Thermophilic Old Yellow Enzyme: Structure and Kinetic Characterisation

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

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 143 5.3.3 Solvent and substrate concentration 143 5.3.4 Solvent type 145 5.4 Discussion ...... 147 6. Temperature-dependence of 1° KIE of TOYE ...... 149 6.1 Transient state traces and interpretation ...... 150 6.2 Coenzyme specificity of TOYE ...... 154 6.2.1 Coenzyme kinetics 154 6.2.2 Binding of nonreactive coenzyme analogues and CT-complex formation 155 6.3 Temperature dependence of the reductive half-reaction ...... 156 6.3.1 Temperature dependences of hydride transfer with NAD(P)H 156 6.3.2 Temperature dependence of 1° KIEs 158 6.3.3 Temperature dependence of 2° KIEs 162 6.3.4 Temperature dependence of 1° KIE in steady-state 164 6.4 Discussion ...... 168

7. Conclusions ...... 170 7.1 Connection between structure and thermal stability of TOYE ...... 170 7.2 Biocatalytical potential of TOYE...... 173 7.3 Effects of structural adaptation on hydrogen tunnelling ...... 175 References ...... 178 Appendix A ...... 196 Appendix B ...... 202 Appendix C ...... 206 Appendix D ...... 208

Word Count: 56,701

Figure list

Figure 1.1. Structural classification in proteins...... 22

Figure 1.2. Structures of selected cofactors; 2Fe-2S cluster and heme B...... 23

Figure 1.3. Structures of selected coenzymes; NAD+ and coenzyme A...... 23

Figure 1.4. Chemical structure of the isoalloxazine ring and flavin cofactors...... 24

Figure 1.5. Redox states of flavin...... 25

Figure 1.6. Amino acid sequence comparison of selected Old Yellow Enzyme family members...... 27

Figure 1.7. The catalytic cycle of Old Yellow Enzymes...... 29

Figure 1.8 Hydride transfer and FMN reduction in the reductive half-reaction of OYEs...... 30

Figure 1.9. Example of reactions catalysed by PETNR...... 31

Figure 1.10. The oxidative half-reaction with 2-cyclohexenone, a common OYE substrate...... 33

Figure 1.11. OYE monomer structures...... 34

Figure 1.12. Comparison of active site residues of OYE1, PETNR and MR...... 35

Figure 1.13. Steroid binding in PETNR...... 38

Figure 1.14. Asymmetric reduction of activated alkenes by OYEs...... 43

Figure 1.15. Reactions catalysed by morphine dehydrogenase (MD) and morphinone reductase (MR). ... 45

Figure 1.16. Isoenzyme stereocontrolled catalysis using OPR1 and OPR3...... 45

Figure 1.17. Asymmetric biocatalysis of (R)-Levodione by enoate reductases...... 46

Figure 1.18. The effect of a catalyst on the transition state diagram of a reaction...... 47

Figure 1.19. Schematic illustration of a potential energy curve of a bond between chemical A and hydrogen/deuterium (H/D)...... 50

Figure 1.20. Reaction coordinate diagram comparison between transition state theory and tunnelling of hydrogen and deuterium...... 52

Figure 1.21. Temperature effect on tunnelling reactions...... 56

Figure 1.22. Structural similarities of nitroreductases adapted to different temperatures...... 61

Figure 2.1. Primers used for removing the His6-tag...... 71

Figure 2.2 Sample UV/VIS spectrum of TOYE around the FMN absorption...... 75

Figure 2.3 Sample redox potentiometric titration of TOYE...... 82

Figure 3.1. Amino acid sequence alignment between TOYE and YqjM...... 90

TM Figure 3.2. SDS PAGE analysis of TOYE-His6 induction trials in E. coli ArcticExpress (DE3) expression strain...... 91

Figure 3.3. SDS PAGE analysis of TOYE-His6 purification...... 92

Figure 3.4. Representative stopped-flow traces and concentration dependences for FMN reduction of 20 µM TOYE at 25 °C...... 94

