Enzyme technologies for pharmaceutical and biotechnological applications

Tai Lieu Chat Luong ISBN: 0-8247-0549-1 This book is printed on acid-free paper Headquarters Marcel Dekker, Inc 270 Madison Avenue, New York, NY 10016 tel: 212-696-9000; fax: 212-685-4540 Eastern Hemisphere Distribution Marcel Dekker AG Hutgasse 4, Postfach 812, CH-4001 Basel, Switzerland tel: 41-61-261-8482; fax: 41-61-261-8896 World Wide Web http:/ /www.dekker.com The publisher offers discounts on this book when ordered in bulk quantities For more information, write to Special Sales/Professional Marketing at the headquarters address above Copyright  2001 by Marcel Dekker, Inc All Rights Reserved Neither this book nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher Current printing (last digit): 10 PRINTED IN THE UNITED STATES OF AMERICA Preface Genes related to human and animal health are being discovered at an ever-increasing rate As gene products, enzymes are being explored for their function and application in a rapidly emerging field that has been termed functional genomics Enzyme Technologies for Pharmaceutical and Biotechnological Applications fills a unique niche for a comprehensive account of certain important enzymes in human and animal health Readers can also gain important insights into enzyme technologies in both the pharmaceutical and biotechnological industries The primary aim of this book is to highlight how, what, and where enzymes have become critically important or are rapidly emerging in these two overlapping and interdependent industries As a state-of-the-art work on enzyme technologies, the book covers four basic principles and applications in (1) antibiotic biosynthesis, (2) biocatalysis, (3) modern screening/optimization, and (4) emerging new technologies In Part I, on biosynthesis, the emphasis is placed on both improvements in antibiotic yield and ways to increase antibiotic structural diversity by modifications of the biosynthetic pathways from diverse microorganisms Here, the emphasis is on using genes to deliver enzymes and to thereby perform metabolic engineering including precursor-directed biosynthesis or mutasynthesis The use of recombinant techniques to generate protein products that are unnatural to the microbial world is also discussed, using specific examples of challenging problems in this area Part II on biocatalysis, covers the direct application of enzymes as chemical tools in manipulating small- to medium-sized synthetic organic compounds Manipulation of the enzyme tools by genetic engineering is described Chapter iii iv Preface discusses how a novel form of enzymes, cross-linked enzyme crystals (CLCs), are especially useful as chemical catalysts An example of a large-scale application of these chemical tools brings the area into focus by leaving the laboratory and entering the manufacturing plant Part III on screening for and optimization of enzyme inhibitors describes integrated approaches in therapeutic discovery research using enzyme targets relevant to human and animal diseases For high-throughput screening, the activity assays for the enzyme targets adopt both conventional (colorimetry, spectrometry, and radioactivity) and contemporary methodologies (fluorescence) A selective enzymatic assay maximizes validated hits from large diversified libraries of samples derived from natural products and synthetic compounds, including those arising from combinatorial chemistry The chapters on screening concentrate on development of effective enzymatic assays, each of which represents specific, kinetic, and molecular interactions between the enzyme and its substrate as well as inhibitors and thus reflects the pharmacological and chemical interplay at the targeted enzyme The primary screening goal is the production of manageable numbers of hits that ultimately generate high-quality lead compounds As a practical rule, optimization of those lead compounds by medicinal chemists is the critical follow-up step required for the discovery of viable drug candidates The process of lead compound optimization for an enzyme inhibitor, often referred to as structure-activity relationship studies or drug design, is dictated by understanding the molecular and kinetic interactions between the enzyme and its inhibitor These insights are typically gained by analyzing X-ray crystallographic depictions and by elucidating the kinetic behavior of the enzyme-inhibitor complex to improve potency and selectivity and to understand mechanisms of interactions The chapters on inhibitor screening/optimization emphasize the synergistic importance of high-throughput screening and structure-function based optimization studies for therapeutic discovery programs Finally, Part IV on emerging technologies examines some non-traditional methods by which enzymes may play important new roles in the drug discovery processes of the future The present ability to completely locate and sequence the gene clusters responsible for the multistep biosyntheses of complex natural products has spawned new technologies Such technologies can precisely and/ or deliberately modify certain parts of gene clusters within organisms