Modern Biooxidation

Enzymes, Reactions and Applications

Edited by Rolf D. Schmid and Vlada B. Urlacher

Modern Biooxidation

Edited by Rolf D. Schmid and Vlada B. Urlacher 1807–2007 Knowledge for Generations

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Enzymes, Reactions and Applications

Edited by Rolf D. Schmid and Vlada B. Urlacher The Editors All books published by Wiley-VCH are carefully produced. Nevertheless, authors, editors, and Prof. Dr. Rolf D. Schmid publisher do not warrant the information Universität Stuttgart contained in these books, including this book, to Institut für Technische Biochemie be free of errors. Readers are advised to keep in Allmandring 31 mind that statements, data, illustrations, 70569 Stuttgart procedural details or other items may Germany inadvertently be inaccurate.

Dr. Vlada B. Urlacher-Kursif Library of Congress Card No.: Universität Stuttgart applied for Institut für Technische Biochemie Allmandring 31 British Library Cataloguing-in-Publication Data 70569 Stuttgart A catalogue record for this book is available from Germany the British Library.

Cover Bibliographic information published by the Crystal structure of human cytochrome Deutsche Nationalbibliothek p450 2C9 (PDB entry 1OG5A) complexed The Deutsche Nationalbibliothek lists this with substrate warfarin (green) [1]. publication in the Deutsche Nationalbibliografi e; The mesh represents the substrate binding detailed bibliographic data are available in the cavity inside the enzyme. The approach of Internet at 〈http://dnb.d-nb.de〉. the substrate (blue) to the heme (red) was observed during molecular dynamics © 2007 WILEY-VCH Verlag GmbH & Co. KGaA, simulations [2]. Weinheim

1 Williams, P.A., Cosme, J., Ward, A., Angove, All rights reserved (including those of translation H.C., Matak Vinkovic, D., Jhoti, H. Crystal into other languages). No part of this book may be structure of human cytochrome p450 2C9 reproduced in any form – by photoprinting, with bound warfarin. Nature 2003, 424, microfi lm, or any other means – nor transmitted 464–468. or translated into a machine language without 2 Seifert, A., Tatzel, S., Schmid, R.D., Pleiss, J. written permission from the publishers. Multiple molecular dynamics simulations of Registered names, trademarks, etc. used in this human p450 monooxygenase CYP2C9: the book, even when not specifi cally marked as such, molecular basis of substrate binding and are not to be considered unprotected by law. regioselectivity toward warfarin. Proteins 2006, 64(1), 147–155. Composition SNP Best-set Typesetter Ltd., Hong Kong

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Contents

1 Biooxidation with PQQ- and FAD-Dependent Dehydrogenases 1 Osao Adachi, Yoshitaka Ano, Hirohide Toyama, and Kazunobu Matsushita 1.1 Introduction 1 1.2 Basic Technical Information Regarding Membrane-bound Enzymes 4 1.2.1 Preparation of Cytosolic Fractions and Membrane Fractions 4 1.2.2 EDTA Treatment of the Membrane Fraction Carrying PQQ as Coenzyme 5 1.2.3 Assays of Enzyme Activity 5 1.3 PQQ-Dependent Dehydrogenases 6 1.3.1 Alcohol Oxidation 6 1.3.1.1 Membrane-Bound Alcohol Dehydrogenase (ADH III) 6 1.3.1.2 Soluble Alcohol Dehydrogenases 9 1.3.1.3 Cyclic Alcohol Dehydrogenase (Secondary Alcohol Dehydrogenase), Membrane-Bound 9 1.3.2 Glucose Oxidation 11 1.3.2.1 Membrane-Bound D-Glucose Dehydrogenase (m-GDH) 11 1.3.2.2 Soluble D-Glucose Dehydrogenase (s-GDH) 12 1.3.2.3 Applications of Quinoprotein GDHs as D-Glucose Sensors 13 1.3.3 Polyol Oxidation 14 1.3.3.1 D-Arabitol Dehydrogenase, Membrane-Bound 14 1.3.3.2 meso-Erythritol Oxidation Dehydrogenase, Membrane-Bound 16 1.3.3.3 D-Gluconate Oxidizing Polyol Dehydrogenase, Membrane-Bound 17 1.3.3.4 Glycerol Dehydrogenase, Membrane-Bound 19 1.3.3.5 D-Mannitol Dehydrogenase, Membrane-Bound 20 1.3.3.6 Ribitol Dehydrogenase, Membrane-Bound 21 1.3.3.7 D-Sorbitol Dehydrogenase, Membrane-Bound 22 1.3.3.8 L-Sorbosone Dehydrogenase, Membrane-Bound 23 1.3.4 Quinate Oxidation. Membrane-Bound Quinate Dehydrogenase (QDH) 24 1.4 FAD-Dependent Dehydrogenase 27 1.4.1 D-Fructose Dehydrogenase, Membrane-Bound 27 VI Contents

