SV.T .... . Reeves pheasant C .. y ...••.•..•. L ...•.............••••••.. H••..••........•..•••..•............. H.S ••.........•.•.......• R••.•••.•••••..•.•• N•••.••.. Copper pheasant .. y •...•.•.... L •..• F .••••..••••••••••••• H••.•••.•••••••••••••..•••••••••••••••••••••••••••••••••••••••.••••••.••• K ••.•••• H •• T •••.•

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. N. . .. AV... • .•. E ... .. KS ..•.. L. .G.. . .•. Y .. P ....• V ... N.. K •. R.IQR .•... T ..•.• K•....•. S•.•. NS •.

RTMDRCSLAREMSNL GVPRDQ- - -LARWACIAEHES SYRTGVV GPENY NGS NDYGIFQINDYYWCAPPS GRFSYNECGL SCNAL LTDDITHSVRCAQKVL S-QQGWSAWSTWHY -CSG- -WLPSIDDCF.NK ..

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. .... T.

10 20 30 40 SO 60 70 BO 120 129 90 100 110 KRFTRCGLVQELRRRGFDETL - - -MSNWVCLVENES GRFTDKIGKV NKNGSRDY GLFQINDKYWCSKGSTPG-KD-CNVTCNQLL TODISVAATCAKKIYK-RHKFDAWYGWKNHCQH --GLPDISDC --

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

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

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SKMKKCEFAKIAKEQHMDGYHGVSLADWVCLVNNESDFNTKAINRNKGI

Figure I. Caption on following 2 pages.

UNCLASSIFIED POSSUM o

. .... G... Q.

. ..• G.•.. K .•.•.•• .... G.•.. K .•• G•.•

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

KVFSKCELAHKLKAQEMDGFGGYSLANWVCMAEYESNFNTRAFNGKNANGSSDYGLFQLNNKWWCKDNK-RSSSNACNIMCSKLLDENIDDDISCAKRVVRDPKGMSAWKAWVKHCKDKDLSEYLASCNL

.1. ... R•.• SMG. . H. Hooded seal m,lk .1. .. R ... TKGL. • YH. .1.T. .... R•. R.EG .•. Cat mllk

CARNIVORES Dog milk

Horse 1'II1lk Donkey mi lk

EQUIDS

Echidna II

CALCIUM-BINDING BIRDS 10 20 30 40 SO 110 120 129 60 70 80 90 100 "HootZl n 5 tom 1m EIIPRCELVKILREHGFE GFE GTTIADWICLVQHESoYNTEAYNNNG -p- SRDYGIFQINSKYWCNDGKTSGAVDGCHISCS ELMTNDLEDDIKCAKKIARDAHGL TPWYGWKNHCEGRDLSSYVKGC -*Hootn n stomach 2 ... S. . ............... . *HoatZln basic 1 n KT.R. . .. K .. L. .. K.. . .. 1< ••• N... K ... 0. . R... N.. R. N .. K. LN. N. . ....... EQ. R. . ..•. K. KN ..•. 1. .. . . o. *Hoatzln baslc 2 K.. . .... K .. l ... K.. . ... K ... N ... K ... O. . D. .R ... N .• R. N•• K. LN. N.. . ....•••• EQ.R. . • ..•. K. KN •.•. 1. ... *Hoatzin baslc 3 KT.R ......... K .. L. .. K. . ... K ... N. . .0. . . R. . N.. R. N .. K. LN. N.. . . .•. EQ. R. . . . . .•. K. KN ••. Pigeon KO.. ..R ...... V K.V.N.V ... K ... G.R.T.F . . . . . N .. . .R. SKNA.N. N •. K .RDDNIA ... Q •...... E.R ..... VA .. KY .Q. K ...... R ... . Blockbird K •.. K .. M..•.. RN .. Q. . .V ... M. MONDTREMES Echidna I KILKKQELCKNLVAQGMNGYQHITLPNWVCTAFHESSYNTRATNHNT -DGSTDYGILQINSRYWCHDGKTPGSKNACNISCSKLLDDDITDDLKCAKKIAGEAKGLTPWVAWKSKCRGHDLSKF-K-C--

*Frul. t fly X *Frul. t fly P

*Frul. t fly S

*Frui. t fly E

-Frui.t fly o,B 1 *Frult fly A,e

DIPTERANS

Cotton leofworm

... S. . . . N.......... A.

... V..... N........ A...... .

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KVY DRCELARALKAS GMDGYAGNSLPNWVCLSKWESSY NTQATNRNT -DGSTDY GIFQINSRYWCDDGRTPGAKNVCGIRCSQLLTDDL TV AIRCAKRVVLDPNGIGAWV AWRLHCQNQDLRSYVAGCGV

. VFKH .. L .. 1. RSSALA .. R .... EN .M.M ...... FD. E. 1. .. ST ... . .V. . .......• A. . .•• R.T ... R. . V. N... L. .1. ..... D.. R.. K. . . R. T. . R.. R..

.1..

· W... A •• K•• E .. M... R. . M . . .. D.•• D. .RY ... N.••. H.• N•. GIN. NV. L. D.. T ..• Q... RV. RDP .. YR ...... N•. EG ..• EQ ..••. D. · YD. . F .. 1. .. S.M •.. R ....... V .. A. .. DF .... 1. R. VG--. .RY. . . . K .. NA .. 1. . KV. LDD. LSQDIE ... RV .RDP ... K. . . RT .• QNK .•. Q. IR .. K. · YD ... F .. 1. .. S.M ... R. . .... V .. A. . OF •... 1. H. VG--. . . . .. RY. . .... K .. NA .. 1. . KV. LoD. LSQDIE. .• RV. RDPL. VK ..... RA .• QNK •.. Q. IR .. K. · YD ... F .• 1. .. S.M ... R. . V .. A .... NF. • . . ... G.Q. . ..• RY. . K •. NA .. 1. . KV. LDD. LSQDIE .•. RV. RDP •.. K •..•.• A .• QNK .•. Q. IR .. K. 10 20 30 40 SO 60 70 80 90 100 110 120 129 KTYERCEFARTLKRNGMSGYVGVSLADWVCLAQHESNYNTQARNYNPGDQSTDYGIFQINSRYWCNDGKTPRAKNACGIPCSALLQDDITQAIQCAKRVVRoPQGIRAWVAIiQRHCKNRDLSGYIRNCGV

·Tobacco hornworm .H.S .. E .. H.... Q.. P.N ..... RD. .S.Y ... V.R. * Si..lkworm k .T. .H ... KH .. E.N ..... R . . . . . . H .. S.D.S.TNT- R ... K. Greater wax moth .T .... E ... A ... Q.... AK ... LRD ..

Gi sllk moth lb

"Gi si I k moth 2) *Gl. silk moth 10

INSECTS LEPIDDPTERANS

-Trout I -Trout II

FISH

·Mouse P

-Mouse M

Rat 10 ·Rat lb -Rat 2'

RODENTS

·Pi.g Z Pig 3

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12

E.M. Prager and P. Jones

Figure 1. Amino acid sequences of Iysozymes c. Of the 75 complete plus 13 partial sequences in this figure, 12 are written out entirely and designated as baseline sequences. The residues present in the other 76 sequences are shown only where different from the closest preceding baseline sequence, with identity indicated by a dot. Dashes denote deletions relative to other sequences, question marks undetermined residues, and blank areas unsequenced regions. Numbering is according to chicken lysozyme; the location of the insertion found in many other sequences between positions 47 and 48 is caned 47a. Major taxonomic categories are indicated with capital letters, including some cases where there is a single sequence or species. Tissues are indicated where it is known or inferred that a different lysozyme c is expressed in other tissues (though for mouse and fruit tly only the descriptive designation of the sequence is used). Asterisks at the left indicate the availability of cDNA and/or gene sequences. Slightly different alignments of the insect to the vertebrate sequences have been proposed (eg., Klysten et aI., 1992; Grobler et aI., 1994). The scientific names of the species in the figure are in order as follows, with underlining for those with baseline sequences: Gallus gallus, Alectoris graeca (with an unpublished correction at position 18 by J.W. Schilling and E.M. Prager), Coturnix coturnix, Meleagris gallopavo, Pavo cristatus, Phasianus colchicus, Chrysolophus pictus, Lophura leucomelana, Syrmaticus reevesi, Syrmaticus soemmerringi, Numida meleagris, Colinus virginianus, Lophortyx californicus, Cyrtonyx montezumae, Ortalis vetula, Anas platyrhynchos, Cygnus atratus, Trionyx gangeticus, Homo sapiens, Papio cynocephalus (with an unpublished correction at position 66 commumcated by W. Messier and C.-B. Stewart), Macaca mulatta, Cercopithecus aethiops, Presby tis eiltellus, Oryctolagus cuniculus, Canis familiaris, Equus cabalIus, Bos taurus, Capra hircus, Ovis aries, Axis axis, Camelus dromedarius, Sus scrofa, Rattus norvegicus, Mus domesticus, Oncorhynchus mykiss, Hyalophora cecropia, Manduca sexta, Bombyx mori, Galleria mellonella, Spodoptera littoralis, Drosophila melanogaster, Opisthocomus hoazin, Columba livia, Agelaius phoeniceus (J. JoBes, E.M. Prager, and P. JoBes, unpublished), Tachyglossus aculeatus multiaculeatus, Tachyglossus aculeatus aculeatus, Equus caballus, Equus asinus, Canisfamiliaris, Cystophora cristata (S. Pervaiz and K. Brew, personal communication), Felis catus, Pseudocheirus peregrinus. Sources of the sequences are Aschaffenburg et al. (1980), Jolles and Jones (1984), Rodriguez et al. (1987), Nicholas et al. (1989), Jolles et al. (1990), Lavoie et al. (1990), Dautigny et al. (1991), McKenzie and White (1991), Swanson et al. (1991), Ito et al. (1993, 1994), Yeh et al. (1993), Araki et al. (1994), Daffre et al. (1994), Grobler et al. (1994), Kornegay et al. (1994), Mulnix and Dunn (1994), Irwin (1995), Lee and Brey (1995), references therein, and unpublished as indicated. aRN, ring-necked: identical sequence in Japanese pheasant, Phasianus versicolor. The Gly at -I retlects a shift in the prelysozyme cleavage site (Weisman et aI., 1986). bIdentical sequence in Lady Amherst pheasant, Chrysolophus amherstiae. CSequence for lysozyme B shown; A, with Asp at 103, likely results from deamidation. dldentical sequence in Gambel (Lophortyx gambeli), Benson (Lophortyx douglasi), scaled (Callipepla squamata), and mountain (Oreortyx pictus) quaillysozymes; the last three are from J.W. Schilling and E.M. Prager, unpublished. "Though all the duck amino acid sequences are reported as Asp-Asn at positions 65-66, they may be Asn-Asp as in all other conventional bird Iysozymes c. fThe sequence was determined for the more common, electrophoretically faster of the two variants characterized from this species. The residues at positions 47 and 48-129 were identified only from X-ray electron density maps, while the other 47 residues were determined also chemically. At position 27 His was inferred from the X-ray data, as opposed to Arg (shown here) from direct protein sequencing. gIdentical sequence in common (Pan troglodytes) and pygmy (Pan paniscus) chimpanzees (G. Maston, W. Messier, and C.-B. Stewart, personal communication). hThe two cow tracheal sequences, which differ only at position 10, may result from alleles at a single locus; sequences I and 2 were determined from cDNA and genomic clones, respectively. The genes encoding cow stomach 2 and kidney lysozymes are also expressed in the trachea (Takeuchi et aI., 1993). iLikely a pseudogene sequence. iGi, giant. k Asn at position 34 in the earlier reported partial protein sequence may be due to intraspecific polymorphism. IThe fruit tly sequences encompass products of seven genes; genes for D and B encode the same mature protein, while A and C likely result from two alleles at one locus.