or, alternatively, can interchange portions of gene clusters between organisms In each instance, new unnatural natural products may be formed by fermentation of the new genetically modified microorganisms The exchange of genetic material can be logically extended into a combinatorial paradigm called combinatorial biosynthesis or combinatorial enzymology, thereby leading to even larger numbers of new natural products Extensive interdisciplinary collaboration between new target identification and screening laboratories, medicinal chemists, and molecular modeling/computational chemists will become even more essential in the future for rapid discovery of useful new entities to evaluate in the field or clinic Preface v The last chapters of Part IV describe essential and overlapping enzyme technologies With the completion of most of the human genome sequence, assigning a precise function to genes ( functional prediction) and redesigning the function of enzymes (enzyme engineering) can play increasingly significant roles in drug discovery Also, the utility of functional genomics in identifying diseaserelevant enzyme targets depends closely on the molecular understanding of these targets under physiological and pathological conditions ( functional proteomics) Enzyme Technologies for Pharmaceutical and Biotechnological Applications is informative, practical, timely, and applicable worldwide to the pharmaceutical and biotechnological industries Real-world examples provided throughout the book are important for discriminating between the use of enzymes solely for academic studies and the practical use of enzymes in industrial applications The reader will acquire a better understanding of applied sciences in the field Areas that have been extensively covered in reviews and the general literature (such as use of natural lipases in organic synthesis) have been minimized here By focusing on real-world applications, the reader will obtain a clearer understanding of what is new and relevant in the field The book is intended primarily for industrial and research scientists with interests in adopting and maximizing enzyme technologies for pharmaceutical discovery, development, and manufacturing The book can also be used by graduate and postdoctoral students in practical enzymology, biochemistry, microbiology, molecular biology, and biochemical engineering, as well as by students in graduate-level courses covering practical enzymology and enzyme biochemistry Herbert A Kirst Wu-Kuang Yeh Milton J Zmijewski, Jr Contents Preface Contributors I iii xi Biosynthesis δ-(L-α-Aminoadipyl)-L-Cysteinyl-D-Valine Synthetase as a Model Tripeptide Synthetase Hans von Doăhren, Wibke Kallow, Mary Anne Tavanlar, Torsten Schwecke, Ralf Dieckmann, and Volker Uhlmann Metabolic Engineering for Cephalosporin C Yield Improvement and Production of Intermediates Joe E Dotzlaf, Steven W Queener, and Wu-Kuang Yeh 39 Bioconversion of Penicillins to Cephalosporins Arnold L Demain, Jose L Adrio, and Jacqueline M Piret Direct Fermentative Production of Acyltylosins by Genetically Engineered Strains of Streptomyces fradiae Akira Arisawa and Hiroshi Tsunekawa 61 89 vii viii II III Contents Engineering Streptomyces avermitilis for the Production of Novel Avermectins: Mutant Design and Titer Improvement Claudio D Denoya, Kim J Stutzman-Engwall, and Hamish A I McArthur Biocatalysis Biocatalytic Syntheses of Chiral Intermediates for Antihypertensive Drugs Ramesh N Patel 137 Cloning, Structure, and Activity of Ketone Reductases from Baker’s Yeast Jon D Stewart, Sonia Rodrı´guez, and Margaret M Kayser 175 Cross-Linked Enzyme Crystals: Biocatalysts for the Organic Chemist Michael D Grim 209 Enzymatic Deacylation of Echinocandins and Related Antifungal Agents Andrew R Cockshott, Adam J Kreuzman, and Wu-Kuang Yeh 227 Screening/Optimization 10 Roles of Enzymes in Antibacterial Drug Discovery Siddhartha Roychoudhury 11 Penicillin-Binding Proteins as Antimicrobial Targets: Expression, Purification, and Assay Technologies Genshi Zhao, Timothy I Meier, and Wu-Kuang Yeh 12 13 113 Development of a High-Throughput Screen for Streptococcus pneumoniae UDP-N-Acetylmuramoyl-Alanine: d-Glutamate Ligase (MurD) for the Identification of MurD Inhibitors Michele C Smith, James A Cook, Gary M Birch, Stephen A Hitchcock, Robert B Peery, Joann Hoskins, Paul L Skatrud, Raymond C Yao, and Karen L Cox Purification and Assay Development for Human Rhinovirus Proteases Q May Wang and Robert B Johnson 245 263 289 307 Contents ix 14 Screening for Parasiticides Using Recombinant Microorganisms Timothy G Geary 323 15 Screening for Inhibitors of Lipid Metabolism ¯ mura Hiroshi Tomoda and Satoshi O 343 16 Design and Development of a Selective Assay System for the Phospholipase A2 Superfamily Hsiu-Chiung Yang, Marian Mosior, and Edward A Dennis IV 379 Emerging Technologies 17 Understanding and Exploiting Bacterial Polyketide Synthases Robert McDaniel and Chaitan Khosla 397 18 Polyketide Synthases: Analysis and Use in Synthesis Kira J Weissman and James Staunton 427 19 Enzymatic Synthesis of Fungal N-Methylated Cyclopeptides and Depsipeptides Mirko Glinski, Till Hornbogen, and Rainer Zocher 471 20 