1.4.2 D-Gluconate Dehydrogenase, Membrane-Bound 28 1.4.3 D-Hexosamine Dehydrogenase, Membrane-Bound 29 1.4.4 2-Keto-D-gluconate Dehydrogenase, Membrane-Bound 31 1.4.5 Sorbitol Dehydrogenase, Membrane-Bound 32 1.5 Miscellaneous 33 1.5.1 Aldehyde Dehydrogenase, Membrane-Bound 33 References 35

2 Catalytic Applications of Laccase 43 Feng Xu, Ture Damhus, Steffen Danielsen, and Lars Henrik Østergaard 2.1 Properties of Classical Laccase 43 2.1.1 Structure 43 2.1.2 Enzymology 44 2.1.3 4As Industrial Catalysts 46 2.1.3.1 Advantages 46 2.1.3.2 Shortcomings 48 2.2 Applications of Laccase for Industrial Oxidation Processes 48 2.2.1 Laboratory-Level Trials 49 2.2.1.1 Delignifi cation 49 2.2.1.2 Dye and Colorant Bleaching 50 2.2.1.3 Bioremediation 50 2.2.1.4 Other Degradation Applications 51 2.2.1.5 Functional Biotransformation 51 2.2.1.6 Biosensing 53 2.2.1.7 Desirable Application Modes 53 2.2.2 Commercialized Applications 55 2.2.2.1 Preventing Taint in Cork Stoppers 56 2.2.2.2 Denim Bleaching 56 2.2.2.3 Paper Mill Effl uent Treatment and Cardboard Strengthening 56 2.2.2.4 Major Hurdles to Further Development from Laboratory Trials 57 2.3 More Recent Developments 57 2.3.1 Novel Laccase Catalytic Systems 57 2.3.1.1 New Laccases 57 2.3.1.2 New Mediators 60 2.3.1.3 Cooperation with Other Enzymes 62 2.3.2 New Leads for Laccase Application 62 2.3.2.1 Laccase-Based Defense Against Biological and Chemical Warfare Agents 62 2.3.2.2 Degradation of PAH, Plastics, or Lipids 63 2.3.2.3 Enzymatic Fuel Cells/Batteries 64 2.3.2.4 Novel Synthetic Applications 65 2.3.2.5 Biorefi nery 66 2.4 Further Developing Laccase Catalysis 66 2.4.1 Laccase Engineering 66 Contents VII

2.4.2 Laccase Production 67 References 68

3 Biocatalytic Scope of Baeyer–Villiger Monooxygenases 77 Marco W. Fraaije and Dick B. Janssen 3.1 Introduction 77 3.1.1 The Baeyer–Villiger Reaction 77 3.1.2 Baeyer–Villiger Biocatalysts: Classifi cation and Occurrence 78 3.1.2.1 Type I Baeyer–Villiger Monooxygenases 78 3.1.2.2 Type II Baeyer–Villiger Monooxygenases 78 3.1.2.3 Alternative Baeyer–Villiger Biocatalysts 79 3.2 Type I Baeyer–Villiger Monooxygenases: Versatile Oxidative Biocatalysts 80 3.2.1 Mechanistic and Structural Properties of Type I BVMOs 80 3.2.2 Diversity 84 3.2.3 Molecular Features 86 3.2.4 Kinetic Characteristics 86 3.2.5 Coenzyme Dependency 87 3.2.6 Uncoupling and Overoxidation 88 3.2.7 Biocatalyst Stability 88 3.2.8 Substrate Specifi city 89 3.2.9 Unexplored Type I BVMOs 90 3.2.10 Mining Genomes for Novel BVMOs 92 3.3 Concluding Remarks 93 References 94