Figure 3.5. Thermal secondary structure unfolding of TOYE...... 96

Figure 3.6. Redox potentiometric titration of TOYE...... 98

Figure 3.7. Temperature dependence of Em during redox potentiometric titration of TOYE...... 98

Figure 4.1. Electron microscopy of negatively stained TOYE...... 104

Figure 4.2. Selected TOYE-His6 crystals from the screening trials...... 106

Figure 4.3. Selected diffraction image of TOYE-His6 crystal from which a dataset was collected...... 107

Figure 4.4. Overall structures of TOYE-His6 and native-TOYE...... 110

Figure 4.5. X-ray crystal structure of holo-TOYE tetramer...... 111

Figure 4.6. Sequence alignment of TOYE and YqjM...... 112

Figure 4.7. Functional interaction between TOYE monomers...... 114

Figure 4.8. Interactions within the non-functional dimer interface of TOYE...... 117

Figure 4.9. Access tunnel to the active site of TOYE...... 118

Figure 4.10. Comparison between TOYE and a classical OYE...... 119

Figure 4.11. Active site of TOYE showing the residues/solvent involved in binding FMN...... 120

Figure 4.12. Active site of TOYE...... 123

Figure 4.13. Structural differences in NADH4 inhibitor binding within the OYE family...... 124

Figure 4.14. Monomer comparison of the thermophilic TOYE and the classical mesophilic OYE PETNR...... 125

Figure 4.15. Thermophilic-like OYE structural comparison...... 126

Figure 4.16. Multi angle laser light scattering of purified TOYE-His6...... 127

Figure 4.17. Sedimentation velocity analysis of TOYE-His6...... 129

Figure 5.1 Proposed reaction mechanism of the reduction of 2-methyl-cyclopentenone...... 136

Figure 5.2. Solvent stability of TOYE and PETNR...... 146

Figure 5.3 Structures of different solvents...... 148

Figure 6.1. Reduction of TOYE by NADPH...... 152

Figure 6.2. Representative traces for FMN reduction of TOYE by NADH at different temperatures...... 153

Figure 6.3. Representative traces for FMN reduction of TOYE by NADPH at different temperatures...... 154

Figure 6.4. Binding titration of TOYE with NAD(P)H4...... 157

Figure 6.5. Temperature dependence of FMN reduction of TOYE-His6 by NADH and NADPH...... 158

Figure 6.6 Temperature dependence on the FMN reduction of TOYE by NADH and (R)-[4-2H]-NADH and the KIE...... 160

Figure 6.7 Temperature dependence on the FMN reduction of TOYE by NADH and (S)-[4-2H]-NADH and the 2° KIE...... 164

Figure 6.8. Stopped-flow studies of 2-methyl maleimide...... 166

Figure 6.9. Concentration dependences at steady-state conditions for NADPH...... 167

Figure 6.10. Temperature dependences at steady-state conditions...... 168

Figure 7.1. Structure of TOYE...... 173

Table list

Table 1.1. Comparison of some members of the Old Yellow Enzyme-family of enzymes...... 28

Table 1.2. Examples of the substrate diversity for the OYE family...... 44

Table 1.3 Types of extremophilic microorganisms ...... 59 Table 2.1. Chemical list...... 66

Table 3.1. Amino acid sequence identity and similarities within the Old Yellow Enzyme family of enzymes...... 89

Table 3.2. Stopped-flow kinetics of the reductive half reaction of TOYE with NADH and NADPH...... 95

Table 3.3 Thermodynamic parameters associated with the two electron reduction of TOYE and selected OYEs...... 99

Table 4.1. Crystallisation screening conditions yielding crystals for TOYE...... 104

Table 4.2. Crystallisation optimisation screen of TOYE-His6 and native TOYE...... 105

Table 4.3 X-ray crystallographic statistics for data collection, processing and refinement of TOYE-

His6 and TOYE-NADH4...... 107

Table 4.4. Interactions between two TOYE-His6 subunits that form a functional dimer and surrounding solvent...... 114

Table 4.5. Interactions between two TOYE-His6 subunits that form a non-functional dimer and surrounding solvent...... 115

Table 4.6. Interactions of the enzyme bound FMN cofactor with the surrounding protein/solvent in the