New Strategies for Target Identification, Validation, and Use of Enzymes in High-Throughput Screening Joaquim Trias and Zhengyu Yuan 499 21 Use of Genomics for Enzyme-Based Drug Discovery Molly B Schmid 515 22 Assigning Precise Function to Genes Ridong Chen 537 23 Redesigning Binding and Catalytic Specificities of Enzymes Ridong Chen 555 24 Proteomics: Chromatographic Fractionation Prior to TwoDimensional Polyacrylamide Gel Electrophoresis for Enrichment of Low-Abundance Proteins to Facilitate Identification by Mass Spectrometric Methods Srinivasan Krishnan, John E Hale, and Gerald W Becker Index 575 597 Index Acetyltransferase (see also Acyltransferase) chloramphenicol, 96 choline, 96 Acinetobacter calcoaceticus, 157, 158 Acremonium chrysogenum, 2–28, 40– 53, 61–65, 75, 76, 81–83 Actin, 364, 365 Actinonin, 505, 506, 509 Actinoplanes utahensis, 230–236, 240 Actinorhodin, 400, 404, 430, 433, 436 Active site (see also Binding site(s)), 51, 119, 120, 264, 290, 345, 348, 402, 407, 416, 433, 483, 526 Aculeacin, 232, 233, 235, 236, 242 Acylase: aculeacin A, 235, 236, 242 cephalosporin, 235, 236 glutaryl, 82, 83 L-amino acid, 139 penicillin, 217, 223, 235, 242 Acyl carrier protein (ACP), 23, 400– 403, 406–414, 433–463 Acyltransferase: acyl-CoA:cholesterol (ACAT), 345, 346, 348, 360, 363, 365, 472 [Acyltransferase] deacetylcephalosporin C, 41–48, 81, 82 diacylglycerol (DGAT), 346–350, 360 dihydrolipoamide, 124 fatty acid, 409 macrolide-3-hydroxy, 91, 93, 96–98, 100, 102, 106 macrolide-4″-hydroxy, 91, 93, 96, 97, 100, 102, 106 malonyl-CoA:ACP (MCAT), 438 polyketide (AT), 402, 403, 407–417, 433–463 Aivlosin (see 3-O-acetyl-4″-Oisovaleryltylosin) ATPase, sarcoplasmic endoreticulum Ca2⫹-, 332 Alamar blue, 328, 331, 333 Albumin, 297–303, 347, 353, 354, 357, 387, 578, 582, 583 Aldolase, fructose 1,6-diphosphate, 220 Allysine ethylene acetal, 140 α-Aminoadipyl-cysteinyl-valine (ACV), 1–31, 40–43, 46, 52, 62, 66 Alpha 1-antitrypsin, 578, 582, 588 Alpha 1-macroglobulin, 582 597 598 Amidase: M neoaurum, 148, 149 penicillin G, 223 Amidepsine, 348 Amino acid sequence, 3, 4, 43, 45, 64, 65, 79, 93, 117–120, 124, 179, 182, 183, 188, 189, 192, 198, 199, 235, 293, 310, 315, 317, 348, 383, 404, 475, 478, 525, 543–549, 555, 556, 562, 563, 569, 576, 577, 587 7-Aminocephalosporanic acid (7-ACA), 45, 53, 81–83, 272 7-Aminodeacetoxycephalosporanic acid (7-ADCA), 48–53, 63, 81–83 3-Amino-5-hydroxybenzoic acid, 408 6-Aminopenicillanic acid, 272 Aminopeptidase, methionine (MAP), 504 Amphotericin B, 227, 430 Angiotensin converting enzyme (ACE), 150–154 Anthracycline, 408 Antibodies, 269, 272, 273, 290, 297– 299, 302–304, 351, 474, 475, 590 Antimicrobial agents, 89, 91, 230, 245– 257, 263–279, 289–304, 326, 346, 367–369, 397, 418, 431, 472, 491, 499–510, 515–520, 523 antifungal compounds, 227–231, 248, 365, 418 beta-lactam(s), 1, 39–54, 61–83, 199, 223, 233, 236, 250, 253, 263– 279, 367, 499, 500, 504, 505, 522 fluoroquinolone(s), 249, 255, 500, 508, 522, 523 glycopeptides, 264, 367, 504 macrolides, 250, 251, 397, 418, 427 sulfonamides, 249, 326 Antisense, 351 APO protein, 353, 588 Arabidopsis, 549 Arachidonic acid, 154, 383, 385 Arachidonyl trifluoromethyl ketone (AACOCF3 or MATFMK), 390, 392 Index Arisugacin, 344 Aromatase, polyketide (ARO), 402, 410–419, 433–437 Aspartame, 218, 219 Aspergillus nidulans, 2–29, 46, 82, 236, 238 Aspergillus niger, 45 Asymmetric reactions (see Stereoselective reactions) Atherosclerosis, 343, 351, 360, 364, 365 Aureol, 354 Avarol, 354 Avermectin, 113–131, 399, 408, 415, 416, 420, 430, 448, 450, 451 Azaphilone, 357, 358 Bacillus brevis, 23 Bacillus licheniformis, 269 Bacillus megaterium, 140 Bacillus stearothermophilus, 124 Bacillus subtilis, 30, 123–125, 128, 269, 271, 523 Bassianolide, 472, 484 Beauveria bassiana, 483, 484 Beauvericin, 472, 483, 484, 486 Beauveriolide, 363, 364 β-Adrenoceptors, 145–150, 164 β-Galactosidase, 20 β-Lactamase, 54, 250, 253, 499, 504, 505, 522 β-Tubulin, 53, 337 Binding site(s) (see also Active site), 3, 17,18, 20, 52, 65, 75, 199, 292, 478, 480, 489–491, 526, 543– 551, 557, 558, 562, 565, 567– 571 Biocatalysis, 12, 20, 137–166, 175– 203, 209–224, 231, 240, 429, 459, 537 Bioconversion: cephalosporin, 44–54, 61, 66 echinocandin B, 227–242 penicillin, 40–54, 61–83 pneumocandin, 229 Bioinformatics, 538 Index Biosynthesis, 457 actinorhodin, 404, 436 avermectin, 114–131 cell wall, bacterial-, 247, 250–252, 263–265, 289–304, 367 cell wall, fungal-, 227, 229 cephalosporins, 30, 39–54, 61–83 cholesterol, 160, 343–365 cyclosporin, 488 6-deoxy sugars, 116, 117 erythromycin, 119, 120, 404, 441, 446, 448 fatty acid, 120, 123, 125, 128, 250, 363, 402, 431, 438, 446, 509 folate, 249 glutathione, 40 isoprenoid, 345, 368, 369 lipid A, 247, 256 lipopolysaccharide, 504 mevalonate, 345, 369 mycolic acid, 368 pentose phosphate pathway, 176, 177 peptide, nonribosomal, 1–31, 40–43, 46, 471–491 polyketide, 91–107, 119–121, 397– 421, 427–464 precursor-directed, 21, 121, 416–418, 420, 450, 451 sterol, 343–346, 365, 368, 369 tetracenomycin F2, 435 triacylglycerol, 347 Biotin, 272, 274, 276, 527 Biotransformation, 67, 91, 147, 148, 154, 156, 163, 179, 189 BLAST search, 192, 195, 475, 541 BOCILLIN