Enzyme Technologies for Pharmaceutical and Biotechnological Applications

628 Pages·2001·5.202 MB·English

by  Herbert Kirst, Wu-Kuang Yeh

ISBN:0-8247-0549-1
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Preface
Genesrelatedtohumanandanimalhealtharebeingdiscoveredatanever-increas-
ing rate. As gene products, enzymes are being explored for their function and
applicationinarapidlyemergingfieldthathasbeentermedfunctionalgenomics.
EnzymeTechnologiesforPharmaceuticalandBiotechnologicalApplicationsfills
a unique niche for a comprehensive account of certain important enzymes in
humanandanimalhealth.Readerscanalsogainimportantinsightsintoenzyme
technologiesinboththepharmaceuticalandbiotechnologicalindustries.Thepri-
maryaimofthisbookistohighlighthow,what,andwhereenzymeshavebecome
criticallyimportantorarerapidlyemerginginthesetwooverlappingandinterde-
pendent 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
yieldandwaysto increaseantibioticstructuraldiversitybymodificationsofthe
biosynthetic pathways from diverse microorganisms. Here, the emphasis is on
using genes to deliver enzymes and to thereby perform metabolic engineering
includingprecursor-directedbiosynthesisormutasynthesis.Theuseofrecombi-
nant techniques to generate protein products that are unnatural to the microbial
worldisalsodiscussed, usingspecificexamplesofchallengingproblemsinthis
area.
PartIIonbiocatalysis,coversthedirectapplicationofenzymesaschemical
toolsinmanipulatingsmall-tomedium-sizedsyntheticorganiccompounds.Ma-
nipulation of the enzyme tools by genetic engineering is described. Chapter 8
iii
iv Preface
discusseshowanovelformofenzymes,cross-linkedenzymecrystals(CLCs),are
especiallyusefulaschemicalcatalysts.Anexampleofalarge-scaleapplicationof
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
integratedapproachesintherapeuticdiscoveryresearchusingenzymetargetsrel-
evanttohumanandanimaldiseases.Forhigh-throughputscreening,theactivity
assaysfortheenzymetargetsadoptbothconventional(colorimetry,spectrometry,
and radioactivity) and contemporary methodologies (fluorescence). A selective
enzymaticassaymaximizesvalidatedhitsfromlargediversifiedlibrariesofsam-
ples derived from natural products and synthetic compounds, including those
arising fromcombinatorialchemistry.Thechaptersonscreeningconcentrateon
development of effective enzymatic assays, each of which represents specific,
kinetic,andmolecularinteractionsbetweentheenzymeanditssubstrateaswell
asinhibitorsandthus reflectsthepharmacologicalandchemicalinterplayatthe
targeted enzyme. The primary screening goal is the production of manageable
numbersofhitsthatultimatelygeneratehigh-qualityleadcompounds.Asaprac-
tical rule, optimization of those lead compounds by medicinal chemists is the
critical follow-up steprequired forthe discoveryofviabledrug 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 under-
standingthemolecularandkineticinteractionsbetweentheenzymeanditsinhibi-
tor.TheseinsightsaretypicallygainedbyanalyzingX-raycrystallographicdepic-
tions and by elucidating the kinetic behavior of the enzyme-inhibitor complex
toimprovepotencyandselectivityandtounderstandmechanismsofinteractions.
The chapters on inhibitor screening/optimization emphasize the synergistic im-
portanceofhigh-throughputscreeningandstructure-functionbasedoptimization
studies for therapeutic discovery programs.
Finally, Part IV on emerging technologies examines some non-traditional
methodsbywhichenzymesmayplayimportantnewrolesinthedrugdiscovery
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/
ordeliberatelymodifycertainpartsofgeneclusterswithinorganismsor,alterna-
tively,caninterchangeportions ofgene clustersbetweenorganisms.Ineachin-
stance, new unnatural natural products may be formed by fermentation of the
newgeneticallymodifiedmicroorganisms.Theexchangeofgeneticmaterialcan
belogicallyextendedintoacombinatorialparadigmcalledcombinatorialbiosyn-
thesis or combinatorial enzymology, thereby leading to even larger numbers of
newnaturalproducts.Extensiveinterdisciplinarycollaborationbetweennewtar-
get identification and screening laboratories, medicinal chemists, and molecular
modeling/computationalchemistswillbecomeevenmoreessentialinthefuture
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, as-
signing a precise function to genes (functional prediction) and redesigning the
functionofenzymes(enzymeengineering)canplayincreasinglysignificantroles
indrugdiscovery.Also,theutilityoffunctionalgenomicsinidentifyingdisease-
relevantenzymetargetsdependscloselyonthemolecularunderstandingofthese
targetsunderphysiologicalandpathologicalconditions(functionalproteomics).
Enzyme Technologies for Pharmaceutical and Biotechnological Applica-
tions is informative, practical, timely, and applicable worldwide to the pharma-
ceuticalandbiotechnologicalindustries.Real-worldexamplesprovidedthrough-
outthebookareimportantfordiscriminatingbetweentheuseofenzymessolely
foracademicstudiesand thepracticaluseofenzymesin industrialapplications.
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 under-
standing 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,andmanufacturing.Thebookcanalsobeusedbygradu-
ate and postdoctoral students in practical enzymology, biochemistry, microbiol-
ogy, molecular biology, and biochemical engineering, as well as by students in
graduate-levelcoursescoveringpracticalenzymologyandenzymebiochemistry.
Herbert A. Kirst
Wu-Kuang Yeh
Milton J. Zmijewski, Jr.