4 The Bacterial Cytochrome P450 Monooxygenases: P450cam and P450BM-3 99 Vlada B. Urlacher, Stephen G. Bell, and Luet-Lok Wong 4.1 Introduction 99 4.2 Biotransformation by Bacterial P450 Enzymes 99 4.3 General Features of P450cam and P450BM-3 102 4.3.1 Aromatic Compounds 105 4.3.2 Alkanes and Alicyclics 109 4.3.3 Terpenoid Compounds 111 4.3.4 Human Metabolites 114 4.4 The Scope of P450 Engineering 116 References 117

5 Cytochrome P450 Redox Partner Systems: Biodiversity and Biotechnological Implications 123 Andrew W. Munro, Hazel M. Girvan, Joseph P. McVey, and Kirsty J. McLean 5.1 Introduction 123 5.2 P450 Redox Partners 124 VIII Contents

5.2.1 A “Historical” Perspective 124 5.2.2 The P450 Catalytic Cycle and Electron Transfer Events 125 5.2.3 P450cam and its Reductase System 127 5.2.4 Adrenodoxin and Adrenodoxin Reductase 128 5.2.5 Cytochrome P450 Reductase 129 5.2.6 P450BM-3 and Related CPR Fusion Enzymes 131 5.2.7 A Novel Class of P450–Redox Partner Fusion Enzymes 136 5.3 Increasing P450–Redox Partner Complexity: Flavodoxins and Diverse Ferredoxins 137 5.4 Natural and Artifi cial P450–Redox Partner Fusion Enzymes and their Biocatalytic Potential 138 5.5 Other Routes to Driving P450 Catalytic Function 140 5.6 Uncoupling, Enzyme Stability and Coenzyme Issues 142 5.7 Future Prospects 143 References 145

6 Steroid Hydroxylation: Microbial Steroid Biotransformations Using Cytochrome P450 Enzymes 155 Matthias Bureik and Rita Bernhardt 6.1 Introduction 155 6.2 Cytochrome P450-Dependent Steroid Hydroxylase Systems 156 6.3 Native Microorganisms in Steroid Biotransformation 159 6.3.1 11α-Hydroxylation 160 6.3.2 11β-Hydroxylation 161 6.3.3 16α-Hydroxylation 162 6.3.4 Conclusions 163 6.4 Genetically Modifi ed Microorganisms in Steroid Biotransformation 163 6.4.1 Soluble Cytochromes P450 164 6.4.2 Membrane-Bound Cytochromes P450 166 6.5 Synopsis and Concluding Remarks 170 References 171

7 A Modular Approach to Biotransformation Using Microbial Cytochrome P450 Monooxygenases 177 Akira Arisawa and Hitosi Agematu 7.1 Introduction 177 7.2 Experimental Outline 180 7.2.1 Gene Sequences 180 7.2.1.1 pT7NS-camAB 180 7.2.1.2 Plasmids to Express Bacterial CYPs 180 7.2.2 Preparation of Whole Cell Catalysts 181 7.2.3 Biotransformation of the CYP Substrates 181 7.2.3.1 Carbomycin A 181 Contents IX

7.2.3.2 Pravastatin 182 7.2.3.3 7-Hydroxycoumarin 182 7.2.4 Biotransformation by CYP Reaction Array 182 7.3 Bacterial CYP Expression System in E. coli 183 7.4 Construction of a Bacterial CYP Library 185 7.5 Construction of a Bacterial CYP Reaction Array 186 7.6 Application of the CYP Reaction Array to Biotransformation Screening 187 References 190