FL, 273–275, 278 Borrelia burgdorferi, 273 Bradykinin, 587 Bromenol lactone (BEL or HELSS), 390, 392 Burkholderia cepacia, 150, 156, 164, 218, 222 Caenorhabditis elegans, 336, 337 Calcium channel blockers, 161–163 599 Candida albicans, 227 Candida boidinii, 140–145, 154 Candida cylindraceae, 164 Candida rugosa, 212–216 Captopril, 150–152 Carbomycin, 91, 93, 97, 102 Carboxylase, propionyl-CoA, 460 Carboxypeptidase, 210, 263 Caspase, 393 Catalase, 75, 76, 140, 154 Catalytic efficiency (performance), 14, 182, 317, 318, 460, 556, 565, 567–571 Celmer model, 442–445 Centrifugation, gradient, 10 Cephalosporin C, 39–54, 61, 233 Cephalosporium acremonium (see Acremonium chrysogenum) Cephamycin, 41, 61, 67, 77, 78 Ceranopril, 154 Cerivastatin, 344 Cerulenin, 344, 363, 441 Chain length factor (CLF), 400, 412, 433, 434, 437, 438 Chiral synthons, 137, 138, 156, 165 ChiroCLEC TM-BL, 217, 218, 220 ChiroCLEC TM-CR, 213, 216 ChiroCLEC TM-EC, 217 ChiroCLEC TM-PC, 218, 222 Chloride channels, 114 Chloropuupehenone, 354 Cholesterol, 156–161, 343–365, 418, 431 Cholesterol ester transfer protein (CETP), 350–354, 357–360 Chromatography, 575, 590 adsorption, 10, 24, 26 hydroxyapatite, 270, 590 affinity, 268–272, 298, 302, 461, 464, 526 ATP-agarose, 390, 391 β-lactam, 268, 271, 272 calmodulin-agarose, 390 dye, 269–271, 569 glutathione, 267–271, 276 heparin, 590 600 [Chromatography] immuno- (antibody), 45, 269, 527, 590 lectin, 590 metal (nickel), 252, 269, 271, 293, 590 moenomycin, 269 cross-linked enzyme crystal, 216 high performance liquid (HPLC), 10, 11, 45, 67, 81, 98, 100, 102, 165, 216, 231, 233, 290, 295, 297, 299, 302, 312, 314, 506, 507 hydrophobic interaction, 270, 590 ion-exchange, 44, 139, 235, 270, 271, 293, 310, 311, 390, 578–584, 588 size exclusion, 310, 311, 439 thin-layer, 11, 128, 347 Chromobacterium viscosum, 152–154 Cilofungin, 229, 230, 233 Clavulanic acid, 61, 250, 253, 504 CLUSTAL W, 4, 541 Collinearity rule, 4, 20, 22 Combinatorial biosynthesis, 30, 31, 365, 366, 412–421, 437, 448–459, 491 Compactin, 343, 363 Conserved domains, 3, 4, 17, 80, 93, 117, 119, 182, 183, 188, 189, 198, 199, 248, 367, 437, 472, 475, 477, 478, 517, 547, 548, 558, 563, 568 Coomassie blue, 310, 576–579 Cooperia oncophera, 126 Coupled enzyme assay, 255, 290–304, 387 Cross-linked enzyme crystals (CLC or CLEC), 209–224 Cross-linked protein crystals (CPC), 209, 212 Cryptosporidium parvum, 324, 332 Cyclase: ACV (see Synthase, isopenicillin N) Aromatic polyketide (CYC), 402, 410–419, 433–437 Oxidosqualene (see Synthase, lanosterol) Cycloguanil, 331 Index Cyclohexane carboxylic acid, 125–128 Cyclooxygenases (COX-1 and COX-2), 383 Cycloserine, 255 Cyclosporin, 11, 28, 472, 486, 490 Cyclothialidine, 249 Cylindrotrichum oligospermum, 490 Cytochalasin, 364, 365 Cytochome P-450, 128, 160, 383, 385 Dabcyl fluorescent quenching acceptor, 315–317 Daptomycin, 230, 231, 233, 236 Daunosamine, 117 Deacetoxycephalosporin C (DAOC), 41–53, 61–63, 81 Deacetylcephalosporin C (DAC), 41– 53, 61, 62 Deacetylase: Lipid A, 247, 256 UDP-3-O-acyl-N-acetyl-glucosamine, 504 Deacylase: ECB, 230–242 Penicillin G, 242 Decarboxylase: α-ketoacid 124 arginine, 331 ornithine (ODC), 331–336 pyruvate, 183 S-adenosylmethionine, 334 Deformylase, peptide or peptidyl, 247, 504–506, 509, 522 Dehydratase: fatty acid, 120 polyketide (DH), 119–121, 402, 403, 410–417, 431–463 Dehydrogenase(s), 557 acyl-CoA (AcdH), 107, 129 alcohol, 29, 46, 183–194, 202, 330, 460 aryl alcohol, 189, 192 medium-chain alcohol, 182, 185– 189 short-chain alcohol, 182, 189–194, 201–203 Index [Dehydrogenase(s)] aldehyde, 330 amino acid, 139 alanine, 141 glutamate, 140, 141 leucine, 141 phenylalanine, 140–145 valine, 107 D-arabinose, 202 β-decarboxylating, 538–551 branched-chain α-ketoacid (BCDH), 107, 121–125, 127, 129 dihydrolipoamide, 124 formaldehyde, 188 formate, 140–145, 154, 199, 460 glucose, 140, 157, 158 glyceraldehyde 3-phosphate (GAP), 53 D-glycerate, 199 D-hydroxyacid, 198, 199 L-2-hydroxyisocaproate, 154 D-2-hydroxyisovalerate, 484 homoisocitrate (HDH), 550, 551 horse liver alcohol (HLADH), 155, 183, 185, 211, 221 inosine monophosphate (IMPDH), 330 isocitrate (IDH), 538–551, 555–571 isopropylmalate (IMDH), 538–551, 555–571 isovaleryl-CoA (IVD), 106 lactate, 183, 216, 220, 290, 556 lipoamide, 216 malate, 556 D-phenyllactate (DPLDH), 486 6-phosphogluconate (6-PGD), 327, 328 pyruvate, 123–125, 183, 251 6-Deoxyerythronolide B (6-dEB), 403, 433, 439, 451, 464 DNA sequencing, 4, 45, 79, 93, 115, 117, 119, 124, 182, 203, 233, 246, 271, 292, 348, 350, 368, 400, 421, 433, 473, 475, 515– 518, 523, 525, 537, 551, 563 dTDP-sugar, 116, 117 601 Destruxin, 484 Diabetes, 146, 343 Diazaquinomycin, 344 Dictyocaulus viviparus, 126 Digoxigenin, 272, 274 3,8-Dihydroxy-1-methylanthraquinone2-carboxylic acid (DMAC), 436 Dilthiazem, 161, 163 Dioxygenase, α-ketoglutarate dependent, 61, 63, 82 Dipeptidase, D-alanyl-D-alanyl, 251, 255, 504 Direct Absorption Scintillation Assay (DASA), 506, 507 Doramectin, 126–128, 130, 131, 448 Doxorubicin, 399, 400, 402, 408 Dynemicin A, 399 Echinocandin B (ECB), 228–242 Ectoparasites, 114, 126, 127, 323, 484 Edans fluorescent donor, 315–317 Eicosanoids, 383 Elanopril, 150 