TOYE-His6 crystal structure...... 120

Table 4.7. Interactions of the enzyme bound NADH4 cofactor with the surrounding protein/solvent in the TOYE-NADH4 crystal structure...... 121

Table 4.8. Comparison of in vitro oligomeric state of TOYE...... 127

Table 5.1. Steady-state kinetics of TOYE with substituted and non-substituted cyclohexenones, cyclopentenones, maleimides, ketoisophorone and thymine...... 133

Table 5.2. Steady-state kinetics of TOYE with oxidative substrates...... 134

Table 5.3. Product determination of the reduction of α,β-unsaturated alkene substrates by TOYE...... 136

Table 5.4 Product determination of the reduction of -unsaturated alkene substrates by TOYE at 50 °C...... 142

Table 5.5. Product determination of the reduction of ketoisophorone by TOYE in the presence of water-miscible solvent systems...... 146

Table 6.1. Substrate specificity of OYEs...... 154

Table 6.2 Parameters from temperature dependence of FMN reduction of TOYE by NAD(P)H and (R)-[4-2H]-NAD(P)H...... 160

Table 6.3. Parameters for 2° KIE temperature dependence studies of TOYE...... 164

Table 6.4. Comparison of concentration dependences performed under steady-state and stopped-flow conditions...... 166

Abstract

The Old Yellow Enzyme (OYE) family of enzymes has been shown to reduce industrially important chemicals and has been used to study quantum tunnelling during enzymatic hydrogen transfer. Though extensively studied, only mesophilic homologues have been studied within the enzyme family. This thesis discusses the characterisation of Thermophilic Old Yellow Enzyme (TOYE), from Thermoanaerobacter pseudethanolicus, and provides the first published crystal structure of a thermophilic OYE-family member. In addition to increased thermostability compared to mesophilic homologues, thermophilic enzymes are important for use in industrial as often they are more stable towards organic solvents used in industry than their mesophilic homologues while catalysing the same reactions. This makes thermophiles and hyperthermophiles interesting targets for investigating the importance of enzyme dynamics during catalysis. They have also been used to study the linking of protein motion to quantum tunnelling during hydrogen transfer in other enzyme systems. In the work for this thesis, the basic characteristics of TOYE were examined. Thermal stability up to 70 °C was shown by CD and fluorescence studies and the preference towards reductive coenzyme was analysed by stopped-flow studies. Structural studies were conducted using X-ray crystallography, electron microscopy and sedimentation velocity studies. The crystal structure revealed a tetrameric enzyme with a relatively large active site. Evidence for higher oligomeric states was also obtained. The potential use of TOYE as a biocatalyst was explored by steady-state reaction, biotransformation and organic solvent resistance studies. The temperature dependences of kinetic isotope effects were used to examine the presence of tunnelling and importance active site geometry during catalysis and compared to previously described enzymes. These studies introduce a new and unique OYE-family member, allowing for more in- depth analysis of TOYE.

Declaration No portion of the work referred to in the thesis has been submitted in support of an application for another degree or qualification of this or any other university or other institute of learning

Copyright i. The author of this thesis (including any appendices and/or schedules to this thesis) owns certain copyright or related rights in it (the “Copyright”) and s/he has given The University of Manchester certain rights to use such Copyright, including for administrative purposes. ii. Copies of this thesis, either in full or in extracts and whether in hard or electronic copy, may be made only in accordance with the Copyright, Designs and Patents Act 1988 (as amended) and regulations issued under it or, where appropriate, in accordance with licensing agreements which the University has from time to time. This page must form part of any such copies made. iii. The ownership of certain Copyright, patents, designs, trade marks and other intellectual property (the “Intellectual Property”) and any reproductions of copyright works in the thesis, for example graphs and tables (“Reproductions”), which may be described in this thesis, may not be owned by the author and may be owned by third parties. Such Intellectual Property and Reproductions cannot and must not be made available for use without the prior written permission of the owner(s) of the relevant Intellectual Property and/or Reproductions. iv. Further information on the conditions under which disclosure, publication and commercialisation of this thesis, the Copyright and any Intellectual Property and/or Reproductions described in it may take place is available in the University IP Policy (see http://www.campus.manchester.ac.uk/medialibrary/policies/intellectualproperty.pdf), in any relevant Thesis restriction declarations deposited in the University Library, The University Library’s regulations (see http://www.manchester.ac.uk/library/aboutus/regulations) and in The University’s policy on presentation of Theses.

