Front matter The characterisation of the flavocytochrome P450-CPR fusion enzymes CYP505A30 from Myceliophthora thermophila and CYP102A1 from Bacillus megaterium. A thesis submitted to The University of Manchester for the degree of Doctor of Philosophy in the Faculty of Life Sciences. 2015 George Baker 1 Front matter List of Contents: List of Contents: ..................................................................................................................... 1 List of Figures: ....................................................................................................................... 8 List of Tables: ...................................................................................................................... 13 List of Abbreviations: .......................................................................................................... 15 Abstract ................................................................................................................................ 17 Declaration ........................................................................................................................... 18 Copyright Statement ............................................................................................................ 18 Acknowledgements .............................................................................................................. 19 Chapter 1: Introduction ........................................................................................................ 20 1.1 Enzyme Classes .......................................................................................................... 20 1.2 Oxidoreductase enzymes ............................................................................................ 20 1.3 Hemoproteins ............................................................................................................. 24 1.4 The Cytochrome P450 Superfamily ........................................................................... 28 1.5 Evolution of the Cytochrome P450 Superfamily ....................................................... 31 1.6 Classification of P450s ............................................................................................... 33 1.7 Major cytochrome P450 reactions .............................................................................. 34 1.8 The Diverse Physiology and Distribution of Cytochromes P450 .............................. 38 1.8.1 Human P450s ....................................................................................................... 38 1.8.2 Plant P450s .......................................................................................................... 39 1.8.3 Bacterial P450s ................................................................................................... 39 1.8.4 Fungal P450s....................................................................................................... 40 1.8.5 Viral P450s .......................................................................................................... 41 1.9 Cytochrome P450 Structure ....................................................................................... 42 1.10 P450 Heme Iron Spin State and Associated Redox Potential Changes.................... 45 1.11 The Cytochrome P450 Catalytic Cycle .................................................................... 48 1.11.1 The Final Solution to the Active Species Question: Elucidation of Compound I ...................................................................................................................................... 52 1.11.2 Rate Limiting Steps in the P450 Catalytic Cycle .............................................. 53 1.11.3 The Peroxide Shunt and Alternative P450 Mechanisms ................................... 54 1.12 Redox Partners ......................................................................................................... 55 1.12.2 Cytochrome P450 Redox Partner Diversity and Classification ......................... 58 1.12.3 P450 electron transfer pathways ........................................................................ 63 1.13 P450 BM3 and other P450 Fusions .......................................................................... 67 2 Front matter 1.14 Biotechnological applications of BM3 and other P450 enzymes ............................ 71 Chapter 2: Materials and Methods ....................................................................................... 77 2.1 Materials and gene constructs .................................................................................... 77 2.1.1 Bacterial cell strains ............................................................................................ 77 2.2 Media and buffer preparation ..................................................................................... 79 2.2.1 Growth media ...................................................................................................... 79 2.2.2 Antibiotics and supplements ............................................................................... 80 2.2.3 Agar plate media ................................................................................................. 80 2.2.4 Agarose gels ........................................................................................................ 80 2.2.5 Buffers ................................................................................................................. 81 2.2.5.1 Phosphate buffer ............................................................................................... 81 2.2.5.2 2’,5’-ADP Sepharose purification buffers ........................................................ 81 2.2.5.3 Imidazole buffer ............................................................................................... 81 2.2.5.4 Enzyme assay buffer ........................................................................................ 81 2.2.5.5 Tris EDTA buffer ............................................................................................. 82 2.3 Experimental procedures............................................................................................ 82 2.3.1 E. coli competent cell preparation ....................................................................... 82 2.3.2 Transformation of plasmid DNA ........................................................................ 83 2.3.3 Manufacture of glycerol stocks of transformed E. coli strains ............................ 83 2.3.4 Plasmid preparation ............................................................................................. 84 2.3.5 Restriction enzyme digests .................................................................................. 84 2.3.6 Ligation reactions ................................................................................................ 85 2.4 Plasmid construct generation ..................................................................................... 85 2.4.1 CYP505A30/pET15b construct generation .......................................................... 85 2.4.2 Generation of G46X heme domain construct by Site-Directed Mutagenesis (SDM) ........................................................................................................................... 86 2.5 Protein expression ...................................................................................................... 88 2.5.1 Small scale expression trials for P450 MT1. ....................................................... 88 2.5.2 P450 MT1 medium scale solubility trials ............................................................ 89 2.5.3 Large scale expression of WT P450 MT1 and the G463X P450 MT1 Heme Domain (HD) ................................................................................................................ 90 2.5.4 Large scale expression of WT P450 BM3, the BM3 HD (Heme Domain) and FAD domain constructs (WT, C773A and C999A Mutants). ...................................... 90 2.7 Enzyme purification ................................................................................................... 91 3 Front matter 2.7.1 Cell lysis by sonication for P450 MT1 and MT1 HD expression cells ............... 91 2.7.2 Ammonium sulfate precipitation ......................................................................... 91 2.7.3 Purification of P450 MT1 .................................................................................... 91 2.7.4 Purification of the P450 MT1 G463X Heme Domain (MT1 HD) and WT P450 BM3 .............................................................................................................................. 92 2.7.5 Purification of the P450 BM3 Heme Domain (BM3 HD) ................................... 93 2.7.6 Purification of WT, C120A and C120A/C999A P450 BM3 FAD domain proteins.......................................................................................................................... 94 2.8 P450 Protein concentration calculation ...................................................................... 95 2.9 Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) of protein samples ................................................................................................................. 96 2.10 Determination of WT P450 MT1 flavin content by High Performance Liquid Chromatography (HPLC) ................................................................................................