Elastase, 354 Electron spin resonance (ESR), 387 ELISA, 290, 291, 297–304 Enantioselective reactions (see Stereoselective reactions) Endoparasites, 114, 126, 127, 323, 325, 328, 332, 484, 537 Endopeptidase, DD-, 152, 263 Enniatin, 11, 28, 472–489 Enolase, glucose 6-phosphate 330 Entamoeba histolytica, 330 Enterobacter cloacae, 256, 504 Enterococcus faecalis, 251, 255 Enterococcus faecium, 251, 255, 269, 327 Enterococcus hirae, 267 Enzyme engineering (see also Genetic engineering; Metabolic engineering), 79–83, 166, 175, 236, 242, 366, 397–421, 427–464, 543, 550, 551, 555–571 Epimerase, 28, 410 isopenicillin N, 41–50, 62, 65 602 Epothilone, 218 Epoxidase, squalene, 345, 365, 366 Erabulenol, 354 Erythromycin, 91, 119, 120, 125, 399, 402–405, 415, 427, 433, 439–464 Escherichia coli, 19, 20, 44, 54, 64–66, 79, 80, 83, 124, 129, 140–145, 235, 247, 250, 252–256, 267– 273, 292, 293, 303, 309, 311, 327–332, 336, 368, 404, 406, 418, 435, 501, 504–509, 516– 525, 537, 541, 546–551, 556– 558, 563–567, 590 Esterase, pig liver, 147, 149, 150 Eukaryotic initiation factor, 308 Evolution, 518, 520, 522 convergent, 235, 236 directed, 107, 240, 555 divergent, 537–541 gene duplication, 18, 538–540 Expandase/hydroxylase (see Synthetase, DAOC; Synthase DAC) Farnesyl pyrophosphate, 160 Fatty acid binding protein, 387 Fermentation: commercial manufacturing, 48, 53, 91, 106, 107, 203, 240–242, 450 cultures for screening, 334, 335, 348, 354, 363, 365 development, 28–31, 39–54, 83, 97– 106, 127–131, 236–242, 460 Ferroverdin(s), 354, 357 FK-506, 399, 415, 420 Fluconazole, 227 Fluorescein, 272–275, 278 Fluorescent assays, 272–275, 292, 314– 318, 328, 333, 353, 387 Formyltransferase, methionyl-tRNA, 522 Fosmidomycin, 369 Fusarium avenaceum, 472, 473 Fusarium lateritium, 480 Fusarium oxysporum, 480 Fusarium sambucinum, 478, 480 Fusarium scirpi, 23, 473, 478, 480 Index γ-Aminobutyric acid (GABA), 114 Gel electrophoresis, 11, 12, 45, 158, 202, 264, 271–274, 309–311, 350, 390, 485–488, 563, 575– 591 Gel filtration, 10–12, 45, 270, 271, 358, 407, 486, 488 Gene: chip technology, 182 complementation, 327, 336 deletion, 125, 202, 203, 331, 412– 416, 518 disruption, 45, 97, 107, 125, 365, 520, 521 essential, 247–250, 263, 289, 473, 499–501, 504, 506, 520–522, 527, 528 expression, 10, 20, 29–31, 44–51, 65, 79–83, 93, 97–106, 117–121, 233, 234, 242, 293, 329–332, 350, 365, 404–406, 418, 434– 437, 473 insertional inactivation, 45, 129, 521 mutational inactivation, 327, 328, 416 nonessential, 124, 202, 263, 501, 506, 520, 521 regulatory, 93, 97, 102 Gene name and function: actinorhodin synthase (act), 400 acyl-CoA:cholesterol acyltransferase (ACAT), 346 acyl-CoA dehydrogenase (acdH), 107, 129, 130 aldehyde (adh), 330 aldo-keto reductase (YPR1), 202 α-acetoxyketone reductase (GRE2), 202 D-arabinose dehydrogenase (ARA1), 202 avermectin biosynthesis (ave), 117, 118, 129 beta-lactam biosynthesis acvA, 29 cefD, 41, 51, 62, 65, 81 cefE, 41, 44, 51–53, 62, 65, 79–82 Index [Gene name and function] cefEF, 41, 44, 45, 51, 53, 64, 65, 81, 82 cefF, 41–51, 62 cefG, 41, 45, 48, 64, 81 ipnA, 29 pcbAB, 41, 43, 46, 62, 64 pcbC, 41, 43, 45, 51, 53, 54, 62– 65, 82 branched-chain α-ketoacid dehydrogenase bfmB, 125 bkd, 124, 125, 127, 130 branched-chain α-ketoacid/pyruvate dehydrogenase (aceA), 125 carbomycin 3-O-acylase (acyA), 93, 96–98, 100, 102, 107 4″-O-acylase (acyB1 or carE), 93, 94, 96–98, 100, 102, 106, 107 regulatory (acyB2), 93, 97, 98, 100, 102, 107 resistance (carB), 93 cell division (yycFG), 248 cell wall ligase murA, 247 murB, 247 murD, 292 murG, 247, 251, 252 mraY, 247 cyclosporin (simA), 475, 488 daunomycin (dnmV), 117 enniatin (esyn), 472, 473, 475 erythromycin eryBIV, 117 RNA methyltransferase (erm), 250 ftsH protease (ftsH), 501 HC-toxin alanine racemase (toxG), 488 methionyl-tRNA formyltransferase (fmt), 522 midecamycin 3-O-acyltransferase (mdmB), 93, 106 penicillin binding protein (pbp), 264– 267 603 [Gene name and function] peptidyl deformylase (def ), 505, 506, 522 phosphofructokinase (pfk), 328 Plasmodium transporter for quinolone resistance (pfmdrI), 332 reductase, enoyl-acyl carrier protein (FabI or inhA), 250 signal peptidase (spsB), 247 spore pigment (whiE), 419 thiostrepton resistance (tsr), 100 two-component kinase (cheA), 254 tylosin regulatory (tylR), 97 vancomycin resistance vanA, 251–253, 255 vanH, 251–253 vanX, 251–253 xylose reductase (GRE3), 202 Genetic engineering (see also Enzyme engineering; Metabolic engineering), 39, 91–107, 113–131, 175, 203, 237, 266–268, 366, 404, 416, 419–421, 429, 439, 464, 491 Gene-to-screen (GTS), 501, 505–509 Genomics, 40, 93, 115, 117, 124, 175, 182, 192, 201–203, 245–246, 289, 307, 336, 337, 368, 400, 501, 515–529, 537, 538, 551, 555, 575 Geotrichum candidum, 156, 157, 160 Giardia lamblia, 330 Gliocladium roseum, 348 Glisopenin, 346 Glutaraldehyde cross-linking, 210–212, 297 Glutathione-S-transferase purification tag, 267–271, 276 Glycolysis, 176, 177, 183, 327–330, 334, 556 Glycosylation, 114, 332, 433, 471, 588 Glycosylphosphatidylinositol (GPI), 332 Gramicidin S, 11, 13, 14, 22, 23, 27, 28, 483 Gyrase, 249, 508, 522 Gyrophoric acid, 348 604 Haemonchus contortus, 328, 331, 334– 336 Haemophilus