Abbreviations and Symbols

% percentage CSS complexation significance score [S] substrate concentration CT charge transfer ° degrees Da Dalton µ reduced mass of a particle dATP deoxyadenosine triphophate µl microlitre dCTP deoxycytidine triphosphate µM micromolar de diastereoisomeric excess 1° primary ΔG activation energy or Gibbs 2° secondary energy

3° tertiary dGTP deoxyguanosine triphosphate

4° quaternary ΔH enthalpy change

A constant of integration DHFR dihydrofolate reductase

Å Ångström DMF Dimethyl formamide

AADH aromatic amine DNA deoxyribonucleic acid dehydrogenase DNP dinitrophenol Abs absorbance unit ΔS entropy change Acet. acetone DSC differential scanning ACN acetonitrile calorimetry

BSA bovine serum albumine dTTP deoxythymidine triphosphate c speed of light E energy c molar concentration E electron potential

C celsius Ea activation energy

CD circular dichroism EDTA ethylenediaminetetraacetic cm centimetre acid

CrS Chromate synthase ee stereoselectivity EIE equilibrium isotope effect

EtOH ethanol kB Boltzmann constant

EWG electron withdrawing group kcal kilocalories

F Faraday constant kcat

F.C. Frank-Condon kcat / KM specificity constant

FAD flavin adenine dinucleotide kD rate constant for a deuterated substrate fH-X force constant of a hydrogen bond kDa kilodalton

FMN flavin mononucleotide kH rate constant for a protiated substrate FPLC fast protein liquid chromatography KIE kinetic isotope effect g gram kJ kilojoules g standard gravity klim limiting rate constant

GC Gas Chromatography KM concentration of half- maximal rate GTN glycerol trinitrate

kmm initial rate of reaction h Planck's constant

kobs observed rate ħ reduced Planck's constant

KS saturation constant HC hydrogen coordinate L ladder HEPES 4-(2-hydroxyethyl)-1- piperazineethanesulfonic L Litre acid l length HPLC high-performance liquid chromatography ℓ width of a rectangular potential energy barrier htADH alcohol dehydrogenase Bacillus stearothermophilus LADH liver alcohol dehydrogenase

IPTG Isopropyl-β-D-1- LB Luria-Bertani thiogalactopyranoside M molar k rate constant mA milliampere

K Kelvin MADH methylamine dehydrogenase K equilibrium constant

MALLS multi angle laser light Opt. optimised scattering Org. Original MD morphine dehydrogenase OYE Old Yellow Enzyme MeOH methanol p momentum of a particle MES 2-(N-morpholino) ethanesulfonic acid P tunnelling probability mg milligram PCR polymerase chain reaction ml millilitre pdb protein data bank mM millimolar PEG polyethylene glycol mm millimetre PETN pentaerythritol tetranitrate

MPD 2-methyl-2,4-pentadiol PETNR pentaerythritol tetranitrate reductase MR morphinone reductase ppm parts per million MRC multiple reaction conformations PrOH propanol mV millivolt psi pounds per square inch n number of electrons R gas constant

NADH nicotinamide adenine RHR reductive half-reaction dinucleotide RNA ribonucleic acid

NADH4 1,4,5,6-tetrahydro NADH RNAse Ribonuclease

NADPH nicotinamide adenine rpm revolutions per minute dinucleotide phosphate s seconds ND Not determined S sedimentation coefficient ng nanogram SAXS small angle X-ray scattering nl nanoelitre SD standard deviation nm nanometres SDS sodium dodecyl sulphate NMR nuclear magnetic resonance SE standard error OD optical density SHE standard hydrogen eletrode OPR 12-oxophytodienoic acid reductases T temperature 16

