Enzyme Technologies for Pharmaceutical and Biotechnological Applications

ISBN: 0-8247-0549-1

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Preface

Genes related to human and animal health are being discovered at an ever-increas￾ing 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 pri￾mary 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 interde￾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

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 recombi￾nant 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. Ma￾nipulation of the enzyme tools by genetic engineering is described. Chapter 8

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 rel￾evant 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 sam￾ples 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 prac￾tical 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 under￾standing the molecular and kinetic interactions between the enzyme and its inhibi￾tor. These insights are typically gained by analyzing X-ray crystallographic depic￾tions 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 im￾portance 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, alterna￾tively, can interchange portions of gene clusters between organisms. In each in￾stance, 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 biosyn￾thesis or combinatorial enzymology, thereby leading to even larger numbers of

new natural products. Extensive interdisciplinary collaboration between new tar￾get 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, as￾signing 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 disease￾relevant enzyme targets depends closely on the molecular understanding of these

targets under physiological and pathological conditions ( functional proteomics).

Enzyme Technologies for Pharmaceutical and Biotechnological Applica￾tions is informative, practical, timely, and applicable worldwide to the pharma￾ceutical and biotechnological industries. Real-world examples provided through￾out 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 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, and manufacturing. The book can also be used by gradu￾ate and postdoctoral students in practical enzymology, biochemistry, microbiol￾ogy, 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 iii

Contributors xi

I. Biosynthesis

1. δ-(L-α-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 Rodrı´guez, 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 A2 Superfamily 379

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

Contributors

Jose L. Adrio Department of Biochemistry, Antibioticos, S.A.U., Leo´n, Spain

Akira Arisawa Biochemistry Laboratory, Central Research Laboratories, Mer￾cian Corporation, Fujisawa, Japan

Gerald W. Becker Lilly Research Laboratories, Eli Lilly and Company, India￾napolis, Indiana

Gary M. Birch Lilly Research Laboratories, Eli Lilly and Company, Indianap￾olis, Indiana

Ridong Chen Department of Biochemistry, University of Saskatchewan, Sas￾katoon, Saskatchewan, Canada

Andrew R. Cockshott Lilly Research Laboratories, Eli Lilly and Company,

Indianapolis, Indiana

James A. Cook Lilly Research Laboratories, Eli Lilly and Company, Indianap￾olis, Indiana

Karen L. Cox Lilly Research Laboratories, Eli Lilly and Company, Indianapo￾lis, Indiana

xi

xii Contributors

Arnold L. Demain Department of Biology, Massachusetts Institute of Tech￾nology, Cambridge, Massachusetts

Edward A. Dennis Department of Chemistry and Biochemistry, Revelle Col￾lege and School of Medicine, University of California, San Diego, California

Claudio D. Denoya Bioprocess Research, Global Research and Development,

Pfizer, Inc., Groton, Connecticut

Ralf Dieckmann Biotechnology Center, Technical University Berlin, Berlin,

Germany

Joe E. Dotzlaf Lilly Research Laboratories, Eli Lilly and Company, Indianapo￾lis, Indiana

Timothy G. Geary Discovery Research, Pharmacia Animal Health, Kalama￾zoo, Michigan

Mirko Glinski Max Volmer Institute for Biophysical Chemistry and Biochem￾istry, Technical University Berlin, Berlin, Germany

Michael D. Grim Westboro, Massachusetts

John E. Hale Lilly Research Laboratories, Eli Lilly and Company, Indianapo￾lis, Indiana

Stephen A. Hitchcock Lilly Research Laboratories, Eli Lilly and Company,

Indianapolis, Indiana

Till Hornbogen Max Volmer Institute for Biophysical Chemistry and Bio￾chemistry, Technical University Berlin, Berlin, Germany

Joann Hoskins Lilly Research Laboratories, Eli Lilly and Company, Indianap￾olis, Indiana

Robert B. Johnson Lilly Research Laboratories, Eli Lilly and Company, India￾napolis, Indiana

Wibke Kallow AnagnosTec GmbH, Luckenwalde, Germany

Margaret M. Kayser Department of Chemistry, University of New Bruns￾wick, Saint John, New Brunswick, Canada

Contributors xiii

Chaitan Khosla Departments of Chemistry and Chemical Engineering, Stan￾ford University, Stanford, California

Adam J. Kreuzman Lilly Research Laboratories, Eli Lilly and Company, Indi￾anapolis, Indiana

Srinivasan Krishnan Lilly Research Laboratories, Eli Lilly and Company, In￾dianapolis, Indiana

Hamish A. I. McArthur Bioprocess Research, Global Research and Develop￾ment, Pfizer, Inc., Groton, Connecticut

Robert McDaniel Kosan Biosciences, Inc., Hayward, California

Timothy I. Meier Lilly Research Laboratories, Eli Lilly and Company, India￾napolis, Indiana

Marian Mosior Lilly Research Laboratories, Eli Lilly and Company, Indianap￾olis, Indiana

Satoshi O¯mura Research Center for Biological Function, The Kitasato Insti￾tute and Graduate School of Pharmaceutical Sciences, The Kitasato Institute and

Kitasato University, Tokyo, Japan

Ramesh N. Patel Process Research and Development, Bristol-Myers Squibb

Company, New Brunswick, New Jersey

Robert B. Peery Lilly Research Laboratories, Eli Lilly and Company, India￾napolis, Indiana

Jacqueline M. Piret Biology Department, Northeastern University, Boston,

Massachusetts

Steven W. Queener Lilly Research Laboratories, Eli Lilly and Company, Indi￾anapolis, Indiana

Sonia Rodrı´guez Department of Chemistry, University of Florida, Gainesville,

Florida

Siddhartha Roychoudhury Discovery-Biology, Procter & Gamble Pharma￾ceuticals, Mason, Ohio

xiv Contributors

Molly B. Schmid Microcide Pharmaceuticals, Inc., Mountain View, California

Torsten Schwecke Biotechnology Center, Technical University Berlin, Berlin,

Germany

Paul L. Skatrud Lilly Research Laboratories, Eli Lilly and Company, India￾napolis, Indiana

Michele C. Smith Lilly Research Laboratories, Eli Lilly and Company, India￾napolis, Indiana

James Staunton Department of Chemistry, University of Cambridge, Cam￾bridge, United Kingdom

Jon D. Stewart Department of Chemistry, University of Florida, Gainesville,

Florida

Kim J. Stutzman-Engwall Bioprocess Research, Global Research and Devel￾opment, Pfizer, Inc., Groton, Connecticut

Mary Anne Tavanlar National Institute of Molecular Biology and Biotechnol￾ogy, University of the Philippines Los Ban˜os, Laguna, Philippines

Hiroshi Tomoda Research Center for Biological Function, The Kitasato Insti￾tute and Graduate School of Pharmaceutical Sciences, The Kitasato Institute and

Kitasato University, Tokyo, Japan

Joaquim Trias Department of Microbiology, Versicor, Inc., Fremont, Cali￾fornia

Hiroshi Tsunekawa Pharmaceuticals and Chemicals Division, Mercian Corpo￾ration, Tokyo, Japan

Volker Uhlmann Department of Histopathology, St. James’s Hospital, Dublin,

Ireland

Hans von Do¨hren Biotechnology Center, Technical University Berlin, Berlin,

Germany

Q. May Wang Lilly Research Laboratories, Eli Lilly and Company, Indianapo￾lis, Indiana