Animal Iysozymes c and g

13

70

Figure 2. Invariant residues in Iysozymes c. The 20 residues invariant in all sequences in Figure I are shown with plain circles; dark circles mark the three residues variant only among the insect sequences. Four disulfide bridges have been characterized in a few Iysozymes c by chemical and crystallographic methods - between half-cystines 6 and 127,30 and 115,64 and 80, 76 and 94; the last three, comprised of invariant residues, are shown here. For the echidna Iysozymes (cf. Fig. I), a 9-127 instead of 6-127 disulfide bridge is readily achievable (Acharya ct aI., 1994). The hydrophobic region around Trp residues 28, 108, and III is denoted as domain A and the hydrophilic region encompassing residues 50- 76 as domain B (see text).

extended the taxonomic distribution of expressed lysozymes c to reptiles and insects. It had also been shown that translation of chicken lysozyme mRNA resulted in a prelysozyme, with an I8-residue amino-terminal signal peptide that is removed in vivo by signal peptidase to yield the mature lysozyme c protein. In 1979-1980 the first lysozyme cDNA and gene sequences, for the chicken, were determined (Irwin et ai., this volume). Development and routine application of molecular biological techniques, improved methods of protein sequencing, and the phenomenon of multiple lysozyme c genes in some species, notably ruminants,

mWhile the hoatzin Iysozymes are clearly phylogenetically allied with the other calcium-binding bird Iysozymes, they may not actually bind calcium in light of the absence of Asp at position 85 (see Tab. 2 and 3). "Expression of the non-stomach, basic hoatzin Iysozymes has not yet been demonstrated, but there is no evidence that the sequences are encoded by pseudogenes (Kornegay, 1994). °The phylogenetic placement of this distinctive marsupial lysozyme c is not clear from the partial, 49-residue sequence.

14

E.M. Prager and P. JoBes

have now raised the total to 75 complete lysozyme c sequences and 13 partial ones (Fig. 1). The sequence of tortoise lysozyme c is remarkable in that 64% of it is known only from X-ray crystallographic data. All the mature proteins have 119-130 residues (Fig. 1), and all the characterized signal pep tides have 15-20 residues. Publication in 1982 of only 20 amino-terminal residues of pigeon egg white lysozyme suggested that this bird had a lysozyme c far more different than would be expected if the same gene encoded the galliform, anseriform, and pigeon enzymes. The complete sequences reported three years later of the pigeon (Rodriguez et al., 1985) and horse milk (McKenzie and Shaw, 1985) enzymes strongly implied that the lysozyme c family tree had one or more additional, deep branches. The calcium-binding property of these lysozymes c (Nitta et al., 1988) gave them their name. Asp residues at positions 85, 90, and 91 were subsequently found to be critical for calcium binding (see, eg., Tab. 3 below; McKenzie, this volume; Acharya et al., 1994). A few years after publication of the chicken lysozyme c sequence, Brew and Campbell (1967) suggested that IX-lactalbumin shared a common ancestor with lysozyme c on the basis of its molecular weight, amino acid composition, and some amino- and carboxy-terminal sequence data. The first complete IX-lactalbumin sequence (Brew et al., 1970) soon provided strong support for this proposal. IX-Lactalbumin, which has to date been found only in the mammary gland (ie. an organ unique to mammals), is a calcium-binding protein that interacts with galactosyl transferase as a specifier or regulatory entity so as to promote lactose synthesis. Any lingering doubts that IX-lactalbumin belonged to the lysozyme c superfamily were dispelled by the finding, first made a decade ago (Qasba and Safaya, 1984), that the genes for vertebrate lysozymes c and IX-lactalbumins all have four exons and, located in the identical places, three introns. However, while the calcium-binding site of IX-lactalbumin uses the same residues as do the calcium-binding lysozymes c (Stuart et al., 1986; Acharya et al., 1994; cf. Tab. 3), there is no consensus as to when and along which lineage IX-lactalbumin arose (see Fig. 3 and discussion below). A radically different lysozyme, called g after the Embden goose, the species in whose egg white it was first discovered (Canfield and McMurry, 1967; Dianoux and Jolles, 1967), was found immunologically to be taxonomically widespread in bird egg whites (Prager et al., 1974). Dramatic contrasts in expression of these distinct lysozyme genes with respect to both species and tissues soon became evident (Hindenburg et al., 1974; Arnheim, 1975). Three of only four lysozyme g sequences known to date were reported in 1980-1983. No evidence has been put forth for the occurrence of lysozyme g or its gene in creatures other than birds.

15

Animal lysozymes c and g Lepidoptera Insects Diptera

I I

Birds

l

Testudine reptiles

CONVENTIONAL LYSOZYMES c

Placental mammals Bony fishes

Equids Placental mammals Carnivores

+

....

CALCIUM-BINDING LYSOZYMES c

r

}

L

Birds

Echidnas (Monotremes) Placenta Is

...... rl

Marsupials

a-LACTALBUMINS In Mammals Only

Monotreme Origin of mammary gland

600

500

400

300

•

200

100

o

Millions of years ago

Figure 3. Protein expression and gene relationships in the lysozyme c superfamily. The figure summarizes the kinds of creatures found to express each type of lysozyme c and ex-lactalbumin, the relationships and estimated divergence times of the organisms along each deep molecular lineage, and a model for the relationships among the present-day molecular lineages and for the gene duplications leading to these lineages. The diamonds arbitrarily placed at 500 million years ago (at about the start of intra-vertebrate divergence) represent early gene duplications, at least two of which appear necessary to account for the known types of vertebrate Iysozymes c. Given the available data, we cannot infer with confidence the relative timing and lineages involved in each of these two duplications - ie. whether, for example, the two calcium-binding lineages arose and diverged from one another after an earlier gene duplication and divergence from the conventional lineage. The large open bar indicates the proposed lineage and likely time period for the origin via gene duplication of ex-lactalbumin from lysozyme c. Two arrows within the figure indicate other candidate lineages for the origin of ex-lactalbumin. Organismal divergence times are based on Carroll (1988), Prager and Wilson (1988), Benton (1990), Labandeira and Sepkoski (1993), and references therein; the intra-insect divergence time shown involves appreciable uncertainty but does not exceed 400 million years ago. The analyses summarized in Figure 4 provide support for several elements of this proposed model.

16

E.M. Prager and P. JoBes

Results and discussion Lysozyme c sequences

At only 20 positions are the amino acid residues invariant in all known lysozyme c sequences (Figs 1 and 2): 28-W, 30-C, 35-E, 36-S, 50-S, 52-D, 53-Y, 54-G, 57-Q, 59-N, 63-W, 64-C, 76-C, 80-C, 94-C, 95-A, 108-W, lll-W, lI5-C, 127-C. Three more positions - 44-N, 96-K, I04-G in vertebrates - are variant only in insect lysozymes c. The invariant residues include the catalytic E35 and D52, a number of residues important for the enzyme's overall three-dimensional structure, and several of the residues lining the active-site cleft. Though only about 20% of all the residues in lysozyme c are internal, about 40% of these conserved residues are fully buried; roughly half are located in the more flexible hydrophilic domain B (Fig. 2) and a quarter in the hydrophobic box (domain A). The deletions and insertions observed (Fig. 1) are situated at the periphery of the molecule; positions 47a-49 are a hotspot for length variations of 1-2 amino acids in vertebrates. Table 1 provides an overview of pairwise sequence differences among animallysozymes c, and Table 2 compares quantitatively all known complete sequences designated as calcium-binding lysozymes c in Figure 1. Even between the most distantly related molecules in Table 1, the number of identical residues is about twice the number of totally invariant positions. This finding can be explained in light of the overall structural and functional requirements of lysozyme c and the observation that at over 40 additional positions (see Tab. 1, in Prager, this volume: Adaptive evolution) few (2-4) alternative amino acids have been recorded. Thus at an appreciable number of positions two highly divergent lineages will have retained the ancestral amino acid, or by chance they will presently have the same amino acid despite multiple hits over eons of time. For the same reasons, the range between orders of insects overlaps with the insect versus vertebrate range (Tab. 1), even though the latter organismal divergence occurred at least 1.5 times as long ago (Fig. 3). Table 1 also implies appreciable differences in rates of amino acid replacement along different molecular lineages. Among the conventional vertebrate lysozymes, for example, there is a threefold range (18-56) between orders of placental mammals (all of which diverged from one another over a relatively short time). Similarly, the range for birds versus mammals is higher than that for fishes versus tetrapods though the relative divergence times (Fig. 3) would predict the opposite. Some of these rate differences have been explained by phylogenetic analysis, for instance the roughly threefold acceleration for some 30 million years along the lineage leading to ruminant stomach lysozymes c (Jolles et aI., 1990; Prager, this volume: Adaptive evolution; Irwin, this volume), and the apparently lower rate along the fish lineage (Dautigny et aI., 1991;

17

Animal Iysozymes c and g Table I. Quantitative comparison of amino acid sequence differences among Iysozymes c Lysozymes compared Conventional vertebrate Among birds Among mammals Within mammalian species Between mammalian orders Birds vs. tortoise Birds vs. mammals Mammals vs. tortoise Fish vs. tetrapods Calcium-binding vertebrate b Among birds Among placental mammals Placental vs. monotreme mammals Birds vs. mammals Vertebrate Conventional vs. calcium-binding Insect Among lepidopterans Within fruit fly Between insect orders Invertebrate vs. vertebrate Insect vs. conventional vertebrate Insect vs. calcium-binding vertebrate

Range of sequence differences

0-31 0-56 1-45 a 18-56 36-47 45-63 42-56 36-56 I 39 3-23 67··70 50-79 56-79 1-30 1- >27 c 69-78 75-90 72-91

Pairwise sequence differences were computed using the alignment in Figure I, with an addition or deletion at any position counted as one amino acid difference. Only complete sequences were considered, with one exception (footnote c). Rat lysozyme 2 was omitted because it is likely a pseudogene sequence. Values of zero mean that the identical lysozyme c sequence was found for more than one species. aGene duplications early in ruminant evolution (irwin, 1995; Irwin, this volume) account for the large intraspecific differences (cf. cow and sheep in Fig. I). bS ee Table 2 for more details. cFruit fly Iysozymes P and X differ at 27 of the 81 positions for which the sequence of lysozyme X is known, which exceeds the maximum of 24 among the complete dipteran sequences in Figure I.

see discussion therein for possible alternative explanations). Overall, the rate of amino acid change is roughly the same among the calcium-binding as among the conventionallysozymes c, as may be seen by comparing pairs of lineages that diverged at about the same time: The difference between orders of placental mammals, 22-23 in Table 2, is within the interordinal range of 18 - 56 in Table 1 discussed above, and the 34-39 between orders of birds in Table 2 is close to values reaching 31 in Table I for conventional bird lysozymes c. The following paragraphs highlight contributions to knowledge made by sequencing and otherwise characterizing the lysozymes from various kinds of creatures; many of these phenomena are discussed in further detail in this book. Advantage was taken of the naturally occurring variation among conventional bird Iysozymes c, especially the very similar phasianoid

HSI

HS2 26 26

HBI 24 25 2

HB2 23 23 3 5

HB3 38 39 36 35 34

Pigeon 60 60 60 59 60 51

EchI 60 60 60 59 60 50 3

EchII

64 67 68 3

73 72 72 72

73 65 68 67 23 22

72

78 77

78

79 78 73 73 73 66 69 70

Dog

77

Donkey

Horse

An addition or deletion at any position was counted as one amino acid difference. See Figure I, Table 3, and text for further details about the expression and likelihood of calcium-binding of the hoatzin Iysozymes.

Hoatzin stomach I Hoatzin stomach 2 Hoatzin basic I Hoatzin basic 2 Hoatzin basic 3 Pigeon Echidna I Echidna II Horse milk Donkey milk Dog milk

Sequences compared

Table 2. Pairwise amino acid sequence differences among II vertebrate calcium-binding Iysozymes c

'"C

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adh lysin seems to be associated with the membranes; when expressed together with the A. holin, it causes E. coli lysis but not when coexpressed with the 4>adh lysin. The interpretation of this observation will require further studies (Henrich et aI., 1995).