TEM transmission electron Tris Tris(hydroxymethyl) microscopy aminomethane

THF tetrahydrofuran TS transition state

Tm melting temperature TST transition state theory tmDHFR DHFR from Thermotoga U unit maritime v mean speed TNT Trinitrotoluene v volume TOYE Thermophilic old yellow enzyme V height of a rectangular potential energy barrier TRC tunnelling ready configuration V volt

Equation list ( ) Eq. 1.1 ( ) ( ) Eq. 2.3 ( )

Eq. 1.2 ( )

( ( ) ) Eq. 2.4 Eq. 1.3 [ ] [ ][ ] ( )

Eq. 1.4 Eq. 2.5

Eq. 1.5 Eq. 2.6

Eq. 1.6 Eq. 2.7 ( )

Eq. 1.7 Eq. 2.8

Eq. 1.8 [ ] Eq. 2.9 [ ] Eq. 1.9

Eq. 2.10 ∑ ( ) Eq. 1.10 [( √ ) ]

Eq. 2.11 ( )

Eq. 2.12 ( ) Eq. 1.11 √

Eq. 2.13

( )

Eq. 1.12 ( ) ( )

( )

Eq. 1.13 ( ) ( ) √(( ) ( ))

( ) ( ) Eq. 1.14 ( ) Eq. 2.13 ( ) [ { }]

( ) ( )

√(( ) ( ) )

Eq. 2.1

A205 Eq. 2.2 P (mg / ml)   A280  27.0 120   A205 

Acknowledgements

The thesis is dedicated to my father, Aðalbjörn Björnsson, uncle, Ásgeir Hilmar Jónsson and great-uncle, Þormóður Eiríksson, who all offered great support during my education but passed away during my years in university.

This thesis would not have been finished without the invaluable support and faith in me from my mother, Ragnhildur Þórólfsdóttir, and grandmother, Þorbjörg Eiríksdóttir.

Many thanks go to Professor Bjarni Ásgeirsson for drawing me into Biochemistry with his contagious enthusiasm towards the beauty of the field in his lectures. Thankfully he allowed me to work in his lab during and after my undergraduate degree where curiosity driven work style was encouraged.

I am very thankful to Professor Nigel Scrutton for taking me in as a PhD student. He showed great trust in me by giving me the freedom to take the research where I wanted to and supporting me in doing so. This allowed me to explore as many different techniques available within his lab as I could. Without the great patience of two of Nigel’s postdocs, Dr. Helen Toogood and Dr. Christopher Pudney, who acted as my guardian angels and made sure that I knew what I was doing.

Finally, I would like to thank my good friends for many years, Indíana Ingólfsdóttir, Örvar Jónsson and Hulda Gunnarsdóttir, in Iceland that cheered me on across the Atlantic and gave me the confidence to finish my degree.

1. Introduction

Table of Contents 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 ...... 45

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 ...... 58

1.7.1 Thermo- and hyperthermophiles ...... 61

1.8 Thermophilic Old Yellow Enzyme: ...... 62

1.9 Aims and Objectives ...... 63

1.1 Enzymes Enzymes are proteins that catalyse chemical reactions. They are found in all living cells and are necessary for life. Enzymes are biomacromolecules capable of great catalytic feats. These typically globular proteins can increase the rate of chemical reactions by up to 21 orders of magnitude compared to uncatalysed reactions (1). This is achieved by locating residues required for substrate binding and/or catalysis in optimal positions to facilitate catalysis. This means that the structure of enzymes dictates their catalytic activity, substrate specificity and interaction with other molecules.

Like other proteins, enzymes are made up of a linear chain of amino acids, which are connected by peptide bonds. They can be quite long and are often monomers with an average of 361 and 267 amino acids for eukaryotes and bacteria, respectively (2). These chains can form local spatial arrangements or secondary (2°) structures, such as α-helices and β-sheets (Figure 1.1). These long peptide chains fold upon themselves forming a complex three dimensional structure (tertiary (3°) structure). Two or more polypeptide chains (subunits) can interact, and the arrangement of these subunits is referred to its quaternary (4°) structure. The amino acid sequence determines the final folding and structure of an enzyme. Environmental factors, like pH and temperature, affect enzyme structures and extreme conditions can cause unfolding.

