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Table of Contents Table of contents Preface .................................................................................................................. v 1 The reaction mechanisms of Groups A and B fl avoprotein monooxygenases ......................................................................................... 1 1.1 Introduction ........................................................................................ 1 1.2 Enzymes acting upon aromatic substrates – Group A........................... 2 1.2.1 Reactions catalyzed ................................................................... 2 1.2.2 Protein structures ....................................................................... 3 1.2.3 Detailed mechanism of PHBH ................................................... 4 1.2.3.1 Reductive half-reaction ................................................. 6 1.2.3.2 Oxidative half-reaction ................................................. 9 1.2.3.3 Hydroxylation chemistry .............................................. 15 1.2.3.4 Summary ...................................................................... 16 1.3 Enzymes acting upon non-aromatic substrates – Group B .................... 16 1.3.1 Reactions catalyzed and subclasses ........................................... 16 1.3.1.1 BVMOs ........................................................................ 17 1.3.1.2 FMOs ........................................................................... 19 1.3.1.3 NMOs .......................................................................... 20 1.3.1.4 YUCCAs ....................................................................... 21 1.3.2 Structural features ...................................................................... 22 1.4 References........................................................................................... 23 2 Flavin-dependent monooxygenases in siderophore biosynthesis .................. 29 2.1 Iron, an essential but scarce nutrient ................................................... 29 2.2 Siderophores ....................................................................................... 30 2.2.1 Siderophores are important virulence factors ............................. 30 2.2.2 Structural diversity of siderophores ............................................ 33 2.3 Flavin-dependent N-hydroxylating monooxygenases........................... 33 2.4 Catalytic cycle of NMOs ..................................................................... 34 2.4.1 Flavin reduction in NMOs ......................................................... 35 2.4.2 Flavin oxidation in NMOs ......................................................... 37 2.5 Three-dimensional structure of NMOs................................................. 37 2.5.1 FAD-binding domain ................................................................. 40 2.5.2 NADPH-binding domain ........................................................... 40 2.5.3 L-Ornithine-binding domain ...................................................... 41 2.6 The structural basis of substrate specifi city in NMOs ........................... 42 2.7 Mechanism of stabilization of the C4a-hydroperoxyfl avin by NADP+ ... 42 2.8 Activation of NMOs by amino acid binding ........................................ 44 2.9 Unusual NMOs ................................................................................... 44 2.10 High-throughput screening assay to identify inhibitors of NMOs ......... 45 2.11 Conclusions ........................................................................................ 46 2.12 References........................................................................................... 47 x Table of contents 3 The fl avin monooxygenases ......................................................................... 51 3.1 Introduction ........................................................................................ 51 3.2 Occurrence and classifi cation ............................................................. 54 3.2.1 Amino acid sequence motifs ...................................................... 54 3.2.2 DNA screening .......................................................................... 55 3.3 Single-component fl avin monooxygenases .......................................... 56 3.3.1 Subclass A ................................................................................. 56 3.3.2 Subclass B ................................................................................. 58 3.3.3 Subclass C ................................................................................. 60 3.3.4 Subclass D ................................................................................. 61 3.3.5 Subclass E .................................................................................. 62 3.3.6 Subclass F .................................................................................. 62 3.4 Conclusions ........................................................................................ 64 3.5 References........................................................................................... 65 4 Structure and catalytic mechanism of NADPH-cytochrome P450 oxidoreductase: a prototype of the difl avin oxidoreductase family of enzymes ............................................................... 73 4.1 Introduction ........................................................................................ 73 4.2 Properties of CYPOR fl avins ................................................................ 75 4.3 Domain structure and function ............................................................ 78 4.4 Membrane binding domain (MBD) ...................................................... 78 4.5 FMN domain ....................................................................................... 79 4.6 Cytochrome P450 binding: role of the FMN domain and connecting domain .......................................................... 80 4.7 FAD domain........................................................................................ 82 4.8 Mechanism of hydride transfer ............................................................ 82 4.9 Interfl avin electron transfer .................................................................. 83 4.10 FMN to heme electron transfer ............................................................ 85 4.11 P450 catalysis ..................................................................................... 85 4.12 Other CYPOR electron acceptors ........................................................ 87 4.13 CYPOR domain movement and control of electron transfer ................. 87 4.14 Physiological functions of CYPOR and effects of CYPOR defi ciency ... 90 4.15 Human CYPOR defi ciency (PORD) ..................................................... 91 4.16 Contribution of CYPOR to inter-individual variation in human drug metabolism ................................................................................. 92 4.17 Unanswered questions and future directions ....................................... 92 4.18 References ........................................................................................... 93 5 The xanthine oxidoreductase enzyme family: xanthine dehydrogenase, xanthine oxidase, and aldehyde oxidase ...................................................... 103 5.1 Introduction ........................................................................................ 103 5.2 Overall structures ................................................................................ 104 5.3 Reaction mechanism ........................................................................... 105 5.4 Electron transfer from the molybdenum center to other redox-active centers ............................................................................ 113 5.5 Reaction of FAD with NAD+ or molecular oxygen ............................... 114 Table of contents xi 5.6 Inhibitors of xanthine oxidoreductase .................................................. 116 5.7 References........................................................................................... 120 6 Assimilatory nitrate reductase ..................................................................... 125 6.1 Introduction and scope........................................................................ 125 6.2 Enzyme structure ................................................................................. 126 6.3 Kinetics and mechanism...................................................................... 131 6.4 Post-translational regulation ................................................................ 133 6.5 Interconversion of sulfi te oxidase and nitrate reductase activities ........ 135 6.6 Conclusions ........................................................................................ 137 6.7 References........................................................................................... 138 7 Succinate dehydrogenase (Complex II) and fumarate reductase .................. 141 7.1 History of Complex II .......................................................................... 141 7.2 Overview of Complex II ...................................................................... 143 7.3 Structure of Complex II........................................................................ 144 7.4 Catalytic assays ................................................................................... 146 7.5 Catalytic mechanism and domain movement .....................................
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