The muramidases of Lactobacillus lactis

The phages Tuc2009 and 4> LC3 encode two lysis proteins associated in an operon. The first protein features characteristics of holins; the second one is a large protein that behaves like muramidases. The genes were cloned; E. coli cells expressing both genes after IPTG addition to the medium were lysed, while those expressing only the lysin were not. Extracts of E. coli in which the Tuc2009 or the 4> LC3 lysins were expressed were able to lyse L. lactis suspensions. A homology search of the data bases again revealed a modular construction of these two very similar lysins: a muramidase domain appears to be associated with a C-terminal domain comprising repeated modules (Arendt et aI., 1994; Birkeland, 1994). The relationship between the putative muramidase domains and the other CH-type enzymes is quite peculiar. A region of high similarity is found between the sequences of the Tuc2009 or 4> LC3 lysins from residues 121 to 223 and those of CPU or CPL9 from residues 91 to 187; this region corresponds approximately to the third domain identified in the structure of the S. erythraeus lysozyme (Harada et aI., 1981). The alignment of the amino-terminal, on the other hand, is difficult; the residues conserved in the other enzymes, particularly the aspartic and glutamic acids separated by 26 residues, are not identifiable; at best, the aspartic acid 67, located in a short sequence with some similarity to those of the other CH lysozymes, could be conserved. As the glutamic acid does not appear to be truly essential, its absence may not be a problem. After another 110 residues with no equivalent in the other CH lysozyme sequences, the C-terminal features a doubly repeated motif already found in the 4>29 lysozyme and present in many functionally unrelated proteins and possibly involved in binding of the enzyme to the cell wall substrate (Birkeland, 1994).

58

J. Fastrez

Conclusions The comparison of the sequences and properties of the many phage lysozymes that have been described in sufficient detail suggest that they all belong to one of two structural families. The V-type, G-type and probably the A-type lysozymes could share the classical lysozyme fold described after studying the structure of HEWL, GEWL and T4L (see Weaver et aI., 1985b). Lysozymes of phages infecting Gram-negative and Gram-positive bacteria are found in this broad family whose members are likely to derive from a remote common ancestor. On the other hand, CH-type lysozymes are encoded by phages infecting Grampositive bacteria. The modular assembly of the lysozyme genes is illustrated by many observations. Several enzymes have substrate-binding domains connected to catalytic domains. Enzymes are found for which homology searches detect similarities only in sequence fragments whose size might correspond to that of a protein domain, the rest of the protein being apparently unrelated (eg., the ," S tlll

I

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Figure 4. Kinetics of refolding of denatured-reduced hen egg white lysozyme at different temperatures. Denatured-reduced lysozyme was diluted into 0.1 M. Tris buffer, pH 8.3, containing 3 mM reduced and 0.3 mM oxidized glutathione. Over a period of 60 min alliquots were withdrawn and assayed for lysozyme activity., The enzyme activity of native lysozyme of identical protein concentration was 100%.... represents the renaturation at 70°C (taken from Fischer et a\., 1993a).

and Wetlaufer, 1976; Fischer et aI., 1992a). Two minutes after initiation of oxidative refolding almost 90% of reduced hen egg white lysozyme was converted to intermediate forms with one or more disulphide bonds, with a lysozyme form with two disu1phide bonds being dominant (Fischer et aI., 1992a). Analysis (Anderson and Wetlaufer, 1976) showed that early disulphide bonds involve cysteine residues 64, 76, 80 and 94, forming the disulphide bonds Cys76_Cys 94 and Cys64_Cys 80, while residues 6, 30, 115 and 127 still remained reduced. Anderson and Wetlaufer (1976) found that formation of disulphide bond between residue 6 and 127 completes the cross-linking of lysozyme. These results were confirmed by energy calculation (Yoshimura et aI., 1991). Early folding intermediates of lysozyme with limited numbers of disulphide bonds formed show a higher hydrodynamic volume when compared with native lysozyme (Acharya and Taniuchi, 1976; Perraudin et aI., 1983). These folding species possess a flexible conformation different from native lysozyme, and tryptophan fluorescence shows an enlarged molecular domain with a disordered conformation (Acharya and Tani-

Folding of lysozyme

155

uchi, 1976). In some respects, these early folding intermediates behave like semi-compact globules predicted by a new interpretation of the protein-folding process, based on Monte Carlo simulation (Sali et aI., 1994). The conversion of a lysozyme molecule with two disulphide bonds to a three disulphide bond-containing intermediate seems to be the rate-limiting step in oxidative folding (Fischer et aI., 1992a). Isolation of incompletely oxidized, partially folded forms of lysozyme (Acharya and Taniuchi, 1976) resulted in a mixture of isomeric structures of lysozyme with three disulphide bonds formed and one of the three disulphide bonds between cysteine residues 6-127, 76-94 and 64-80 being open. Twenty-four percent of the isolated intermediates contained incorrect disulphide bonds involving cysteine residues 6, 30, 115 and 127. However, the lysozyme isomer having the open disulphide bond between Cys30 and CysllS was not found (Acharya and Taniuchi, 1982). An explanation would be that formation of the disulphide bond Cys30_CysllS is extremely fast. Folding intermediates with three disulphi de bonds already possess a native-like, compact structure but reduced activity (Acharya and Taniuchi, 1978; Fischer et aI., 1992a). Renaturation of hen egg white lysozyme with one carboxymethylated cysteine residue showed that no single one of the four disulphide bonds of native lysozyme is essential to the formation of the other three native disulphide bonds, establishing the intrinsic flexibility of the pathways for formation of disulphide bonds (Acharya and Taniuchi, 1977; Acharya and Taniuchi, 1982). Similar results have been obtained from in vitro folding studies of four derivatives of genetically engineered hen egg white lysozyme, each lacking one disulphide bond of the four in authentic lysozyme (Sawano et aI., 1992). All four derivatives resulted in stable folding prdducts after oxidative refolding, but showed different secondary structures as analyzed by CD spectroscopy. However, the limited number and pattern of folding intermediates and disulphide pep tides isolated indicate a restricted search of structures in the formation of the three-dimensional structure and a nucleation in the folding of denatured-reduced lysozyme (Ristow and Wetlaufer, 1873). Analysis of catalytic properties of lysozyme during oxidative refolding showed that generation of molecules with native-like catalytic properties depends on the final formation of all four disulphide bonds (Fischer et aI., 1992a). Evidence for the existence of nonpolar surfaces in early folding intermediates during oxidative refolding of hen egg white lysozyme has been obtained from fluorescence studies of the transient binding of a hydrophobic dye, 8-anilino-l-naphalene sulfonate (ANS), to the protein (Fischer et aI., 1993a). A significant enhancement of ANS fluorescence was observed within a few seconds of addition of denatured-reduced lysozyme to renaturation buffer. A similar increase in fluorescence intensity has been observed during interaction of ANS and reduced-car-

156

B. Fischer

Table 2. Oxidative folding of denatured/reduced hen egg white lysozyme in the glutathion renaturation system. Time (s)

Probe

Structure formation

Phe» Trp > Leu> Ala (Muraki et al., 1992). A similar phenomenon was seen in the mutations of Trp62 in hen lysozyme (Kumagai et al., 1993). These results suggest that the interaction of substrate and Trp62, which is presumed to be interacting with only Be sugars, cooperates with the interaction of the substrate at the A site. This was previously suggested from the computer simulations of reaction rate analyses of chemically modified lysozyme (Fukamizo et al., 1986). The results of the X-ray analyses mentioned above also support this cooperation because the movement of Trp62 is different depending on whether or not the A site is occupied. The mutation of Trp63 in human lysozyme to Tyr or Phe caused a drastic decrease in both activities (Muraki et al., 1987b). Trp108 in hen lysozyme participated in binding with the substrate by its hydrophobicity and the mutation of Tyr or GIn led to weak binding of the substrate according to its decreased hydrophobicity (Inoue et al., 1992b). Similarly, the mutation of Trp108 in human lysozyme to Tyr or Phe caused a considerable decrease in the activity (Muraki et al., 1987b). AsplOl, which lies on the top of the active site cleft, was variously modified, and the modifications led to the decreased binding ability of

Engineering of lysozyme

167

the substrate (Kuroki et aI., 1986b). The Asp101Gly mutant showed a binding ability to (NAG)3 which was decreased to one-tenth (Kumagai and Miura, 1989). The Asp101Gly102 sequence easily forms a succinimide ring and the derivative shows 160% lytic activity (Tomizawa et aI., 1994a). The following results were obtained from the experiments employing (NAG)s-PNP. When the binding ability was decreased at residues 62 and 101, the B-F binding mode became dominant. On the other hand, the A-E binding mode became dominant when Asn37, which seems to participate in binding at the F site, was mutated to Gly (Kumagai et aI., 1993). The triple mutant Trp62HisjAspl0lGlyjAsn37Gly showed a tripled lytic activity over that of the wild type (Kumagai et aI., 1992b). Arg114 in human lysozyme was mutated to Lys, His, GIn and Glu, and the positive charge at this position was shown to be important for the manifestation of the lytic activity (Muraki et aI., 1989). On the other hand, it was found that the active site cleft become narrower in the mutant Argl14Glu in human lysozyme (Harata et aI., 1993). The interaction between the net positive charge on lysozyme and the net negative charge on the cell surface is a dominant factor in the lytic activity, as was proved by the acetylation of amino groups on lysozyme (Yamasaki et aI., 1968). The double mutant Val73ArgjGlnl25Arg in human lysozyme showed higher lytic activity than the wild type at higher ionic strength and higher pH. On the other hand, the double mutant Arg41GlnjArglOOSer showed this behavior at lower ionic strength and lower pH (Muraki et aI., 1988). Thus, lysozyme might be designed to have an optimum balance of charges so as to express a proper lytic activity under the conditions where it is set to work. It is clear that interactions other than that with sugar residues are operative in the lytic activity. As for the binding of the substrate to lysozyme, we can conclude that for the binding of sugars to lysozyme, many interactions are operative and these are particularly important for the simple substrates. For the lytic activity, ionic interactions are another dominant factor, and sometimes weaker interactions with sugars are preferred for effective turnover. This can be confirmed from the comparative biochemistry shown in Table I.

Catalytic action We can estimate the catalytic residues from the X-ray analysis of an enzyme-substrate complex. However, in order to confirm the identity or to elucidate the mechanism and degree of participation of these residues, kinetic analyses with site-specifically modified enzymes are required.

168

T. Imoto

Table 1. Activities and substrate binding abilities of lysozymes of various origins

Lytic activity

Activity against glycol chitin

Lysozyme

(%)

(%)

Binding ability against (NAGh KA (M- 1)

Hen Japanese pheasant Turkey Rabbit Pig I Rat Human

100 123 176 204 245 255 396

100 82 80 99 45 99 110

71400 55600 8300 17500 8300 9200 10000

It was deduced from a detailed analysis of the complex between lysozyme and (NAG)6 by X-ray crystallography that Glu35 and Asp52, which lie close to the susceptible glycosidic bond between D and E, should be catalytic residues. The reaction mechanism was also proposed as follows. Glu35, which lies in a hydrophobic environment, participates in catalysis in prontonated form and Asp52, which lies in a hydrophilic environment, does so in dissociated form. As shown in Figure 2, Glu35 donates its proton to the oxygen linking DE sugars. The bond between this oxygen and CIon the D sugar is cleaved. A carbonium ion is formed on the D sugar and this ion is stabilized by the formation of an oxocarbonium ion. The distortion of the D sugar from the chair form to the sofa form favors this process. The negative charge on Asp52 stabilizes the formation of a positive charge on the D sugar. Thanks to these efforts to stabilize the transition state, lysozyme can hydrolyze glycosidic bonds smoothly (Phillips, 1966). Catalytic participations of Glu35 and Asp52 were confirmed by chemical mutations, where the residues were converted to the respective amides after specific esterifications and ammoniolyses. Purified Gln35 and Asn52 lysozyme showed little activity at pH 3.4, 5.5 and 8.0., although their binding abilities against (NAG)6 and (NAG)3 were almost equivalent to that of native lysozyme (Kuroki et aI., 1986a). The mutation of Glu35 to Asp or Asp52 to Glu in hen (Imoto, 1990) or human (Muraki et aI., 1991) lysozyme caused inactivation of the enzyme. Moreover, the double mutant Glu35Asp/ Asp52Glu was also inactive (Imoto, 1990). These observations mean that the catalytic groups are strictly located and that the movement of even one methylene unit is not allowed. Malcolm et aI. (1989) have shown that the mutant Asp52Asn exhibits approximately 5% of the wild-type activity, although the mutant Glu35Gln exhibits no measurable activity. This finding is consistent with the activity profiles of the above-mentioned derivatives modified at the catalytic residues. A careful examination of the activity of the mutant of Glu35 to GIn, His or Ala

169

Engineering of lysozyme

!