8 Selective Microbial Oxidations in Industry: Oxidations of Alkanes, Fatty Acids, Heterocyclic Compounds, Aromatic Compounds and Glycerol Using Native or Recombinant Microorganisms 193 Albrecht Weiss 8.1 Introduction 193 8.2 Selective Oxidation of Hydrocarbons and Fatty Acids 194 8.2.1 Alkane Oxidation to Medium-Chain Alcohols [11] 194 8.2.2 Alkane and Fatty Acid Oxidation to Dicarboxylic Acids 196 8.2.2.1 Alkanes 197 8.2.2.2 Dicarboxylic Acids 197 8.3 Aromatic Compounds/Fine Chemicals 198 8.3.1 Conversion of Toxic Compounds: Catechols 198 8.3.2 Production of (R)-2-(4-Hydroxyphenoxy)propionic Acid 199 8.3.3 Selective Oxidation to Aromatic Aldehydes with Recombinant Cells 200 8.3.4 Styrene Oxide Production in a Two-Liquid Phase System 200 8.4 Heterocyclic Compounds 200 8.4.1 Enzymatic Oxidation of Methyl Groups in Aromatic Heterocycles 201 8.4.2 Preparation of 6-Hydroxynicotinic Acid 202 8.4.3 Preparation of 5-Hydroxypyrazinecarboxylic Acid 202 8.4.4 Preparation of 6-Hydroxy-(S)-nicotine and 4-[6-Hydroxypyridin-3-yl]4-oxobutyrate 202 8.4.5 Bulk Chemicals/Indigo 203 8.5 Glycerol Conversion to Dihydroxyacetone 206 8.6 Perspectives 207 References 207

9 Preparation of Drug Metabolites using Fungal and Bacterial Strains 211 Oreste Ghisalba and Matthias Kittelmann 9.1 Introduction 211 9.2 Phase I Drug-Metabolizing Enzymes 212 9.3 Needs and “Platforms” for the Generation of Drug Metabolites 214 X Contents

9.3.1 Recombinant Human Cytochrome P450 (rhCYP) Systems (acquired from British Technology Group/University of Dundee) 215 9.3.2 Microbial Strains Performing Oxidative Reactions (in-house technology) 215 9.4 Microbial Models for Oxidative Drug Metabolism 215 9.4.1 2Prokaryotic P450s 218 9.4.2 Microbial Eukaryotic P450s 218 9.5 Correlation of Microbial and Mammalian Oxidative Drug Metabolism 221 9.6 Correlation of Microbial Reactions with Human CYP Isozyme-Specifi c Reactions 221 9.7 Novartis Research Examples of Microbial Hydroxylations 225 9.7.1 Preparation of 10,11-Epoxy-carbamazepine and 10,11-Dihydro-10-hydroxy-carbamazepine 225 9.7.2 Preparation of 4-(4′-Hydroxyanilino)-5-anilinophthalimide and 4,5-Bis-(4′-hydroxyanilino)-phthalimide by Microbial Hydroxylation 227 9.8 Microbial Oxidation of Natural Products 228 9.8.1 Microbial Hydroxylation and Epoxidation of Milbemycins 229 9.9 Conclusions 229 References 231

10 Recombinant Yeast and Bacteria that Express Human P450s: Bioreactors for Drug Discovery, Development, and Biotechnology 233 Steven P. Hanlon, Thomas Friedberg, C. Roland Wolf, Oreste Ghisalba, and Matthias Kittelmann 10.1 Background 234 10.1.1 Importance of Recombinant P450s for Drug Development 234 10.1.2 Fundamentals of Heterologous Expression in Bacteria 235 10.1.3 Fundamentals of Heterologous Expression in Yeast 236 10.2 Comparison of P450 Levels and Enzymic Activities in Various Models 237 10.3 Use of E. coli P450 Expression Systems in Bioreactors 240 10.3.1 General Considerations 240 10.3.2 The Roche Experience 240 10.3.2.1 Background and Utility of P450 Systems in Pharma Research 240 10.3.2.2 Fermentation of Recombinant E. coli 241 10.3.2.3 Biotransformations Catalyzed by Recombinant CYP450 241 10.3.2.4 Preparation of N-Desethyl Amodiaquine 242 10.3.3 The Novartis Experience 244 10.3.3.1 Introduction 244 10.3.3.2 Production of E. coli Cells with CYP Activity 244 10.3.3.3 Whole Cell Biotransformation 246 10.3.3.4 Recent Developments 246 Contents XI

