Thermophilic Old Yellow Enzyme: Structure and Kinetic Characterisation
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THERMOPHILIC OLD YELLOW ENZYME: STRUCTURE AND KINETIC CHARACTERISATION A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Life Sciences 2012 Björn Vidar Adalbjörnsson Table of Contents Table of Contents ................................................................................................................ 2 Figure list ............................................................................................................................ 7 Table list ............................................................................................................................ 10 Abstract ............................................................................................................................. 12 Declaration ........................................................................................................................ 13 Copyright .......................................................................................................................... 13 Abbreviations and Symbols .............................................................................................. 14 Equation list ...................................................................................................................... 18 Acknowledgements ........................................................................................................... 19 1. Introduction ............................................................................................................ 20 1.1 Enzymes ................................................................................................................. 21 1.2 Flavoproteins .......................................................................................................... 24 1.3 The OYE-family of flavoproteins .......................................................................... 25 1.3.1 OYE subclasses 26 1.3.2 Reaction mechanism of OYEs 26 1.3.3 Structural features of the OYE family 34 1.3.3.1 Structural features of classical OYE ............................................................ 39 1.3.3.2 Structural features of the thermophilic-like OYEs ...................................... 40 1.4 Biocatalysis ............................................................................................................ 41 1.4.1 Biocatalysis potential of the OYE-family 42 1.5 Quantum mechanical tunnelling ............................................................................ 46 1.5.1 Transition state theory 46 1.5.2 Theory of Quantum Mechanical Tunnelling 48 1.5.2.1 The wave/particle duality ............................................................................. 49 1.5.3 Hydrogen tunnelling 51 1.5.4 Comparison of transition state and tunnelling 51 1.5.3.2 Kinetic isotope effect ................................................................................... 53 1.6 Temperature studies on tunnelling and enzyme dynamics .................................... 55 1.7 Extremophilic Enzymes ......................................................................................... 59 1.7.1 Thermo- and hyperthermophiles .................................................................. 61 2 1.8 Thermophilic Old Yellow Enzyme: ....................................................................... 62 1.9 Aims and Objectives .............................................................................................. 63 2. Materials and Methods ........................................................................................... 64 2.1 Materials ................................................................................................................. 66 2.1.1 Media and solutions 68 2.2 Synthesis and Cloning of TOYE ............................................................................ 69 2.2.1 Transformation of TOYE into expression strains 69 2.2.2 Plasmid synthesis and purification 70 2.2.3 Removal of the His6-tag from TOYE-His6 70 2.3 Expression trials of the recombinant TOYE-His6 .................................................. 72 2.4 Large scale production and purification of TOYE ................................................. 72 2.4.1 Pretreatment of dialysis membranes 72 2.4.2 Large scale of production of TOYE 72 2.4.3 Purification of TOYE-His6 73 2.4.4 Purification of native TOYE 73 2.5 Protein purity and molecular mass determination .................................................. 74 2.5.1 Polyacrylamide gel electrophoresis 74 2.5.2 Staining and destaining of polyacrylamide gels 74 2.6 Determination of enzyme concentration ................................................................ 75 2.7 Re-flavination of TOYE ......................................................................................... 76 2.8 Temperature stability studies on TOYE-His6 ........................................................ 76 2.8.1 Circular dichroism studies on TOYE-His6 76 2.8.2 Fluorescence spectroscopy 77 2.9 Crystallisation of TOYE ........................................................................................ 77 2.9.1 Screening for crystallisation conditions of TOYE 77 2.9.2 Crystallisation optimisation trials of TOYE-His6 and TOYE-NADH4 78 2.9.3 X-ray diffraction data collection and processing 78 2.10 Sedimentation velocity and bead modelling of TOYE .......................................... 79 2.11 Transmission Electron Microscopy of TOYE ....................................................... 79 2.12 Multi angle laser light scattering studies on TOYE ............................................... 79 2.13 Isotopologues for kinetic studies and crystallisation ............................................. 80 2.13.1 Isotopologue synthesis 80 3 2.13.2 Isotopologue purification 80 2.14 Preparation of anaerobic solutions ......................................................................... 81 2.15 Potentiometric titrations of TOYE ......................................................................... 81 2.16 Enzyme kinetics studies ......................................................................................... 84 2.16.1 Steady-state kinetic measurements of TOYE-His6 84 2.16.2 Stopped-flow kinetic studies of the reductive half-reaction of TOYE. 85 2.17 Biotransformations of TOYE-His6 ......................................................................... 86 2.18 Temperature-dependence of observed kinetics ...................................................... 86 2.19 Error propagation of observed rates and KIEs ...................................................... 87 3. Enzyme production and general properties ............................................................ 88 3.1 Plasmid synthesis and transformations .................................................................. 89 3.2 Induction trials for TOYE ...................................................................................... 91 3.3 Production and purification of TOYE .................................................................... 92 3.4 Initial transient kinetics of the reductive half-reaction of TOYE .......................... 93 3.5 Thermal stability of TOYE .................................................................................... 95 3.6 Redox potentiometry of electron transfer in TOYE ............................................... 97 3.7 Discussion ............................................................................................................ 100 4. Structural studies of TOYE .................................................................................. 101 4.1 Transmission Electron Microscopy of TOYE 102 4.2 Generation of the crystal structure of TOYE ....................................................... 102 4.2.1 Crystallisation of TOYE 102 4.2.2 Crystallographic data collection 104 4.2.3 Data processing and refinement 106 4.3 Structural analysis of TOYE ................................................................................ 108 4.3.1 Overall structure 108 4.3.2 Structural analysis of the crystal structures of TOYE-His6 and TOYE-NADH4 ....... 108 4.3.2 Functional dimer interactions 112 4.3.3 Interaction between non-functional dimers 112 4.3.4 Active site of holo-TOYE-His6 117 4.3.5 NADH4 binding 119 4.3.6 Overall structural comparison between mesophilic and thermophilic OYEs 124 4.4 Multi Angle Laser Light Scattering ..................................................................... 126 4 4.5 Sedimentation velocity and hydrodynamic bead model of TOYE ...................... 127 4.6 Discussion ............................................................................................................ 129 5. Substrate profiling of TOYE ................................................................................ 130 5.1 Specific activity of substrates.................................................................................... 131 5.1.1 2-Cyclohexanone and derivatives 132 5.1.2 Maleimide related substrates 132 5.1.3 Other substrates 132 5.2 Biotransformation studies with TOYE................................................................ 134 5.2.1 2-Methyl-cyclopentenone 135 5.2.2 Carvone 135 5.2.3 Maleimide related substrates 137 5.2.4 2-Methylpentenal 138 5.2.5 Citral 138 5.2.6 α,β-Unsaturated nitroalkene 139 5.2.7 Ketoisophorone 140 5.3 Optimisation of biotransformations ..................................................................... 140 5.3.1 Biocatalysis at elevated temperature 140 5.3.2 Time