＠ｾ o

80

> !::: i= ()

«

60

> i= « ...J

40

It

20

w

w 0 2

3

4

5

6

7

8

pH

Figure 3. pH-dependence of hydrolytic activity of native (0) and Asp52Ala (.) lysozyme against (NAG)6 at 40°C (Hashimoto et aI., 1996).

mutant. Thus, it was clearly demonstrated that Asp52 enhances the catalytic activity four hundred times in hen lysozyme, although it was estimated from the reaction mechanism of goose lysozyme that Asp52 may not be required for the lysozyme activity (Weaver et aI., 1995). If Asp52 has participated as a nucleophile for a covalent intermediate, severe effects on the activity should have been observed. However, in the mutant Asp52Ser, minor peaks corresponding to covalent complexes between the mutant and oligosaccharides arose with electro spray mass spectrometry (Lumb et aI., 1992). In the mutant of Asp52Glu, the carboxylate came closer to the carbonium ion intermediate and, in the reaction of the mutant and (NAG)6' a stable covalent intermediate of a lysozyme-(NAG)4 complex could be obtained. However, because there is no system to remove the sugar intermediate, the hydrolysis of the intermediate was very slow (Ito et aI., 1995). The result of X-ray analysis of the complex between lysozyme and NAM-NAG-NAM revealed that Glu35 lies at 2.9 Afrom 01 ofthe D sugar and that they form a hydrogen bond. On the other hand, Asp52 is fixed with hydrogen bonding networks with Asn46 and Asn59 and the carboxyl oxygens cannot approach within 2.3 A of Cion the D sugar. Therefore formation of the covalent intermediate is improbable (Strynadka and James, 1991). This was also supported by recent X-ray analyses (Hadfield et aI., 1994; Song et aI., 1994; Weaver et aI., 1995). In chicken type lysozyme, the following mechanism seems to be plausible: Glu35 donates a proton to the glycosidic oxygen as a general base catalyst: Asp52 bearing a negative charge stabilizes the carbonium ion intermediate at the closest position that is not so close as to form a covalent intermediate. The distortion of the D sugar was reconfirmed by two recent X-ray analyses (Strynadka and James, 1991; Hadfield et aI., 1994). It was also

Engineering of lysozyme

171

confirmed in the T4 lysozyme (Kuroki et aI., 1993). The mutant Val109Pro in human lysozyme lost activity, although its binding ability to the substrate was not affected (Kikuchi et aI., 1988). This result supported the finding in X-ray analysis that the hydrogen bond of the main chain amide proton of Val109 stabilizes the binding of the sofa type D sugar. This is a good example of stable binding with a transition state substrate being an important factor in effective enzyme reaction. Two types of binding mode were presented for the binding of (NAg)6 from the energy calculation (Pincus and Scheraga, 1979). One is a left-sided binding mode in which EF sugars extended to the left side from the active site cleft, and which is a non-productive (ground state) binding mode. The other is a right-sided binding mode, which is similar to the binding mode presented by the X-ray analysis (Phillips, 1966), and which is a productive (transition state) binding mode with a distorted D sugar ring conformation. More than one binding mode was also estimated from the kinetic analyses of reaction pathways (Holler et aI., 1975; Banerjee et aI., 1975). Asn47 lies in the left-side binding site and the mutant Asn47Asp showed enhanced binding to the ground state substrate leading to a decreased activity. On the other hand, Asn36 lies in the bottom of the right-sided binding site and the mutant Asn36Gly showed increased activity (Inoue et aI., 1992c). This clearly showed that the stabilization of the ground state complex is not essential to the enzyme reaction and that the formation of a stable complex with the transition state substrate is. As you can see, the original proposal of the mechanism based on the X-ray analyses (Phillips, 1966) almost exactly predicted the known reaction mechanism of lysozyme. Glu35, which participates in catalysis in protonated form, has an abnormally high pKa of 6.1 and this enhances the catalytic efficiency. This abnormality is produced by the negative charge of Asp52 and by the surrounding hydrophobic environment, especially of Trp108 (Inoue et aI., 1992a,b). Fluctuation of protein conformation is considered to be important in enzyme reaction. This was elucidated by employing the Argl4Hisl5deleted lysozyme. The temperature-activity curve of the mutant was shifted to a lower temperature than that of the wild-type, and the H - D exchange of the NH-proton of indole of Trp63 was faster in the mutant than in the wild-type, although the structure around Trp63 was identical in each case (Imoto et aI., 1994).

Engineering to improve protein properties Proteins are the products of evolution over 3.5 billion years and have evolved almost to their maximum levels. However, the levels are based on their normal requirements and we can improve their characteristics

172

T.Imoto

according to new aims, for example if we use them for industrial purposes. Recent advances in gene engineering and cell technology have enabled us to produce proteins in large quantities. If you can produce a protein, you should produce one which has optimum properties. Alteration of pH optimum

Sometimes we can improve the enzyme properties by altering its pH optimum. As mentioned before, we can alter the pH dependence of lysozyme activity by modifying the surface charges. Lysozymes which can function at low pH in the stomach have evolved in the course of evolution by lowering their isoelectric points (Dobson et aI., 1984; Jolles et aI., 1984; Jolles et aI., 1989; Ito et aI., 1993). Alteration of specificity

As mentioned before, we can alter the cleavage pattern of oligosugars by modification of the binding site. However, neither the change from endo-type to exo-type, nor the production of an enzyme which acts on a substrate without an N-acetyl group, has been achieved. Lysozyme loses lytic activity with acetylation and becomes a simple chitinase. When lysine residues were modified with palmitic acid (Ibrahim et aI., 1991) or a hydrophobic peptide (PhePheVaIAlaPro) was attached to the C-terminal (Ibrahim et aI., 1992; Ibrahim et aI., 1994), the lysozyme derivative suppressed the growth of E. coli, a gram negative bacterium. Improving activity

The activity of the enzyme has evolved to almost its maximum and it is hard to improve it by modifying its binding or catalytic residues. However, as mentioned before, considerable success in improving the lytic activity of lysozyme has been achieved. Stabilization

Proteins are usually labile owing to their physiological requirements, and we can engineer more stable versions. We can improve the function of the proteins by stabilization because stabler proteins can work at elevated temperature where the functional efficiencies are high. Examples of stabilized proteins which showed improved functionality are shown in Figure 4.

173

Engineering of lysozyme

5

4 >-

I-

> IU

e:(

3

w

>

le:(

-l

w

2

0:::

1

-3

A-

30

40

50

80 90 TEMPERATURE (OC) 60

70

Figure 4. Temperature dependence of hydrolytic activity against glycol chitin. 0, native; e, cross-linked lysozyme as Lysl-CH zCONH(CH 2 )nNHCOCHz-HisI5, n = 2; 1'::., n = 4; D, n = 6. (Ueda et aI., 1985).

A hydrophobic group such as a phenyl or sugar derivative was covalently introduced on AsplOl and the resulting derivatives showed enhanced stabilities (Imoto et aI., 1987). Trp108 is a buried residue. When this residue was mutated to Tyr or GIn, the stability of the protein decreased according to the decreased hydrophobicity (Inoue et aI., 1992b). The combinations of the mutations Thr40Ser, Ile55Vai and Ser91 Thr which lie in the hydrophobic core were investigated. The stability of the protein was increased according to the increase in methyl groups in the hydrophobic core. The double mutant Thr40Ser and Ser9IThr showed a slight increase in stability while no increase in the methyl group was seen. This was explained by the fact that the strain on Ile55 was released by the mutation (Wilson et aI., 1992). A pair of two cysteines, 76 and 94, in human lysozyme were variously mutated and the stability was increased by filling the cavity or forming a hydrogen bond. The activities of the mutants against the synthetic substrate, (NAGkPNP, were higher than that of the wild-type human lysozyme, although the lytic activities against M. lysodeikticus were

174

T.lmoto

decreased in Cys76Leu/Cys94Ala and Cys76Ile/Cys94Ala and increased in Cys76Ala/Cys94Leu (Yamada et aI., I 994a). Electrostatic interactions stabilize proteins. Hisl5, which lies in a field of positive charges, was carboxymethylated and the derivative was stabilized (lmoto et aI., 1987). The C-terminal Leu129, which forms a salt bridge with Lys13, was deleted with carboxypeptidase and the derivative was destabilized (lmoto et aI., 1987). Glu35 lies in a constrained environment and showed an abnormal pKa of 6.1. This strained carboxyl residue was removed by site-directed mutagenesis and the mutants showed enhanced stabilities (Inoue et aI., 1992a). Proteins can be stabilized by introducing new ligand binding sites. The mutant human lysozyme Gln85Asp/Ala91Asp which was constructed imitating a-Iactoalbumin, a calcium binding protein, showed strong binding to calcium (KA = 5.0 X 106 M- 1) and it was more stable at 85°C than the wild-type by 1.9 kcal/mol (Kuroki et aI., 1992c). It was found that the enthalpic contribution for the Ca 2 + binding reaction was small and that the entropy release (10 kcal/mol) upon the binding of Ca2 +, which arises primarily from the release of bound water molecules hydrating the free Ca 2 +, was essential (Kuroki et aI., 1992b). The calcium-binding constants of wild type human lysozyme and of the mutants Ala91Asp and Ala82Lys/Gln85Asp/Asn87Asp/Ala9IAsp measured at 25°C and pH 7.5 were 2 x 102 , 8 X 103 and 9 x 106 M- 1, respectively (Haezebrouck et aI., 1993). Covalent cross-linkings can stabilize proteins by causing entropy losses in the denatured state of proteins. Various cross-lin kings were introduced at Glu35-Trp108 (lmoto et aI., 1987). Lysl-Hisl5 (Ueda et aI., 1985) and Trp62-AsplO1 (Ueda et aI., 1991) with chemical modifications and the derivatives showed enhanced stabilities. On the other hand, the cross-linking between Lysl3 and the C-terminal carboxyl of Leu129, which formed a salt bridge in the wild-type, caused some instability of the protein (lmoto et aI., 1987). These results showed that we should cross-link proper residues with a bridge of the proper length for effective stabilization. Cys6-CysI27, the most outside SS bridge of the four, was reduced and carboxymethylated. The derivative became unstable by about 30°C more than native (Cooper et aI., 1992). One SS bond was removed in human lysozyme by the mutation of Cys76Ala/Cys94Ala, and the mutant was less stable than the wild-type by 4.6 kcal/mol (Kuroki et aI., I 992a). The structures of the mutants Cys76Ala and Cys76Ala/ Cys94Ala in human lysozyme were investigated by differential scanning calorimetry and X-ray analysis, and it was shown that the effect of cross-linking on the stability of protein is not solely explained by the entropy change in denaturation (Kuroki et aI., 1992a). The dynamic structures of the wild type and the mutant Cys76Ala/Cys94Ala in human lysozyme were determined by normal mode refinement of the

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175

1.5 A resolution X-ray data. The mutant showed an increase in apparent fluctuations at most residues. It was postulated that the disulfide bond does not have any substantial role to play in the dynamic structure (Kidera et aI., 1994). The introduction of proline destabilizes the denatured state of proteins and stabilizes proteins. Rabbit lysozyme has five prolines while chicken-type lysozymes normally have two, and shows 6 degrees higher melting temperature than hen or human lysozyme (Ito et aI., 1990). To investigate the effects of proline, the mutants Pr070Gly, Pro102Gly, Asp90Pro, Ala47Pro and Vall09Pro in human lysozyme were constructed. A reasonable change in stability was obtained only in Pro70Gly, and this was because the other changes in stability overlapped these effects (Heming et aI., 1992). In the course of a trial of introducing Proline at Asp 10 1Gly 102 in hen lysozyme, the mutants Asp 10 1Pro 102 and Pro 101 Gly 102 could not attain stabilization owing to their strain, while the mutant Gly 10 1Pro 102 attained stabilization with no strain. The conformational restraint around proline is severe, particularly at the N-terminus of proline. Thus, we can introduce proline without severe strain if we bring glycine, which is the most restriction-free amino acid, to this position (Veda et aI., 1993). Coating a protein with high polymers is another way to stabilize it. Coating with polyethylene glycol is widely employed, and attaching polysaccharides is another choice. Amino acid residues 22, 67 or 117 in human lysozyme were mutated to Asn to produce the N-linked glycoslation signal sequence, and the glycosylation was tested using a cultured hamster cell expression system. A partial glycosylation was seen in 22 and 67 but not in the 117 mutant (Horst et aI., 1991). We can modify the glycosylation pattern by fusing the 22Asn-mutant with a special protein such as cathepsin D (Horst et ai., 1993). Residues 48, 67, 70 or 105 in hen lysozyme were mutated to produce the signal sequence, and the glycosylation was investigated using a yeast secretion system. Only the 48 mutant was expressed in the two types of glycosylated forms, a small oligomannose chain form and a large polymannose chain form, whereas other mutants were not glycosylated (Nakamura et aI., 1993b). The polymannosyl lysozyme exhibited excellent emulsifying properties superior to those of commercial emulsifiers in addition to heat stability, although the oligomannosyl lysozyme did not (Nakamura et aI., 1993a). Polymannosylation was also seen in the mutant Arg21Thr (AsnI9Tyr20Thr21) in hen lysozyme (Kato et aI., 1994). Sometimes it is important to stabilize proteins against irreversible denaturations. Proteolysis is one of the most important. Proteolysis usually proceeds via the denatured state (lmoto et aI., 1986). We must decrease the denaturation rate for protection from this sort of denaturation, that is, kinetic stabilization is essential. For kinetic stabilization, we should stabilize the protein at the site where the structure is most

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disrupted in the transItIon state for the denaturation. The way to determine this site was also described using lysozyme (Yamada et aI., 1993b). Multiple chemical reactions are accumulated in a protein at elevated temperature or during prolonged storage and the protein ultimately becomes denatured (Tomizawa et aI., 1994b). Some additives such as trifluoro ethanol were found to prevent the chemical reactions (Tomizawa et aI., 1995b), and thermodynamic stabilization helps to retain the native conformation of deteriorated proteins (Tomizawa et aI., 1994b). The mutations of chemically labile sequences, particularly AspGly, to stable sequences were found to be effective in lysozyme stabilization (Tomizawa et aI., 1995a).