influenzae, 271, 273, 303, 520, 576, 590 Hansenula anamola, 147 Hansenula polymorpha, 165 HC toxin, 488 Heat shock protein (GroEL), 76, 77 Helminths (see Endoparasites) Herbimycin, 344 High density lipoprotein (HDL), 351, 353, 357, 359 High-throughput screen, 247, 250, 253– 256, 265, 274–278, 289–304, 311–318, 326–337, 351, 365, 366, 491, 499–510, 515, 521, 526–529 Histidine purification tag, 252, 267– 271, 292, 293, 461, 464 Humulus lupulus, 348 Hydratase, enoyl-CoA, 106 Hydrolase, 459 acetyl, 47 amidino, formaldehyde-glutathione, 188 PAF acetyl, 360 metallo 500–506, 509 NTP, 525 phosphatidic acid phospho-, 392 6-Hydroxynorleucine, 138–140 Hymeglusin, 344, 345, 363 Hypercholesterolemia, 343, 344, 357 Hypertension, 137–166, 343 Hyphomicrobium methylovorum, 199 Immobilized enzymes, 3, 29, 77, 78, 83, 138, 145, 147, 152, 156–159, 166, 178, 209, 211, 238, 239, 459–461, 464 Immobilized pH gradient, 576–579 Immunoglobulin, 298, 302, 578, 582, 583 Inhibitors, 178, 247, 254–256, 277, 290, 303, 304, 318, 327–337, 343– 369, 391, 392, 483, 488–490, 504–508, 519, 527 Index Insects (see Ectoparasites) In silico, 520, 526 In vivo expression technology (IVET), 247 Isoelectric focusing, 576, 578 Isomerase: DXP reducto-, 369 glucose, 222 glucose 6-phosphate, 330 triosephosphate, 330 Isoniazid, 250, 346, 368 Isopenicillin N, 2, 41–54, 62–65 Isopropyl thiogalactoside, 141 Itraconazole, 227 Jadomycin, 76 Josamycin, 96, 102 Ketoprofen, 212, 213 Keyhole limpet hemocyanin, 297 Kinase: histidine protein, 248 phosphoenol pyruvate carboxy (PEPCK), 328 phosphofructo (PFK), 328–330, 332– 337 phosphoglycerate, 330 pyruvate, 290 two-component, 247, 248, 254–256 VanS, 248 VncS, 248 Kinetics, 18, 26, 232, 269, 274–276, 294–297, 312–316, 387, 439, 567, 569, 570 Kinnogens, 587 Klebsiella pneumoniae, 256, 504 Knockout mutants, 131, 179, 202, 203, 499, 501 Lactobacillus casei, 327 Lactobacillus confusus, 154 Lactocystin, 344 Lead identification, 251, 332, 336, 526– 528 Leishmania donovani, 330 Leucomycin, 91, 96, 102, 106 Leukotrienes, 383 Index Ligase: acyl-CoA, 402, 408 D-alanine-D-alanine, 247 aminoacyl, 522 4-coumarate, 474 cytosolic, 290 UDP-MurNAc-Ala:Glu (see MurD) VanA, 251, 255 Lipase: BMS, 150, 158 Burkholderia cepacia, 150, 164, 218, 222 Candida cylindraceae, 164 Candida rugosa, 212–216 Chromobacterium viscosum, 152–154 PS-30, 150–153, 156, 158, 159 Porcine pancreatic, 152 Rhizopus oryzae, 150 Serratia marcescens, 163 Lipid A, 247, 256 Liposomes, 361 Lipoxygenase, 383 Lovastatin, 343, 430 Low density lipoprotein (LDL), 343, 351, 353, 360, 364, 385 Luciferase, firefly 474 Lysobacter lactamgenus, 65 Lysophospholipids, 383, 385 Macrophage-derived foam cells, 345, 346, 360–365 Malic enzyme, 331 Maltose-binding protein, 267, 271, 461 Manoalide, 391 Mass spectrometry, 22, 163, 293, 310, 311, 314, 439, 575–591 Matrilysin, 505 Metabolic engineering (see also Enzyme engineering; Genetic engineering), 19, 20, 29–31, 39–54, 81, 82, 107, 179, 421 Metabolism: amino acid, 101, 105–107, 123, 125, 129 lipid, 343–369 phospholipid, 361, 379–393 605 Methyl arachidonyl fluorophosphate (MAFP), 392 Methymycin, 405 6-Methylsalicylic acid, 405, 408, 409 Mevastatin, 159 Mevinolin, 343 Micelles, 387, 388 Michaelis complex, 544, 546, 550, 556, 558, 570 Michaelis-Menton kinetics, 18, 26 Microbial resistance, 63, 89, 245, 246, 250, 251, 264, 265, 289, 367, 499, 504, 505, 509, 510, 516, 518, 522 Midecamycin, 93, 96, 106 Milbemycin, 114 Modular enzymes, 3, 4, 12–31, 116– 125, 397–421, 431–464, 471–491 Moenomycin, 264, 269 Monacolin K, 343 Monensin, 399, 430, 450 Monopril, 152 Moraxella catarrhalis, 303 Mortierella ramanniana, 163 Multienzyme complex, 2, 3, 12–31, 76, 121, 124, 433, 448, 459–464, 472, 480, 487, 488, 523 Multifunctional (bifunctional) enzyme, 43, 44, 48, 51, 61, 64, 116, 249, 330, 379, 400, 431, 473, 478, 487, 538 MurA, 247, 367, 506, 508 MurB, 247, 506 MurC, 290, 506 MurD, 289–304, 506 MurE, 290–300, 304, 506 MurF, 290–300, 304, 367, 506 MurG, 247 Mutagenesis: chemical, 113, 117, 118, 123, 128, 130 insertional, 45, 129 random, 39, 51, 53, 97, 107, 131, 166, 555, 564, 565, 567, 568 site-directed, 51, 52, 166, 412, 543, 547, 555–558, 563–565 606 Mutasynthesis, 113, 121, 125, 130, 131 Mycelia sterilia, 484–486 Mycobacterium leprae, 267, 269 Mycobacterium neoaurum, 147, 148 Mycobacterium smegmatis, 269 Mycobacterium tuberculosis, 250, 264, 271, 346, 368, 523 Mycolic acid, 368 Mycoplasma genitalium, 516, 520, 521 Mycoplasma pneumoniae, 516 N-acetylcysteamine (NAC), 409, 416, 459, 460 Neisseria gonorrhoeae, 267 Nematodes (see Endoparasites) Niddamycin, 455 Nitrocefin, 277 Nocardia globerula, 156 Nocardia lactamdurans, 2–16, 51, 65, 79–83 Nocardia salmonicolor, 163, 165 NMR, 163, 251, 334, 387, 439, 571 Nutrient-dependent viability screen, 327–331, 333–335 3-O-Acetyl-4″-O-(3-hydroxyisovaleryl)tylosin, 106 3-O-Acetyl-4″-O-isovalerylleucomycin, 102 3-O-Acetyl-4″-O-isovalerylspiramycin, 106 3-O-Acetyl-4″-O-isovaleryltylosin (AIVtylosin), 89–107 3-O-Acetyltylosin, 93, 98, 102, 105 4″-O-Acetyltylosin, 100, 101 4″-O-Butyryltylosin, 100, 101 4″-O-Isovaleryltylosin, 93, 98, 100, 101 Oleandrose, 114, 116 Omapatrilat, 140 OmpA signal peptide, 