10.4 Conclusion 246 References 247

11 Human Cytochrome P450 Monooxygenases – a General Model of Substrate Specifi city and Regioselectivity 253 Jürgen Pleiss 11.1 Introduction 253 11.2 What Can We Learn From Sequence? 254 11.2.1 The Cytochrome P450 Engineering Database (CYPED) 254 11.2.2 The Effect of Mutations on Activity 255 11.3 What Can We Learn from Structure? 258 11.3.1 2The Role of Flexibility 258 11.3.2 The Role of Binding Site Shape 259 11.4 Conclusion 261 References 262

12 Approaches to Recycling and Substituting NAD(P)H as a CYP Cofactor 265 Dirk Holtmann and Jens Schrader 12.1 Introduction 265 12.2 Chemical Substitution of Cofactors 266 12.3 Enzymatic Regeneration of Cofactors 267 12.4 Photochemical Approaches to Substituting or Regenerating Cofactors for P450 Systems 271 12.5 Electrochemical Systems for Substitution or Regeneration of Cofactors 272 12.5.1 Electrochemical Regeneration of Natural Cofactors 273 12.5.2 Electrochemical Regeneration of Artifi cial Cofactors 274 12.5.3 Electrochemical Generation of Hydrogen Peroxide 275 12.5.4 Electrochemistry of P450 at Modifi ed Electrodes 275 12.5.5 Electrochemistry of P450 in Surfactant Films 276 12.5.6 Incorporation of Cytochrome P450 in Conducting Polymers 278 12.6 Redox Mediators 278 12.7 Molecular Biological Approaches 280 12.7.1 Peroxide Shunt 280 12.7.2 Artifi cial Electron Transfer Systems 281 12.7.3 Changing the Cofactor Specifi city of P450 Systems 281 12.7.4 Intracellular Cofactor Regeneration 282 12.8 Conclusion and Outlook 282 References 284

Index 291

XIII

Preface

In recent decades, biochemical science and technology has made tremendous progress. A recent survey among US universities has revealed that nearly half of academic funding is devoted to the life sciences [1], and a similar situation pre- vails in most other industrialized countries. In spite of this massive investment, the share of biotechnology in the production of energy and chemicals is small – educated estimates are in the range of 3% (R Diercks, BASF AG, personal com- munication). Thus, much remains to be done to develop the “biorefi nery” concept (see, e.g. ref. [2]) from vision into reality. To date, enzyme technology in industry has been mostly restricted to selective hydrolyses or ester/amide bond formations. Important unit operations in chemi- cal synthesis such as C-C bond formation or selective oxidations have remained the domain of the synthetic organic chemist, who can fi nd in his or her textbooks a vast number of protocols for the transformation of many, if not most types of possible structures. Enzymatic methods have been less successful in these do- mains, major stumbling blocks being the lack of enzymes with the required se- lectivity and the need for expensive cofactors such as NADH or ATP. More recently, techniques such as protein engineering, directed evolution, co- factor regeneration, and metabolic engineering have opened up new avenues to remove these bottlenecks. In addition, new types of enzymes have appeared on the stage of enzyme technology. Thus, among the oxidizing enzymes cytochrome and fl avine monooxygenases, a class of enzymes hitherto unknown in the context of enzyme technology, are now being hotly investigated for use in fi ne chemical selective oxidation. Also laccases, mostly known for their potential as bleaching agents for paper pulp, are just being rediscovered as candidates for selective biooxidation. Research in advanced enzyme technology is global. In fact, a Japanese program on “Green Biotechnology”, inaugurated as early as 2000 and now in its second 5- year cycle (S Shimizu, Coordinator of METI project “Green Biotechnology”, per- sonal communication), has made signifi cant advances towards the design of se- lective oxidative enzymes. It is thus our great privilege to have collected 12 contributions from leaders in this area of research with pharmaceutical or chemical backgrounds, from