Engineering a novel protein

To design and create a protein with a novel function is the ultimate goal of protein engineering. It is important to establish the fundamental knowledge needed for this purpose using well-characterized proteins such as lysozyme. In addition, lysozyme is a small and stable protein with a well-characterized active site cleft and we can employ this protein as a template for designing functionally novel proteins. To investigate the functional properties of the ArgGlyAsp (RGD) sequence in a cell adhesion protein, mutant proteins were constructed by inserting 4-12 amino acid residues from the RGD region of human fibronectin between Va173 and Asn74 of human lysozyme. The mutants with an 8-12 amino acid insertion showed about 10% of the adhesion activity of human fibronectin (Yamada et aI., 1993a). The activity was improved by introducing conformational constraint by forming a short loop SS bond like Cys(Gly) ArgGlyAsp(Ser) Cys (Yamada et aI., 1994b). a-Lactalbumin is homologous to lysozyme but does not exhibit lysozyme activity. Endowing a-lactalbumin from goats with lysozyme activity was attempted. Ten amino acids were mutated so as to resemble the active site of lysozyme, but no activity was detected. However, when the second exon of a-lactalbumin was exchanged with that of lysozyme (Trp28-Ser86) and Tyrl07 was mutated to Ala, or when the second and third exons were exchanged with those of lysozyme, the derivatives showed slight lysozyme activity (Kumagai et aI., 1992a). Constructing a smaller protein is desirable because a small protein can be synthesized chemically and because it is less antigenic. The exon 2 of hen lysozyme (residues 28-81) fused to the N-terminus of f)-galactosidase of E. coli showed about 1/40000 of the activity of wild-type lysozyme (Kuchinke, 1989). The loop region of 65-79 was replaced with a short loop of AsnGlySerAsn which was obtained from a data bank, and a smaller lysozyme was constructed. The truncated lysozyme showed 25% of the lytic activity of the wild-type, and the pH depen-

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dence of the activity was similar to that of the wild-type (Pickersgill et aI., 1994). The peptide fragment 36-105 was prepared by chemical and protease cleavage. It was shown that this peptide can form a stable higher-order structure including helix 88-98 (Ueda et aI., 1994). As a chemical approach to designing a protein, a template-assembled synthetic peptide (T ASP) is a good way to construct a novel protein framework because in this manner we can escape an entropic disadvantage in the folding of a long peptide (Mutter and Vuilleumier, 1989). Four copies of helix 87 -97 were assembled on a short template peptide, and the characteristics were examined (Vuilleumier and Mutter, 1993). If we are engineering proteins for medical uses, we should be very careful. Engineered proteins might be sometimes harmful to human bodies. Only one amino acid substitution (Ile55Thr or Asp66His) turned human lysozyme to an amyloid fibril protein (Pepys et aI., 1993).

References Banerjee, S.K., Holler, E., Hess, G.P. and Rupley, J.A. (1975) Reaction of N-acetylglucos amine oligosaccharide with lysozyme: temperature, pH and solvent deuterium isotope effect, equilibrium, steady state. pre-steady state measurement. J. Bioi. Chem. 250: 43554367. Blaber, M., Lindstrom, J.D., Gassner, N., Xu, J., Heinz, D.W. and Matthews, B.W. (1993) Energetic cost and structural consequences of burying a hydroxyl group within the core of a protein determined from Ala -- Ser and Val-- Thr substitutions in T4 lysozyme. Biochemistry 32: 11363 -11373. Cheetham, J.c., Artymiuk, P.J. and Phillips, D.C. (1992) Refinement of an enzyme complex with inhibitor bound at partial occupancy. Hen egg-white lysozyme and tri-N-acetylchitotriose at 1.75 A resolution. J. Mol. Bioi. 224: 613-628. Cooper, A., Eyles, SJ., Radford, S.E. and Dobson, C.M. (1992) Thermodynamic consequences of the removal of a disulphide bridge from hen lysozyme. J. Mol. BioI. 225: 939-943. Dobson, D.E., Prager, E.M. and Wilson, A.C. (1984) Stomach Iysozymes of ruminants. 1. Distribution and catalytic properties. J. BioI. Chem. 259: 11607-11616. Fukamizo, T., Hayashi, K. and Goto, S. (1986) The role of binding subsite A in reactions catalyzed by hen egg-white lysozyme. Eur. 1. Biochem. 158: 463-467. Haezebrouck, P., De-Baetselier, A., Joniau, M., Van-Dael, H., Rosenberg, S. and Hanssens, I. (1993) Stability effects associated with the introduction of a partial and complete Ca2+-binding site into human lysozyme. Protein Eng. 6: 643-649. Hadfield, A.T., Harvey, D,J., Archer, D,B., MacKenzie, D.A., Jeenes, D,J., Radford, S,E., Lowe, G" Dobson, C.M. and Johnson, L.N. (1994) Crystal structure of the mutant D52S hen egg white lysozyme with an oligosaccharide product. J. Mol. Bioi. 243: 856-872. Harata, K., Muraki, M. and Jigami, Y. (1993) Role of Argl15 in the catalytic action of human lysozyme. X-ray structure of Hisl15 and Glul15 mutants, J, Mol. BioI. 233: 524-535. Hashimoto, Y., Yamada, Y., Motoshima, H., Omura, T., Yamada, H., Yasukochi, T., Miki, T,. Veda, T. and Imoto, T, (1996) A mutational study of catalytic residue Asp52 in hen egg lysozyme. J. Biochem. 119: 145150. Hayashi, K., Imoto, T., Funatsu, G. and Funatsu, M. (1965) The position of the active tryptophan residue in lysozyme. J. Biochem. 58: 227-235. Heinz, D.W" Baase, W.A., Zhang, X.I., Blaber, M., Dahlquist, F.W. and Matthews, B.W. (1994) Accommodation of amino acid insertions in an alpha-helix of T4 lysozyme. Structural and thermodynamic analysis. J. Mol. BioI. 236: 869-886.

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Heming, T., Yutani, K., Inaka, K., Kuroki, R., Matsushima, M. and Kikuchi, M. (1992) Role of proline residues in human lysozyme stability: a scanning calorimetric study combined with X-ray structure analysis of proline mutants. Biochemistry 31: 7077-7085. Holler, E., Rupley, J.A. and Hess, G.P. (1975) Productive and nonproductive Iysozymechitosaccharide complexes. Biochemistry 14: 2377-2385. Horst, M., Harth, N. and Hasilik, A. (1991) Biosynthesis of glycosylated human lysozyme mutants. J. Bioi. Chem. 266: 13914-13919. Horst, M., Mares, M., Zabe, M., Hummel, M., Wiederanders, B., Kirschke, H. and Hasilik, A. (1993) Synthesis of phosphorylated oligosaccharides in lysozyme is enhanced by fusion to cathepsin D. J. Bioi. Chem. 268: 19690-19696. Ibrahim, H.R., Kato, A. and Kobayashi, K. (1991) Antimicrobial effects of lysozyme against gram-negative bacteria due to covalent binding of palmitic acid. J. Agric. Food Chem. 39: 2077-2082. Ibrahim, H.R., Yamada, M., Kobayashi, K. and Kato, A. (1992) Bacteriocidal action of lysozyme against gram-negative bacteria due to insertion of a hydrophobic pentapeptide into its C-terminus. Biosci. Biotech. Biochem. 56: 1361-1363. Ibrahim, H.R., Yamada, M., Matsushita, K., Kobayashi, K. and Kato, A. (1994) Enhanced bactericidal action of lysozyme to Escherichia coli by inserting a hydrophobic pentapeptide into its C terminus. J. Bioi. Chem. 269: 5059-5063. Imoto, T., Yamada, H. and Ueda, T. (1986) Unfolding rates of globular proteins determined by kinetics of proteolysis. J. Mol. Bioi. 190: 647-649. Imoto, T., Yamada, H., Okazaki, K., Ueda, T., Kuroki, R. and Yasukochi, T. (1987) Modifications of stability and function of lysozyme. J. Protein Chem. 6: 95-107. Imoto, T. (1990) Strategy to examine the function of hen lysozyme by protein engineering. In: Ikehara, M. (ed.): Protein Engineering. Japan Scientific Societies Press & Springer-Verlag, pp 153-158. Imoto, T., Ueda, T., Tamura, T., Isakari, Y., Abe, Y., Inoue, M., Miki, T., Kawano, K. and Yamada, H. (1994) Lysozyme requires fluctuation of the active site for the manifestation of activity. Protein Eng. 7: 743-748. Inoue, M., Yamada, H., Hashimoto, Y., Yasukochi, T., Hamaguchi, K., Miki, T., Horiuchi, T. and Imoto, T. (1992a) Stabilization of a protein by removal of unfavorable abnormal pKa: substitution of undissociable residue for glutamic acid-35 in chicken lysozyme. Biochemistry 31: 8816-8821. Inoue, M., Yamada, H., Yasukochi, T., Kuroki, R., Miki, T., Horiuchi, T. and Imoto, T. (l992b) Multiple role of hydrophobicity of tryptophan-I08 in chicken lysozyme: structural stability, saccharide binding ability, and abnormal pKa of glutamic acid-35. Biochemistry 31: 5545-5553. Inoue, M., Yamada, H., Yasukochi, T., Miki, T., Horiuchi, T. and Imoto, T. (l992c) Left-sided substrate binding of lysozyme: evidence for the involvement of asparagine-46 in the initial binding of substrate to chicken lysozyme. Biochemistry 31: 10322-10330. Ito, Y., Yamada, H., Nakamura, S. and Imoto, T. (1990) Purification, amino acid sequence, and some properties of rabbit kidney lysozyme. J. Biochem. 107: 236-241. Ito, Y., Yamada, H., Nakamura, M., Yoshikawa, A., Ueda, T. and Imoto, T. (1993) The primary structures and properties of non-stomach Iysozymes of sheep and cow, and implications for functional divergence of lysozyme. Eur. J. Biochem. 213: 649-658. Ito, Y., Ogata, Y., Hashimoto, Y., Kuroki, R. and Imoto, T. (1995) Formation and decomposition of a stable covalent-linked intermediate with substrate in hydrolytic reaction of D52E hen lysozyme. Protein Eng. 8: 962. Jolles, P., Schoentgen, F., Jolles, J., Dobson, D.E., Prager, E.M. and Wilson, A.C. (1984) Stomach lysozyme of ruminants II. Amino acid sequence of cow lysozyme 2 and immunological comparisons with other Iysozymes. J. Bioi. Chem. 259: 11617-11625. Jolles, J., Jolles, P., Bowman, B.H., Stewart, c.B. and Wilson, A.C. (1989) Episodic evolution in the stomach Iysozymes of ruminants. J. Mol. Evol. 28: 528-535. Kato, A., Takasaki, H. and Ban, M. (1994) Polymannosylation to asparagine-19 in hen egg white lysozyme in yeast. FEBS Lett. 355; 76-80. Kidera, A., Inaka, K., Matsushima, M. and Go, N. (1994) Response of dynamic structure to removal of a disulfide bond: normal mode refinement of C77AjC95A mutant of human lysozyme. Protein Sci. 3: 92-102.