267 3-O-Propionyl-4″-Oisovalerylspiramycin, 106 4″-O-Propionyltylosin, 100, 101 Ortholog(s), 501, 517, 518, 523, 525, 537–551 Index Ostertagia ostertagi, 126 Oxidase, 52 D-amino acid, 139, 140 L-amino acid, 154 Oxidative deamination, 154 Oxidative phosphorylation, 176 Oxidoreductases, 157, 166, 185, 459, 484 Oxygenase, non-heme, 65 p-Aminobenzoic acid, 249, 316 Paralog(s), 501, 522–525, 537–551 Paraquat, 331 Parasiticide(s), 323–337, 418, 484, 486 Pathway assays (screens), 367, 500, 506–509 Penicillin(s), 1, 30, 39–54, 61–83, 233, 235, 245, 264, 272–278, 290, 326, 367 Penicillin-binding protein (PBP), 250, 263–279, 500, 522 Penicillium chrysogenum, 2–30, 39–54, 63 Penicillium citrinum, 159 Penicillium patulum, 408 Pepticinnamine, 344 PeptiCLEC TM-BL, 219 PeptiCLEC TM-CR, 214 PeptiCLEC TM-TR, 218, 223 Peptidase: lipoprotein leader, 508, 509 signal, 247, 253 VanX, 251, 255 PeptideSearch, 580, 587 Peptidoglycan, 247, 250–252, 263–265, 290, 506, 522 Peptidomimetic, 217, 219, 220 Peroxidase: horseradish, 299, 302 selenium CLC, 222 streptavidin-conjugated, 272 PF-1022, 480, 484–486 Phage display, 527, 528 Phenochalasin, 363–365 Pheromone transporter, 332 Phosalacine, 344 Index Phosphatidylinositol phosphate (PIP), 385 Phospholipase A2 (PLA2), 360, 379–393 Phospholipase C, 393 Phospholipids, 347, 348, 361, 379, 383–388 4′-Phosphopantetheine, 3, 13, 20, 22, 23, 472, 473, 478 Photoaffinity labeling, 477 Pichia pastoris, 141–145 Picromycin, 405, 410, 415 Pigment epithelium-derived factor (PEDF), 585 Plasmodium falciparum, 325, 330–332, 369 Platelet-activating factor (PAF), 385 Pneumocandin, 227, 229 Pneumocystis carinii, 227, 230, 332 p-Nitroanilide chromogenic peptide, 312–314, 318 Polyketide(s), 2, 22, 114, 119–121, 397–421, 427–464, 491 Polymerase: DNA, 523 RNA, 249, 250, 254, 522 Polymerase chain reaction (PCR), 20, 119, 124, 267, 292 Posttranslational events, 13, 20, 23, 30, 116, 350, 404, 405, 509, 522, 575 Potassium channel, 163 Pravastatin, 159, 160, 343, 344 Pregnenolone, 363 Presqualene pyrophosphate, 160 Promoter: ethanol dehydrogenase (alcAp), 29 galactose-responsive (GAL), 332 gpdA, 82 icd, 563 PA, 509 PBAD, 501, 508 pcbC, 82 penDE, 51, 82 tet regulon, 509 Prontosil, 326 Propranolol, 164 Prostaglandins, 383 607 Protease: aspartyl, 509 cysteine, 307, 308 HIV, 315, 500 HRV 2A, 307–312, 314, 316, 318 HRV 3C, 307–309, 311, 312, 314, 316, 318 HtrA, 509 metallo, 139, 223 serine, 247, 308, 441, 509 Proteinase K, 18 Protein Data Bank (PDB), 525 Protein engineering (see Enzyme engineering) Protein kinase C (PKC), 393 Protein Prospector, 580, 584, 587 Proteomics, 201–203, 246, 529, 575–591 Providencia alcalifaciens, 154 Pseudomonas aeruginosa, 250, 267, 273, 504, 516 Pseudomonas cepacia (see Burkholderia cepacia) Pseudomonas putida, 124, 516 Pseudomonas syringae, 488 Pseudomycin, 229, 233 Purpactin, 346 Puupehenone, 354 Pyrimethamine, 331 Pyripyropene, 344, 346 3-Quinuclidinol, 217 Racemase, 44, 487 alanine, 488 Radiometric assay, 10, 14, 22, 128, 254, 255, 264, 272–274, 276, 290, 347, 353, 361, 387, 506, 507, 527, 576 Rapamycin, 402, 414, 415, 420, 430, 454–456 Rate-limiting enzyme reaction, 46, 290, 294, 328, 383 Recombination: heterologous, 117 homeologous, 79, 83 homologous, 79, 97, 404 608 Reductase, 179 aldose, 194–198 aldo-keto, 194, 195, 202 α-acetoxyketone, 189, 192, 202 α-keto ester, 199 β-ketoester, 189 cinnamoyl-CoA, 192 dihydroflavinol, 192 dihydrofolate (DHFR), 249, 331 enol-acyl carrier protein, 250 enoyl (ER), 402, 403, 410–417, 431– 463 hydroxymethylglutaryl CoA (HMGCoA), 156–159, 343, 345, 363 3-keto-ACT/CoA, 118 keto (KR), 402, 403, 407–419, 431– 463 ketone, 175–203 3-oxo-ACP, 201 xylose, 198, 202 Reduction, 146–149, 156–158, 162, 176, 179, 220 Reductive amination, 139, 140, 142 Regulatory function, 46, 337, 385, 501, 509, 516, 547–551, 575 Resolution of racemates, 138, 148, 158, 159, 164, 217, 218, 222 Response regulator, 248 Restriction map, 119, 124 Reverse genetics, 40, 43, 45, 47, 54, 404 Rhizopus oryzae, 150 Rhodococcus rhodochrous, 147, 165 Rifampicin, 254 Rifamycin, 249, 408, 416, 430 Roselipin, 348 Rossmann fold, 188, 199, 525, 539, 556–558 Saccharomyces cerevisiae, 45, 156, 175–203, 331–337, 528, 550, 551, 576 Saccharopolyspora erythraea, 117, 415, 433, 455 Salmonella typhimurium, 250, 330, 518, 521 SANDIMMUN TM, 486 Index Scanning radiography, 128 Schistosoma mansoni, 330, 332 Schizosaccharomyces pombe, 365 Scintillation proximity assay (SPA), 274, 276, 353, 526, 527 Scleroderolide, 354 Sclerotioramine, 358 Sclerotiorin, 354, 357–360 SELEX, 527, 528 Sequest, 587 Serpin, 588 Serratia marcescens, 163, 504 Shigella dysenteriae, 264 Signal transduction, 247, 522 Simvastatin, 344 Sinefungin, 483, 489, 490 Sotalol, 164 Specificity determinants (see also Substrate specificity), 543–548, 551, 557, 558, 567 Spectrophotometric assay, 272–278, 290, 312–318, 328, 333, 387, 563 Sphingomonas paucimobilis, 147 Spiramycin, 94, 106, 415 Squalene, 160, 345 Squalestatin, 345 Staphylococcus aureus, 247, 251, 255, 256, 264, 267, 269–271, 273, 276, 277, 289, 303, 505, 508, 509, 527 Staurosporine, 344 Stem-cell factor receptor, 588 Stereoselective acylation, 158, 159, 164, 217, 218 Stereoselective