The enzyme structure is important for catalysis as the location of hydrophilic, charged and hydrophobic amino acids in the active site influence the substrate binding and reaction mechanism of the catalysed reaction. Enzyme specificity was originally described with the ‘lock and key’ model where the enzyme active site and substrates have a complementary shape and fit together (3). However, this model did not take into account that enzymes are flexible and their active sites are constantly undergoing conformational motions. This mobility of the active site allows the enzyme to mould around the substrate (see Section 1.4.1 for more details regarding the transition state theory). On the other hand, it has been suggested that in at least some cases this state is too fleeting, lasting on a femtosecond to picosecond time scale, for it to be considered a stabilised situation (4). The understanding and theories of enzyme catalysis are still evolving as new discoveries are being made.

A) C)

…-Met-Ala-Pro-Leu-Thr-Arg-Leu-Arg-Ser-Ile-…

B) α -helix

β-sheet

Figure 1.1. Structural classification in proteins. A) Primary structure of pentaerythritol tetranitrate reductase (PETNR), residues Met22-Iso31 (pdb accession code: 2ABA). B) Examples of secondary structure from PETNR, α-helix (Ser76-Glu83) and β-sheet (Ile97-Trp102). C) Tertiary structure of PETNR. D) Quaternary structure of TOYE (pdb accession code: 3KRU). Residues are shown as atom coloured sticks with green carbons and green ribbons.

Some enzymes utilise chemicals to assist with catalysis, namely cofactors and coenzymes. Cofactors are additional chemical compounds that are often non-covalently bound to enzymes and are required for catalysis. These chemicals can be either organic (for example hemes or flavins) or inorganic (for example metal ions such as Mg2+ and Cu+ or iron-sulfur clusters; Figure 1.2). Cofactors take part in forming the active site and enzymes with a cofactor referred to as holoenzymes, but if a cofactor is removed from an enzyme it becomes an inactive apoenzyme. As cofactors take part in the catalysed reaction, they undergo a chemical change and are regenerated during a catalytic cycle, often by additional coenzymes or secondary substrates. Coenzymes are a group of organic compounds that can facilitate a catalytic cycle of enzymes (Figure 1.3). Vitamins can be precursors to coenzymes, like riboflavin (B2) for flavin mononucleotide (FMN) (Figure 1.2). Vitamins can also be coenzymes themselves, such as vitamin C which is a coenzyme for Cu+- dependent monooxygenases (5) and Fe2+-dependent dioxygenases (6). They can be involved in transfer of various groups/atoms: for example acyl group transfer for coenzyme A and alkyl group transfer for vitamin B12, or single hydrogen atom transfer, in the case of nicotinamide adenine dinucleotide (NADH). They often require regeneration by a

secondary enzyme system like a glucose 6-phosphate dehydrogenase-NADP+/NADPH cofactor system (7).

Enzymes are classified into 6 broad groups according to the reactions they catalyse; namely oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases. Enzymes with high sequence similarity are considered to be within the same family. The work in this thesis will focus on a flavoprotein family within the oxidoreductase category, the Old Yellow Enzyme family.

H C 3 CH2

N H3C CH3 2+ N Fe N O Enzyme S S Enzyme N S H2C OH Fe Fe

CH Enzyme S S S Enzyme 3 HO O 2Fe-2S cluster Heme B

Figure 1.2. Structures of selected cofactors; 2Fe-2S cluster and heme B.

NH - 2 O + N NH2 O P O O N N H C O O 3 CH3 O O N OH P O P O N O OH HS O NH NH NH OH OH 2 OH N N O HO O P O N N HO P O Coenzyme A - O O OH

OH OH Nicotinamide adenine

dinucleotide (NAD+)

Figure 1.3. Structures of selected coenzymes; NAD+ and coenzyme A.