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Kikuchi, M., Yamamoto, Y., Taniyama, Y., Ishimaru, K., Yoshikawa, W., Kaisho, Y. and Ikehara, M. (1988) Secretion in yeast of human Iysozymes with different specific activities created by replacing valine-1I0 with proline by site-directed mutagenesis. Proc. Natl. Acad. Sci. USA 85: 9411-9415. Kuchinke, W. (1989) 50 residues coded by ex on 2 of chicken lysozyme carry residual catalytic activity. Biochem. Biophys. Res. Commun. 159: 927-932. Kumagai, I. and Miura, K. (1989) Enhanced bacteriolytic activity of hen egg-white lysozyme due to conversion of Trp62 to other aromatic amino acid residues. 1. Biochem. 105: 946-948. Kumagai, I., Takeda, S. and Miura, K. (1992a) Functional conversion of the homologous proteins alpha-lactalbumin and lysozyme by exon exchange. Proc. Nat!. A cad. Sci. USA 89: 5887 -5891. Kumagai, I., Sunada, F., Takeda, S. and Miura, K. (1992b) Redesign of the substrate-binding site of hen egg white lysozyme based on the molecular evolution of C-type Iysozymes. 1. BioI. Chem. 267: 4608-4612. Kumagai, I., Maenaka, K., Sunada, F., Takeda, S. and Miura, K. (1993) Effects of subsite alterations on substrate-binding mode in the active site of hen egg-white lysozyme. Eur. 1. Biochem. 212: 151-156. Kuroki, R., Inaka, K., Taniyama, Y., Kidokoro, S., Matsushima, M., Kikuchi M. and Yutani, K. (I 992a) Enthalpic destabilization of a mutant human lysozyme lacking a disulfide bridge between cysteine-77 and cysteine-95. Biochemistry 31: 8323-8328. Kuroki, R., Kawakita, S., Nakamura, H. and Yutani, K. (1992b) Entropic stabilization of a mutant human lysozyme induced by calcium binding. Proc. Natl. A cad. Sci. USA 89: 6803-6807. Kuroki, R., Nitta, K. and Yutani, K. (l992c) Thermodynamic changes in the binding ofCa2 i to a mutant human lysozyme (086/92). Enthalpy-entropy compensation observed upon Ca2 + binding to proteins. 1. Bioi. Chem. 267: 24297 -24301. Kuroki, R., Weaver, L., H. and Matthews, B.W. (1993) A covalent enzyme-substrate intermediate with saccharide distortion in a mutant T4 lysozyme. Science 262: 20302033. Kuroki, R., Yamada, H., Moriyama, T. and Imoto, T. (l986a) Chemical mutations of the catalytic carboxyl groups in lysozyme to the corresponding amides. 1. BioI. Chem. 261: 13571-13574. Kuroki, R., Yamada, H. and Imoto, T. (l986b) Specific carbodiimide-binding mechanism for the selective modification of the aspartic acid-l OJ residue of lysozyme in the carbodiimideamine reaction. 1. Biochem. 99: 1493-1499. Lumb, K.J., Aplin, R.T., Radford, S.E., Archer, D.B., Jeenes, D.J., Lambert, N., MacKenzie, D.A., Dobson, CM. and Lowe, G. (1992) A study of D52S hen lysozyme-GlcNAc oligosaccharide complexes by NMR spectroscopy and electrospray mass spectrometry. FEBS Lett. 296: 153-157. Maenaka, K., Kawai, G., Watanabe, K., Sunada, F. and Kumagai, I. (1994) Functional and structural role of a tryptophan generally observed in protein-carbohydrate interaction. TRP-62 of hen egg white lysozyme. 1. Bioi. Chem. 269: 7070- 7075. Malcolm, B.A., Rosenberg, S., Corey, M.J., Allen, J.S., DeBaetselier, A. and Kirsch, A.F. (1989) Site-directed mutagenesis of the catalytic residues Asp-52 and Glu-35 of chicken egg white lysozyme. Proc. Natl. A cad. Sci. USA 86: 133-137. Muraki, M., Harata, K., Hayashi, Y., Machida, M. and Jigami, Y. (1991) The importance of precise positioning of negatively charged carboxylate in the catalytic action of human lysozyme. Biochim. Biophys. Acta. 1079: 229-237. Muraki, M., Harata, K. and Jigami, Y. (1992) Dissection of the functional role of structural elements of tyrosine-63 in the catalytic action of human lysozyme. Biochemistry 31: 9212-9219. Muraki, M., Jigami, Y., Morikawa, M. and Tanaka, H. (l987a) Engineering of the active site of human lysozyme: conversion of aspartic acid 53 to glutamic acid and tryosine 63 to tryptophan or phenylalanine. Biochim. Biophys. Acta 911: 376·-380. Muraki, M., Morikawa, M., Jigami, Y. and Tanaka, H. (1987b) The roles of conserved aromatic amino-acid residues in the active site of human lysozyme: a site-specific mutagenesis study. Biochim. Biophys. Acta 916: 66- 75.

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Muraki, M., Morikawa, M., Jigami, Y. and Tanaka, H. (1988) Engineering of human lysozyme as a polyelectrolyte by the alteration of molecular surface charge. Protein Eng. 2: 49-54. Muraki, M., Morikawa, M., Jigami, Y. and Tanaka, H. (1989) A structural requirement in the subsite F of lysozyme. The role of arginine 115 in human lysozyme revealed by site-directed mutagenesis. Eur. J. Biochem. 179: 573-579. Mutter, M. and Vuilleumier, S. (1989) A chemical approach to protein design-template-assembled synthetic proteins (TASP). Angew. Chem. Int. Ed. Engl. 28: 535-554. Nakamura, S., Kobayashi, K. and Kato, A. (1993a) Novel surface functional properties of polymannosyl lysozyme constructed by genetic modification. FEBS Lett. 328: 259-262. Nakamura, S., Takasaki, H., Kobayashi, K. and Kato, A. (1993b) Hyperglycosylation of hen egg white lysozyme in yeast. J. Bioi. Chem. 268: 12706-12712. Pepys, M.B., Hawkins, P.N., Booth, D.R., Vigushin, D.M., Tennent, G.A., Soutar, A.K., Totty, N., Nguyen, 0., Blake, C.C.F., Terry, C.J., Feest, T.G., Zalin, A.M. and Hsuan, J.J. (1993) Human lysozyme gene mutations cause hereditary systemic amyloidosis. Nature 362: 553-557. Phillips, D.C. (1966) The three-dimensional structure of an enzyme molecule. Sci. Amer. 215: 78-90. Pickersgill, R., Varvill, K., Jones, S., Perry, B., Fischer, B., Henderson, I., Garrard, S., Sumner, I. and Goodenough, P. (1994) Making a small enzyme smaller; removing the conserved loop structure of hen lysozyme. FEBS Lett. 347: 199-202. Pincus, C.R. and Scheraga, H.A. (1979) Conformational energy calculations of enzyme-substrate and enzyme-inhibitor complexes of lysozyme: 2. Calculation of the structures of complexes with flexible enzyme. Macromolecules 12: 633-644. Pjura, P., Matsumura, M., Baase, W.A. and Matthews, B.W. (1993) Development of an in vivo method to identify mutants of phage T41ysozyme of enhanced thermostability. Protein Sci. 2: 2217-2225. Song, H., Inaka, K., Maenaka, K. and Matsushima, M. (1994) Structural changes in human lysozyme co-crystallized with hexa-N-acetyl-chitohexaose at pH 4.0. J. Mol. Bioi. 244: 522-540. Strynadka, N.C. and James, M.N. (1991) Lysozyme revisited: crystallographic evidence for distortion of an N-acetylmuramic acid residue bound in site D. J. Mol. Biol. 220: 401-424. Tomizawa, H., Yamada, H., Ueda, T. and Imoto, T. (1994a) Isolation and characterization of 101-succinimide lysozyme that possesses the cyclic imide at AsplOl-GlyI02. Biochemistry 33: 8770-8774. Tomizawa, H., Yamada, H. and Imoto, T. (1994b) The mechanism of irreversible inactivation of lysozyme at pH 4 and 100 degrees C. Biochemistry 33: 13032-13037. Tomizawa, H., Yamada, H., Hashimoto, Y. and Imoto, T. (1995a) Stabilization of lysozyme against irreversible inactivation by alterations of Asp-Gly sequences. Protein Eng. 8: 1023- 1028. Tomizawa, H., Yamada, H., Wada, K. and Imoto, T. (1995b) Stabilization of lysozyme against irreversible inactivation by suppression of chemical reactions. J. Biochem. 117: 635-640. Ueda, T., Nakashima, A., Hashimoto, Y., Miki, T., Yamada, H. and Imoto, T. (1994) Formation of alpha-helix 88-98 is essential in the establishment of higher-order structure from reduced lysozyme. J. Mol. Bioi. 235: 1312-1317. Ueda, T., Tamura, T., Maeda, Y., Hashimoto, Y., Miki, T., Yamada, H. and Imoto, T. (1993) Stabilization of lysozyme by the introduction of Gly-Pro sequence. Protein. Eng. 6: 183-187. Ueda, T., Yamada, H., Hirata, M. and Imoto, T. (1985) An intramolecular cross-linkage of lysozyme. Formation of cross-links between lysine-I and histidine-IS with bis(bromoacetamide) derivatives by two-stage reaction procedure and properties of the resulting derivatives. Biochemistry 24: 6316-6322. Ueda, T., Yamada, H., Sakamoto, N., Abe, Y., Kawano, K., Terada, Y. and Imoto, T. (1991) Preparation and properties of a lysozyme derivative in which two domains are cross-linked inttamolecularly between Trp62 and AsplOI. J. Biochem. 110: 719-725. Vuilleumier, S. and Mutter, M. (1993) Synthetic peptide and template-assembled synthetic protein models of the hen egg white lysozyme 87-97 helix: importance of a protein-like framework for conformational stability in a short peptide sequence. Biopolymers 33: 389-400.

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Weaver, L.H., Grutter, M.G. and Matthews, B.W. (l995) The refined structures of goose lysozyme and its complex with a bound trisaccharide show that the 'goose-type' lysozyme lacks a catalytic aspartate residue. J. Mol. Bioi. 145: 54-68. Wilson, K.P., Malcolm, B.A. and Matthews, B.W. (1992) Structural and thermodynamic analysis of compensating mutations within the core of chicken egg white lysozyme. J. Bioi. Chem. 267: 10842-10849. Yamada, H., Kanaya, E., Inaka, K., Ueno, Y., Ikehara, M. and Kikuchi, M. (1994a) Stabilization and enhanced enzymatic activities of a mutant human lysozyme C77/95A with a cavity space by amino acid substitution. Bioi. Pharm. Bull. 17: 192-196. Yamada, T., Matsushima, M., Inaka, K., Ohkubo, T., Uyeda, A., Maeda, T., Titani, K., Sekiguchi, K. and Kikuchi, M. (1993a) Structural and functional analyses of the Arg-GlyAsp sequence introduced into human lysozyme. J. Bioi. Chem. 268: 10588-10592. Yamada, H., Ueda, T. and Imoto, T. (1993b) Thermodynamic and kinetic stabilities of hen-egg lysozyme and its chemically modified derivatives: analysis of the transition state of the protein unfolding. J. Biochem. 114: 398-403. Yamada, T., Uyeda, A., Kidera, A. and Kikuchi, M. (1994b) Functional analysis and modeling of a conformationally constrained Arg-Gly-Asp sequence inserted into human lysozyme. Biochemistry 33: 11678-11683. Yamasaki, N., Hayashi, K. and Funatsu, M. (1968) Acetylation of lysozyme: Part II. Mechanism of lysis by lysozyme. Agric. Bioi. Chem. 32: 64-68.