Contents
Preface iii
Contributors xi
I. Biosynthesis
1. d -(L-a -Aminoadipyl)-L-Cysteinyl-D-Valine Synthetase as a
Model Tripeptide Synthetase 1
Hans von Do¨hren, Wibke Kallow, Mary Anne Tavanlar, Torsten
Schwecke, Ralf Dieckmann, and Volker Uhlmann
2. Metabolic Engineering for Cephalosporin C Yield Improvement
and Production of Intermediates 39
Joe E. Dotzlaf, Steven W. Queener, and Wu-Kuang Yeh
3. Bioconversion of Penicillins to Cephalosporins 61
Arnold L. Demain, Jose L. Adrio, and Jacqueline M. Piret
4. Direct Fermentative Production of Acyltylosins by Genetically
Engineered Strains of Streptomyces fradiae 89
Akira Arisawa and Hiroshi Tsunekawa
vii
viii Contents
5. Engineering Streptomyces avermitilis for the Production of
Novel Avermectins: Mutant Design and Titer Improvement 113
Claudio D. Denoya, Kim J. Stutzman-Engwall, and Hamish
A. I. McArthur
II. Biocatalysis
6. Biocatalytic Syntheses of Chiral Intermediates for
Antihypertensive Drugs 137
Ramesh N. Patel
7. Cloning, Structure, and Activity of Ketone Reductases from
Baker’s Yeast 175
Jon D. Stewart, Sonia Rodr´ıguez, and Margaret M. Kayser
8. Cross-Linked Enzyme Crystals: Biocatalysts for the Organic
Chemist 209
Michael D. Grim
9. Enzymatic Deacylation of Echinocandins and Related
Antifungal Agents 227
Andrew R. Cockshott, Adam J. Kreuzman, and Wu-Kuang Yeh
III. Screening/Optimization
10. Roles of Enzymes in Antibacterial Drug Discovery 245
Siddhartha Roychoudhury
11. Penicillin-Binding Proteins as Antimicrobial Targets:
Expression, Purification, and Assay Technologies 263
Genshi Zhao, Timothy I. Meier, and Wu-Kuang Yeh
12. Development of a High-Throughput Screen for Streptococcus
pneumoniae UDP-N-Acetylmuramoyl-Alanine: d-Glutamate
Ligase (MurD) for the Identification of MurD Inhibitors 289
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
13. Purification and Assay Development for Human Rhinovirus
Proteases 307
Q. May Wang and Robert B. Johnson
Contents ix
14. Screening for Parasiticides Using Recombinant Microorganisms 323
Timothy G. Geary
15. Screening for Inhibitors of Lipid Metabolism 343
Hiroshi Tomoda and Satoshi O¯mura
16. Design and Development of a Selective Assay System for the
Phospholipase A Superfamily 379
2
Hsiu-Chiung Yang, Marian Mosior, and Edward A. Dennis
IV. Emerging Technologies
17. Understanding and Exploiting Bacterial Polyketide Synthases 397
Robert McDaniel and Chaitan Khosla
18. Polyketide Synthases: Analysis and Use in Synthesis 427
Kira J. Weissman and James Staunton
19. Enzymatic Synthesis of Fungal N-Methylated Cyclopeptides and
Depsipeptides 471
Mirko Glinski, Till Hornbogen, and Rainer Zocher
20. New Strategies for Target Identification, Validation, and Use of
Enzymes in High-Throughput Screening 499
Joaquim Trias and Zhengyu Yuan
21. Use of Genomics for Enzyme-Based Drug Discovery 515
Molly B. Schmid
22. Assigning Precise Function to Genes 537
Ridong Chen
23. Redesigning Binding and Catalytic Specificities of Enzymes 555
Ridong Chen
24. Proteomics: Chromatographic Fractionation Prior to Two-
Dimensional Polyacrylamide Gel Electrophoresis for Enrichment
of Low-Abundance Proteins to Facilitate Identification by Mass
Spectrometric Methods 575
Srinivasan Krishnan, John E. Hale, and Gerald W. Becker
Index 597