1.2 Flavoproteins Flavoproteins are enzymes that contain a yellow coloured flavin cofactor (Figure 1.4). They take part in many biological processes from energy synthesis, degradation of aromatic compounds by hydroxylation, biosynthesis of ubiquinone to detoxification of reactive oxygen species, for example (8-10). They catalyse both one- and two-electron transfer reactions (11) to and from substrates. In flavoenzymes there is a bound flavin mononucleotide (FMN) or flavin adenine dinucleotide (FAD) cofactor (Figure 1.4) (12). During a catalytic cycle, the flavin shifts between oxidised, one-electron reduced (semiquinone) and two-electron reduced (dihydroquinone) (Figure 1.5). The word flavoprotein is derived from their yellow (latin: flavus) coloured cofactor with identifiable spectroscopic properties that enable detailed studies of these enzymes (11). The properties of flavin and the readily separated half-reactions makes flavoproteins an excellent subject for enzymological research, this has led to several members of this family being studied in great detail (13-16).

The interactions between the apoenzyme and its cofactor are highly conserved within each enzyme family (17). The reactive site of the enzyme is the tricyclic isoalloxazine ring- system of the flavin cofactor in combination with the active site residue side chains. As substrates are often bound in one position in the active site near the coenzyme, these enzymes are often stereo- and enantio-selective and catalytically efficient (18).

H3C

C CH3 HC 7 C 6 8 C CH 9 N 5a C NH2 5 9a C O C N CH2 OH N C 4a C 10 C N 4 10a CH HC HC C CH N N HN HO CH CH2 O O N 3 C 1 2 HO O P O P O CH2 O O OH OH HC CH Riboflavin CH CH OH OH FMN

FAD

Figure 1.4. Chemical structure of the isoalloxazine ring and flavin cofactors. Ring atoms of the isoalloxazine ring are numbered. 24

Red anionic semiquinone

H3C N NH O - - O e e + 2H H - H3C N H3C N N O H3C N NH NH R

H3C N N O O H3C N N O - H - H R e e + 2H R H3C N C NH Oxidised flavin Two-electron or

H3C N N O fully reduced flavin R (dihydroquinone)

Blue neutral semiquinone

Figure 1.5. Redox states of flavin.

While the precise interactions within the active site of an enzyme dictates its activity, flavoproteins with similar overall structure can catalyse different reactions, while family members that catalyse similar reactions can have different topology (17). Flavoproteins can be divided into three groups depending on their physiochemical characteristics (11). ‘Simple flavoproteins’ are individual proteins that are again divided into subcategories based on their reactivity to oxygen (oxidases, electron transferases and monooxygenases). More complex flavoproteins have ‘auxiliary redox centers’ and can be divided into three subcategories (flavoprotein disulphide oxidoreductases, flavocytochromes and metal containing flavoproteins). The third group consists of flavoproteins with ‘unknown function’ like the family of Old Yellow Enzymes.

1.3 The OYE-family of flavoproteins The Old Yellow Enzyme family (OYE-family) consists of a variety of flavoproteins of unknown function exhibiting high sequence similarity that contain the cofactor FMN (19). Despite being extensively researched, the physiological function(s) of most of these enzymes still eludes researchers. While many OYE-family members catalyse the reduction of steroids, it has not been proven that they take part in sterol metabolism, although their ability to bind steroids might serve as a clue to their physiological role (15). The 12- oxophytodienoic acid reductases in plants have been shown to take part in the biosynthesis of jasmonic acid (20-22). The first flavoprotein isolated was Old Yellow Enzyme 1 (OYE1). It was isolated from Saccharomyces pastorianus (formerly Saccharomyces carlsbergensis) or ‘Brewer’s Bottom Yeast’ (19). Later, when the second (‘the new’) Old 25

Yellow Enzyme 2 (OYE2) from Saccharomyces cerevisiae was isolated in 1938, the previous enzyme was termed ‘old’ yellow enzyme, a name which has been used to this day to describe this family of closely related enzymes. OYE1 is the archetypical enzyme for the growing OYE-family.