Lysozyme: A model enzyme in protein crystallography

Lysozymes: Model Enzymes in Biochemistry and Biology

ed. by P. Jolles

© 1996 Birkhauser Verlag Basel/Switzerland

Lysozyme: A model enzyme in protein crystallography N.C.J. Strynadka and M.N.G. James MRC Group in Protein Structure and Function, Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada Summary. The review concentrates on the crystal structure results from several protein crystallography laboratories on three different Iysozymes, the type-c Iysozymes such as hen egg-white lysozyme (HEWL), the type-g lysozyme, such as goose egg-white lysozyme (GEWL), and the lysozyme from T4 bacteriophage (T4L). The crystallographic studies on HEWL in several different crystal forms have shown that the lysozyme molecule is relatively rigid with the residues of the active site Glu35 and Asp52 adopting almost identical conformations in all structures and species variants. The NMR results also confirm the presence of a similar conformation of HEWL in solution. All three enzymes, HEWL, GEWL and T4L are composed of two domains, one that is predominantly IX-helical and a smaller domain that is mainly Ii-sheet in nature. The general acid/general base residue in each lysozyme (Glu35 in HEWL, Glu73 in GEWL and Glull in T4L) is contributed by the larger ex-helical domain. The Ii-sheet domains of HEWL and T4L contribute an aspartate to their respective active sites, which is likely involved in electrostatic stabilization of the oxycarbonium ion intermediate of the site D sugar on the hydrolytic pathway of oligosaccharides. There is no analogous aspartate carboxylate group in GEWL although minor conformational changes could position one or other of Asp86 or Asp97 for such a stabilization role. The binding of substrate analogues, transition state mimics and oligosaccharide products of hydrolysis to HEWL, GEWL and T4L have contributed greatly to our understanding of sugar binding to proteins. The observed subtle conformational differences of the free vs bound forms of these enzymes are best described by a narrowing of the active site clefts in the presence of the inhibitors. Details of the binding interactions of those residues lining the oligosaccharide binding clefts of the three enzymes HEWL, GEWL and T4L with the sugar residues in sites A, B, C and D are presented and discussed. Oligo saccharides of (GlcNAc)n and alternating MurNAc-GlcNAc-MurNAc have been bound to these three enzymes and the structures determined at high resolution. These binding studies have contributed greatly to our understanding of the catalytic mechanism of the lysozyme glycosidase activity. The currently accepted view of this mechanism is presented and discussed in this review.

Introduction Lysozyme from hen egg white (HEWL) was the first enzyme to have its three-dimensional structure determined by X-ray crystallography (Phillips, 1966; Blake et aI., 1965, 1967a,b). This structure provided an extraordinary insight into the realm of protein architecture that had been dominated until then by the structures of myoglobin (Nobbs et aI., 1966) and hemoglobin (Perutz et aI., 1968). Thus, lysozyme gave the first view of a region of f3-sheet in a globular protein, a feature not found in the structures of the two all IX-helical 02-binding proteins. In addition, the structure of HEWL provided a paradigm for the binding

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of substrate analogues to an enzyme's active site (Johnson and Phillips, 1965; Ford et al., 1974). The structures of the complexes of oligosaccharides of N-acetylglucosamine [(GLcNAc)n, where n = 1,2, or 3] with HEWL showed that interactions between a substrate and its cognate enzyme were relatively few in number and comprised non-covalent weak forces such as van der Waals interactions, hydrogen bonds and a few electrostatic attractions. These structural studies paved the way for a possible catalytic mechanism for HEWL that has withstood the tests of time and kinetics, albeit some of the fine details of the mechanism are still debated (Sinnott, 1990; Strynadka and James, 1991). The molecular structure of HEWL and its determination by the methods of X-ray crystallography have been truly inspirational. One of us (MNGJ) had the pleasure of being present at the Royal Society Discussion meeting held at the Royal Institution of Great Britain on February 3, 1966. Following an introductory lecture by Sir W.L. Bragg, the audience was led through the chemical determination of the aminoacid sequences of the lysozymes by Professor Jolles, to the unveiling of the HEWL model structure by David Phillips, Colin Blake and Tony North. The model was beautifully highlighted to show hydrophobic residues on the inside of the molecule, hydrophilic residues on the outside, the disposition of acidic and basic residues, and key residues lining the active site. Dr Louise Johnson presented her results showing how oligo saccharides bound in lysozymes' cleft. The return to Oxford that night passed rapidly with animated discussions of whether static snapshots of enzyme structures contributed to our understanding of how rapidly they catalyzed reactions. HEWL has been the subject of many additional studies centred on the elucidation of its catalytic mechanism. Chemical mimics of proposed transition state structures have been bound to lysozyme and the structures analyzed (Ford et al., 1974). The structure of HEWL with the trisaccharide MurNAc-GLcNAc-MurNAc bound in the activity site has provided direct experimental evidence of distortion of the sugar residue in site D likely contributing to the catalytic rate enhancement (Strynadka and James, 1991). The structure of HEWL has been determined at high pressures (Kundrot and Richards, 1987). This work showed that protein structures have compressibilities similar to those of organic solids. Theoretical studies of protein structures have used HEWL as a prototype for many calculations. It has been the subject of Monte Carlo simulations which showed how water molecules behave near protein surfaces (Hagler and Moult, 1978). Molecular dynamics simulations have used the distribution of crystallographically determined B-factors as a benchmark to evaluate the simulations (Levitt et al., 1985). These methods are capable of giving similar distributions. More recent molecular dynamics simulations of HEWL in the presence of a hexasaccha-

Lysozyme: A model enzyme in protein crystallography

187

ride substrate have given rise to suggested alternative catalytic mechanisms involving cleavage of the pyranose ring of the saccharide (GlcNAc) bound in site D (Post et aI., 1986; Post and Karplus, 1986). This mechanism has been hotly contested by the structural results from high resolution structures of HEWL with bound products (Strynadka and James, 1991). The structures of Fab fragments of monoclonal antibodies raised against HEWL have provided a paradigm for the recognition of a protein antigen by an antibody (Mariuzza et aI., 1987). In the several complexes that exhibit different epitopes on the surface of lysozyme there are no large changes in the structures of the complementarity determining regions of the antibodies or in lysozyme upon forming the tight-binding molecular complexes. Crystallographic studies of other muramidases have been inspired by the structural work on lysozyme. The phage T4 lysozyme (T4L) structure has become a playground for the analysis of the effects of mutations on the structural stability of that molecule (Matthews, 1993). Other lysozyme structures such as goose egg-white lysozyme (GEWL) have suggested that a second ionized carboxylate (analogous to Asp52 of HEWL or of Asp20 in T4 lysozyme) may not be required for catalytic activity (Weaver et aI., 1995). Notwithstanding these results on a related lysozyme, mutations of HEWL (Malcolm et aI., 1989) and of human lysozyme (HL) (Harata et aI., 1992) confirm that Asp52 is an important residue in the mechanism. The present chapter will review some of the more recent crystallographic results on HEWL and related muramidases. It will also review how the several structures of substrate analogues bound to these lysozymes have contributed to furthering our understanding of their catalytic mechanisms. The only uncontested role for a residue in the active site is the general acid-general base role of Glu35 (Glu73 in GEWL or Glul1 in T4L) in providing a proton to the bridging oxygen atom between the saccharide residues bound in sites D and E. Transition state stabilization of the oxycarbonium intermediate by a specific residue may only be required by some lysozymes. Lysozyme - The archetypal crystal The wonderful ability of HEWL to crystallize easily and reproducibly has made it the focus of a massive number of studies designed to probe the physical processes involved in the formation of protein crystals (Giege and Ducruix, 1992). In addition, there has been recent work aimed at utilizing HEWL as a catalyst for the crystallization of a secondary peptide or protein of interest. For example, creation of a HEWL-integrin construct successfully led to crystals in which the conformation of the attached integrin peptide could be discerned (Donahue et aI., 1994).

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HEWL, HL and T4L are part of a small group of proteins for which there has been high resolution determinations of the same structure in several different crystal forms (Kurachi et al., 1976; Jensen, 1992; Zhang and Matthews, 1994; Song et al., 1994). These studies have provided valuable information regarding the effects of crystal packing forces in X-ray crystallographic structures and regarding the conservation of the positions of ordered solvent molecules. In T4L, for example, site-directed mutants often crystallize in unique crystal forms. An analysis of ordered solvent molecules of T4L mutants from ten different crystal forms showed that despite different crystallization conditions, variable crystal contacts, changes due to mutation, and varying attention to solvent during crystallographic refinement, 62% of the 20 most frequently occupied sites were conserved (Zhang and Matthews, 1994). How comparable is lysozyme in the crystalline state to that in solution? A number of studies have now verified that various lysozymes retain their activity within the constraints of the crystal lattice (Hadfield et al., 1994; Howell et al., 1992). A comparison of the three-dimensional structure of HEWL via X-ray crystallography and NMR (Smith et al., 1993) indicates a high degree of structural similarity with a root mean square deviation (rmsd) on the 516 main chain atoms of 1.8 A. Finally, HEWL crystals have been used to follow collective motions in proteins through the modeling of X-ray diffuse scattering effects (Mizuguchi et al., 1994; Clarage et al., 1992). These studies suggest that the global low-order motions one would expect of the protein in solution are sustained even within the crystalline lattice. Overall folds of various lysozyme species

A summary of the currently determined three-dimensional structures of various lysozymes having the HEWL fold is given in Table 1. As previously determined from their primary sequences, catalytic activity and immunologic cross-reactivity (Jolles and Jolles, 1984), these structures fall into three distinct classes of endo-N-acetylmuramidases; that for which hen egg white lysozyme (HEWL) is the prototype, that of goose egg white lysozyme (GEWL), and that of the bacteriophage T4 lysozyme (T4L). Very recently the three-dimensional structure of a novel bacterial muramidase, the periplasmic, soluble lytic transglycosylase (SLT) from Escherichia coli has been determined (Thunnissen et al., 1994). The C-terminal domain (residues 451-618) of this 70000 dalton protein exhibits a lysozyme-like fold that can be successfully superimposed onto the structures of HEWL and T4L. As a result of this superposition and biochemical data, Glu478 has been identified as the probable catalytic general acid analogous to Glu35 of HEWL. Coordinates of SLT have

Lysozyme: A model enzyme in protein crystallography

189

Table I. X-ray crystallographic structures of native Iysozymes Protein name

Space group

Resolution

----------------

-

R-factor (%)2 ---

---

P4 3 2,2 P4 3 21 2

1.6 1.7

16.1 (6-1.6) 14.8 (8-1.7)

P4 3 2 1 2

2.1

16.2 (10-21)

P2 1 PI

2.5 2.0

26.0 (5-2.5) 12.4

Pheasant Guinea hen Japanese quail Human (HL)

P2 1 2 1 2, P2 1 P6 1 22 P4 3 2,2 P6 1 22 C2 P2 1 2,2,

2.0 1.5 2.2 2.1 1.9 1.4 1.5

23.7 18.4 19.2 17.8 17.0 16.5 17.7

Goose (GEWL) T4L

P2 1 P3 2 21

1.6 1.7

15.0 15.7 (6-1.7)

HEWL

Turkey

pdb code l

Reference

(5-2.5) (10-2.2) (6.0-2.1) (6.0-1.9) (6.0-1.4) (10-1.5)

Maenaka et aI., 1995 Kundrot & Richards, 1987 Kodandapani et aI., 1990 Rao et aI., 1983 Ramanadham et aI., 1990 Berthou et aI., 1983 Harata et aI., 1993 Parsons, 1988 Lescar et aI., 1994 Lescar et aI., 1994 Houdousse, 1992 Artymiuk and Blake, 1981 Weaver et aI., 1995 Weaver and Matthews, 1987

ILZA 2LYM 4LYM ILYM 2LZT ILZ3 2LZ2 1GHL IHHL 2IHL 1LZ1 153L 3LZM

- - -

'Brookhaven National Laboratory Protein Data Bank Code for coordinate deposition (Bernstein et aI., 1977). 2Where R = l:llFol-IFcll/l:IFol; the range of data used in the calculation of the R-factor is given if quoted in the reference.