esterification 151, 152 Stereoselective hydrolysis, 147, 149, 150, 152, 156, 163, 212, 222 Stereoselective oxidation, 155, 156, 160, 163 Stereoselective reduction, 146, 147, 156–158, 162, 163, 175–203, 221 Sterigmatocystin, 236 Stilbamidine isethionate, 334 Streptococcus pneumoniae, 246, 248, 264, 265, 267, 269–271, 273– 275, 289–304, 509 Index Streptococcus salivarius, 548 Streptomyces ambofaciens, 94, 96 Streptomyces avermitilis, 107, 113–131, 448 Streptomyces carbophilus, 160 Streptomyces clavuligerus, 3, 4, 10–19, 26, 29, 40–54, 61–83 Streptomyces coelicolor, 107, 405, 421, 436, 439, 454, 455 Streptomyces fradiae, 89–107 Streptomyces glaucescens, 80, 435 Streptomyces griseus, 234 Streptomyces kasugaensis, 98 Streptomyces kitasatoensis, 102, 106 Streptomyces lividans, 10, 16, 65, 80, 81, 93, 233, 234, 236, 242 Streptomyces mycarofaciens, 93, 106 Streptomyces narbonensis var josamyceticus, 102 Streptomyces peucetius, 117 Streptomyces thermotolerans, 91, 93, 97, 101 Stromal cell derived factor, 585, 588 Strongylin A, 354 Suberitenone, 354 Substrate-assisted stabilization, 527, 570 Substrate specificity (see also Specificity determinants), 14, 43, 52, 63, 178, 182, 185, 192, 203, 231, 232, 236, 265, 318, 387, 409, 480, 483–486, 490, 544–546, 550, 551, 557, 558, 562, 565, 567–571 Subtilisin, 18, 210, 217–220, 222 Sulbactam, 253, 504 Superoxide dismutase, 76, 330, 556 Surfactin, 30 SwissProt database, 584, 585 SynthaCLEC TM-PA, 223 Synthase: acyl-CoA, 387 aromatic polyketide, 400–402, 406, 409, 412–418, 431–439 β-keto acylthioester, 402 609 [Synthase] deacetylcephalosporin C, 41–53, 62– 63, 79–83 6-deoxyerythronolide B (DEBS), 407–421, 433–463 dihydropteroate, 249 fatty acid (FAS), 199–202, 363, 400, 407, 431, 437, 438 geranyl-geranyl pyrophosphate, 160 1,3-glucan, 227 glucosamine 6-phosphate, 247 HMG-CoA, 345, 363 isopenicillin N, 2, 41–54, 62 keto (KS), 119, 400–403, 407–418, 431–463 lanosterol, 345 6-methylsalicylic acid (MSAS), 405, 406, 408 polyketide (PKS), 22, 116, 119–121, 125, 397–421, 427–464, 488 rapamycin (RAPS), 454–456 squalene (oxidosqualene cyclase), 160, 345 Synthetase: acetyl-CoA, 363, 460 actinomycin II, 491 α -aminoadipyl-cysteinyl-valine (ACV), 1–31, 40–43, 46, 62 beauvericin, 480, 483 cyclosporin (Cysyn), 11, 28, 473, 475, 478, 483, 487–490 deacetoxycephalosporin C, 41–53, 61–63, 79–83 enniatin (Esyn), 11, 28, 473–489 gramicidin S, 11, 13, 14, 22, 23, 27, 28, 483 isopenicillin N (IPNS), 46 nonribosomal peptide, 1–31, 290, 402, 415, 471–491 PF1022 (Pfsyn), 480, 484, 485 SDZ 214–103, 490 surfactin, 30 syringomycin, 488 tRNA, 522 tyrocidine, 13, 22, 23 Syringomycin, 229, 488 610 Target identification/validation, 245– 257, 278, 289, 290, 307, 318, 327–337, 343–345, 351, 365– 369, 499–510, 515–529, 575 TATA-binding protein, 309 Taxol TM, 201 Tazobactam, 250, 253 Teicoplanin, 233 Terbinafine, 365 Terpendole, 346 Tetracenomycin, 400, 404, 435, 436 Tetracycline, 309, 399 Thermoactinomyces intermedius, 140– 143 Thermolysin, 211, 212, 214, 215, 218, 223, 505 Thermus thermophilus, 539, 546, 547, 550, 557, 558, 563, 565 Thioesterase, 3, 27, 28 polyketide (TE), 30, 402, 410–417, 433–463 Thiostrepton, 100, 233 Thiotemplate, 2, 3, 23, 472, 473, 486 Thrombospondin, 588 Thromboxane, 154–156, 383 Tolnaftate, 365 Tolypocladium niveum, 23, 486 Topoisomerase, 249, 500, 508, 522 Toxoplasma gondii, 324, 330, 332, 337 Transaminase, branched amino acid, 121 Transcriptional processes, 182, 248–250, 308, 309, 331, 337, 501, 509, 577 Transferase: AdoMet-dependent methyl-, 475, 477, 478 cholesterol acyl, 472 C-methyl, 409 DNA methyl-, 475, 477 erythromycin RNA methyl- (Erm), 250, 251 farnesyl, 345 fatty acid acyl, 409 glycosyl, 332 guanidinoacetate N-methyl, 477, 488 hypoxanthine phosphoribosyl (HPRT), 330, 332 Index [Transferase] indolethylamine N-methyl, 488 malonyl (MAT), 400 N-methyl, 472–483, 488, 489 O-methyl, 117, 130 peptidyl, 23 4′-phosphopantetheine-protein (PPT), 23 Transferrin, 578 Transglycosylase, 263–266 Transit peptides, 549 Transpeptidase, 250, 251, 263–266, 278 Triacsin, 344, 363 Triacylglycerol (TG), 346–348, 350, 360–365 Triclosan, 250 Trigonopsis variabilis, 140 Trimethoprim, 249 Tritrichomonas foetus, 330 Trypanosoma brucei, 327, 330 Trypanosoma cruzi, 330 Trypanosomes, 326, 332 Trypsin, 577–579, 583–587 Tylactone, 418 Tylosin, 89–107, 430 Tyrocidine, 13, 23 Tyrosinase, 80 UDP-MurNAc-L-Ala (UMA), 291–298, 303 UDP-MurNAc-L-Ala-D-Glu (UMAE), 293, 295, 297 UDP-MurNac-L-Ala-D-iso-Glu-Lys (UMAEK), 295, 297 UDP-MurNAc-pentapeptide (stem peptide), 264–265, 290–304 UDP-N-acetyl muramoyl-alanine:Dglutamate ligase (see MurD) Ureohydrolase, agmatine 331 Valinomycin, 484 Vancomycin, 248, 251, 252, 255, 264, 265 Vasopeptidase, 138, 140 Virulence, bacterial, 247, 248, 250, 256, 509 Index 611 Virus: human immunodeficiency (HIV), 227, 315, 324, 325, 500 human rhino- (HRV), 307–318 vaccinia, 477 Xanthohumol, 348 X-ray crystallography, 64, 65, 182, 195, 198, 199, 252, 264, 308, 312, 392, 464, 491, 539, 543, 547, 556, 557, 567, 571 Whole-cell target-directed screens, 246, 251, 255, 256, 325, 336, 345, 508–510, 521, 526, 528 Wiedendiol, 354 Zaragozic acid, 345 Zeolites, 212, 216 Zofenopril, 151, 152