Table 2. Root mean square superpositions of the HEWL-related Iysozymes l Hen ------

Hen Turkey Quail Pheasant Guinea hen Human

--

Turkey -

0.53 0.53 0.97 0.51 0.88 0.77

Quail

Pheasant --

--

1.00 0.75 0.78 0.89

0.97 1.00 1.0 1.27 1.09

0.51 0.75 1.0 0.99 0.81

Guinea hen -

---

0.88 0.78 1.27 0.99

Human --------

0.77 0.89 1.09 0.81 1.06

1.06

IBased on 516 common main chain atoms; values are in A units. Coordinates used are hen (ILZA), turkey (I LZ3), quail (2IHL), pheasant (lGHL), guinea hen (IHHL) and human (lLZI) (see Tab. I).

not been released to the Protein Data Bank and thus this structure will not be discussed in detail in this review. It has long been recognized that lysozymes share significant sequence identity with the a-lactalbumin family of proteins suggesting that they have common structures (Browne et aI., 1969; McKenzie and White, 1991). This suggestion has been subsequently borne out through the determination of the X-ray crystallographic structure of a-lactalbumin (Acharya et aI., 1989, 1990). More recently, Holm and Sanders (1994) have identified significant structural similarity between a representative

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from the chitinase family of N-acetylglucosidase enzymes (Hart et aI., 1993) and all three classes oflysozymes HEWL, GEWL and T4L (Holm and Sanders, 1994). This structural homology arises despite the relative lack of any sequence identity between the chitinases and the other families of enzymes. Hen egg white lysozyme

This group of lysozyme structures includes those enzymes having from 127 to 130 amino acids that have been isolated from hen, turkey, pheasant, guinea hen, quail, pigeon, horse, and human (see Tab. 1 for references). The mixed al fJ fold common to all of these structures is shown diagrammatically in Figure la and b. A deep cleft containing the active site divides the molecule into two domains; one of them is almost entirely fJ-sheet structure (encompassing residues 40-85), whereas the other is comprised of the N- and C-terminal segments (residues 1-39, and 101-129) and is more helical in nature. The two domains are linked by an a-helix (residues 89-99). HEWL has disulfide bridges formed by the cysteine residue pairs (6,127), (30,115), (76,94), and (64,80). The molecular dimensions of the enzyme are approximately 36 A x 45 A x 42 A. The two proposed catalytic residues, Glu35 and Asp52, protrude from opposite sides of the active site. Figure 2a gives an electrostatic surface potential diagram of HEWL. It is clear that the active site cleft is largely electronegative in character. The overall fold in this class of Iysozymes is highly conserved (Tab. 2). Superimpositions of the common main chain atoms betwene each of the variant species and that from HEWL range from 0.51 to 0.97 A. The largest root mean square (rms) deviation on the 516 equivalent atoms is between that of hen and quail (0.97 A). Figure 3 shows an overlap of all the currently available, refined, high resolution lysozyme structures that fall within the HEWL class. Note the high degree of conservation in the position of the catalytic glutamic acid 35. The regions of greatest variability in the structures are in the vicinity of residues 45-50, 66-73, 100-102,118-122 and at the C-terminus, residues 126-129. Flexibility in several of these regions was predicted from the calculated atomic fluctuations derived from molecular dynamics simulations (Post et aI., 1986). The crystal structure determinations of the calcium binding lysozyme from horse (Tsuge et aI., 1992) and pigeon (Yao et aI., 1992) have

Figure 1. Molscript ribbon diagrams (Kraulis, 1991) depicting the backbone folds of the lysozyme families. (a) HEWL, view facing into the active site. (b) HEWL (Maenaka et aI., 1995); the view is approximately 90' from (a) and highlights the four disulfide bridges. (c) GEWL (Weaver et aI., 1995). (d) T4L (Grutter et aI., 1983).

Lysozyme: A model enzyme in protein crystallography (a)

(b)

(e)

(d)

191

192

N.C.l. Strynadka and M.N.G. lames

Figure 2(a). Surface depiction (GRASP; Nicholls et aI., 1991) of the lysozyme families. HEWL with the tetra-NAG oligosaccharide bound (Maenaka et aI., 1995).

recently been reported. These structures closely maintain the expected HEWL tertiary fold. The only significant structural difference lies in the surface loop (86 to 92) which binds the calcium ion. This calcium binding loop is analogous to that found in the a-lactalbumin family of proteins (Acharya et aI., 1989). Coordinates for these two structures are currently unavailable from the PDB and thus are not included in Table 1.

Lysozyme: A model enzyme in protein crystallography

193

Figure 2(b). Surface depiction (GRASP; Nicholls et aI., 1991) of the lysozyme families. Human lysozyme with two saccharide fragments bound (NAG)4 and (NAGh in sites A-D and E, F (Maenaka et aI., 1995).

The crystallographic structure of HEWL has been multiply-determined under a wide variety of crystallization conditions. These include a tetragonal form (Blake et aI., 1965), a triclinic form (Joynson et aI., 1970; Hodgson et aI., 1990; Moult et aI., 1976), a monoclinic form (Hogle et a1., 1981) and an orthorhombic form (Berthou et aI., 1983) of the structure (see Tab. 1). In addition, the structure of HEWL has been determined from tetragonal crystals under a variety of conditions of temperature (Young et aI., 1994), pressure (Kundrot and Richards,

194

N.C.J. Strynadka and M.N.G. James

Figure 2(c). Surface depiction (GRASP; Nicholls et aI., 1991) of the lysozyme famili es. GEWL with the trisaccharide (NAGh bound (Weaver et aI. , 1995).

1987), and humidity (Kachalova et aI., 1991; Kodandapani et aI., 1990). The structural basis for the interaction of urea with HEWL (Pike and Acharya, 1994), and the effects of reductive methylation of lysine residues in HEWL (Rypneiwski et aI., 1993) have also been examined at the atomic level using X-ray crystallography. All of these studies indicate that the conformation of lysozyme is essentially the same under the different conditions and crystalline environments. The molecular structure of the high-temperature (40°C) orthorhombic crystal form of HEWL was determined at 2.0 A resolution in an attempt to understand the differences in catalytic behaviour and inhibitor binding of the high-temperature (physiological) form relative to the low-temperature ("" 20°C) form of enzyme (Berthou et aI., 1983). The temperature of the intensity data collection was not reported, however; it is assumed to be ambient ( "" 20°C). Comparisons between the refined orthorhombic and tetragonal structures showed the hightemperature and low-temperature forms were quite similar with an rms difference on the 129C"-atom positions of 0.46 A. Three regions of the

Lysozyme: A model enzyme in protein crystallography

195

Figure 2(d). Surface depiction (GRASP; Nicholls ct aI. , 1991) of the lysozyme families. T4L with the cell wall product (NAG-NAM-L-Ala-D-Glu-Diaminopimellic acid-DAlal bound (Kuroki et aI. , 1993).

molecule exhibited C"-atom differences greater than two times the rms difference: the f3 -hairpin loop from Asn46 to Ser50, the loop Thr69 to Leu75, and the extended chain segment from Val99 to Ala107. Two of these regions are important in substrate binding, 46 to 50 and 99 to 107. The former is important for interactions in subsite D; the latter spans subsites A, Band C. These conformational differences may be responsible for the altered activity at high temperature (Jolles and Jolles, 1984), although the authors also attributed the observed differences to crystal packing interactions in the regions of subsites A and B.

196

N.C.J. Strynadka and M.N.G. James

Figure 3. A stereographic ball-and-stick diagram of the superimposed main chain structure of HEWL (white; Maenaka et ai., 1995), human (blue; Artymiuk and Blake, 1981), pheasant (purple; Lescar et ai., 1994), guinea hen (green; Lescar et ai., 1994), and turkey (yellow; Harata, 1993). The side chains of Glu35 and Asp52 are shown in red. Areas of greatest structural variation between the different species are labeled.

Although site specific mutagenesis of the framework of HEWL and related lysozymes has not been characterized as fully as that of T4L, there are several reported works of interest. Hill et al. (1993) have shown that site specific mutagenesis of one of the four disulfide bridges in HEWL (ie. disruption of the cysteine pair 6, 127; see Fig. Ib) yields a mimic of the ubiquitin-degradation substrate form of the protein. The 1.9 A refined structure of this HEWL mutant showed multiple conformers for the C-terminal residues that were distinct from the native enzyme. This work provided evidence that the initial step in the ubiquitination process, the reduction of the Cys6/Cysl27 disulfide, would create a flexible C-terminus which would expose the ubiquitination target sites in HEWL, Lysl and Lys13. Based on the sequence homology with IX-lactalbumin, Inaka et al. have introduced a calcium binding site into human lysozyme via sitespecific mutagenesis (lnaka et aI., 1991). The resulting X-ray crystallographic structures indicate that the calcium site was successfully introduced without significant change to the overall protein structure or molecular rigidity (based on an analysis of crystallographic temperature factors). Human lysozyme has also been used as a template for protein engineering studies designed to probe protein translocation (addition of

Lysozyme: A model enzyme in protein crystallography

197

various signal sequences; Kikuchi and Ikehara, 1994), cell adhesion (creation of mutants with an inserted Arg-Gly-Asp sequence; Yamada et al., 1995) and protein folding and dynamics (introduction of disulfide bridges; Kukuchi and Ikehara, 1994).

Goose egg white lysozyme

The overall tertiary fold of GEWL, another ex/ f3 structure, is given in Figure lc. The 185 amino acid polypeptide again folds to form a pronounced active-site cleft separating two distinct domains, one small f3 -strand domain (spanning residues 75 to 110), and a second much larger, all-helical domain. GEWL contains two disulfide bridges between cysteine residues at positions (18/29) and (4/60). The overall dimensions of the enzyme are 40 A x 44 A x 39 A. A surface representation of GEWL is shown in Figure 2c. The sequence identity between GEWL and HEWL can be considered, at best, very weak (Simpson et al., 1980; Schoentgen et al., 1982). However, it has been shown that apart from the amino-terminal region (residues 1-46), there are 90 spatially or structurally equivalent residues between GEWL and HEWL with an rms of 3.2 Aon main chain atoms. (Fig. 4, Grutter et al., 1983). In the superposition of HEWL or GEWL, these authors found that the ex-carbon of Glu73 in GEWL coincided closely with the ex-carbon of Glu35 in HEWL (rms deviation is 0.8 Afor these two groups). The closest structural counterpart to the Asp52 of HEWL was speculated to be Asp86 in GEWL, but it was noted that the position of these two groups in the superposition was relatively far apart, ｾＵＮＷ＠ A. T4lysozyme

The overall fold of bacteriophage T4 lysozyme is shown in Figure lc. Like HEWL and GEWL, the 164 amino acids of T4L fold into a two-domain structure with the interface of the domains forming a pronounced active site cleft (Fig. 2d). One domain of T4L has five strands of f3 sheet from residues 13 to 35 and from 51 to 61. There is also a helix from residues 39 to 49 in this small domain. The second larger domain contains the Nand C termini and is predominantly helical in nature. The two domains are linked by the long helix from residues 61 through 81. There are no cysteine disulfide bridges in T4L. The overall dimensions of the molecule are 38 A x 38 A x 48 A. The structure of T4L can be superimposed onto that of both HEWL and GEWL (Matthews et al., 1981; Grutter et al., 1983; Weaver et al.,

198

N.C.J. Strynadka and M.N.G. James

1985; see Fig. 4). T4L shares 74 common amino acids with HEWL (rms deviation of 3.8 A on common main chain atoms) and 91 common residues with GEWL (rms deviation of 3.2 A on common main chain atoms; Grutter et aI., 1983). Glull and Asp20 in T4L lie on opposite sides of the activity site cleft and superimpose closely with the analogous Glu35 and 052 in HEWL (Matthews et aI., 1981). T4 lysozyme undoubtedly represents one of the most well-probed of all crystallographic three-dimensional structures. The native form of the enzyme has been determined from a number of varying crystallization conditions (Bell et aI., 1991). A comparison of the crystallographic models determined in crystals at low, medium and high ionic strength indicated very little change in the formation of salt bridges in the enzyme. The conclusion from this paper was that a crystal structure determined at high salt concentrations is thus a good representation of the structure at lower ionic strengths, and that models electrostatic interactions in proteins that are based on crystal structures determined at high salt concentrations are likely to be relevant at physiological ionic strengths. A very important body of work concerning T4L has come from the laboratory of Brian Matthews in the past decade. This work uses a £xon I -- Ｍｴｾ

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