1.3.1 OYE subclasses Aligning selected OYEs reveals two subclasses (23) within the enzyme family, each with their own sequence and structural features (Figure 1.6). This subclass has been termed the ‘thermophilic-like’ subclass as the sequences are shorter than in the classical OYEs, a feature frequently seen in thermophilic homologues (Section 1.7.1), and contains enzymes from thermophilic organisms as well as the mesophilic YqjM. Among conserved residues within the thermophilic subclass are C-terminal residues R312, Q333, Y334 and R336 (YqjM numbering) which take part in formation of active dimers (24) and Cys25 (YqjM numbering) in the active site which is Thr38 (OYE1 numbering).

The basic monomer structure (α/β-barrel) and reaction mechanism are conserved within the OYE-family but there are differences within and outside the active site that translate to different preferences towards coenzymes and substrate range. OYEs generally prefer NADPH as a reducing coenzyme, with the exception of MR (Table 1.1). The thermophilic- like OYE YqjM has been shown to form a tetramer. There are a variety of oligomeric states within the enzyme family (Table 1.1), with the classical OYEs being either monomeric (PETNR or OPR1 for example) or dimeric (OYE1 and MR for example).

1.3.2 Reaction mechanism of OYEs Hydrogen transfer reactions are common and important enzymatic reactions for life, spanning a wide range of reactions, for example acid-base catalysis (as a movement of protons), radical transfer and hydride transfer (redox catalysis). In the case of the OYE family, the reaction proceeds via hydride transfer mechanisms involving two half reactions; a NAD(P)H-dependent flavin reductive step followed by flavin reoxidation concomitant with activated alkene reduction (Figure 1.7) The half-reactions of OYEs have been studied, both in steady-state, stopped-flow and computationally, to enable a detailed understanding of the reaction mechanism (14, 25, 26).

1 10 20 30 40 50 60 70 | | | | | | | | OYE1 MSFVKDFKPQALGDTNLFKPIKIGNNELLHRAVIPPLTRMRALHPGNIPNRDWAVEYYTQRAQRPGTMII PETNR ------MSAEKLFTPLKVGAVTAPNRVFMAPLTRLRSIEPGDIPTPLMGE-YYRQRASA--GLII MR ------MPDTSFSNPGLFTPLQLGSLSLPNRVIMAPLTR--SRTPDSVPGRLQQI-YYGQRASA--GLII Classic OYEs OPR1 -MENKVVEEKQVDKIPLMSPCKMGKFELCHRVVLAPLTRQRSY---GYIPQPHAILHYSQRSTNG-GLLI TOYE ------MSILHMPLKIKDITIKNRIMMSPMCMYSAST-DGMPNDWHIV-HYATRAIGGVGLIM nemA ------MSKLFTPLQIKGLSLKNRIGMSPMCMYCAGE-DGLATDWHFV-HYSTRAVGGVGLIL Thermophilic- Geo ------MNTMLFSPYTIRGLTLKNRIVMSPMCMYSCDTKDGAVRTWHKI-HYPARAVGQVGLII YqjM ------MARKLFTPITIKDMTLKNRIVMSPMCMYSSHEKDGKLTPFHMA-HYISRAIGQVGLII like OYEs <-αA-> <->βA<->βB <->β1 <----->α1 β2<-- 80 90 100 110 120 130 140 | | | | | | | OYE1 TEGAFISPQAGGYDNAPGVWSEEQMVEWTKIFNAIHEKKSFVWVQLWVLGWAAFPD-NLARDGLRYDSAS PETNR SEATQISAQAKGYAGAPGLHSPEQIAAWKKITAGVHAEDGRIAVQLWHTGRISHSSIQPGGQAPVSASAL MR SEATNISPTARGYVYTPGIWTDAQEAGWKGVVEAVHAKGGRIALQLWHVGRVSHELVQPDGQQPVAPSAL Classic OYEs OPR1 GEATVISETGIGYKDVPGIWTKEQVEAWKPIVDAVHAKGGIFFCQIWHVGRVSNKDFQPNGEDPISCTDR TOYE QEATAVESRGRITDHDLGIWNDEQVKELKKIVDICKANGAVMGIQLAHAGRKC----NISYEDVVGPSPI nemA QEATAVEKRGRITANDLGLWDDNQIAPLKHIVDFVHSVECKIGVQLGHAGRKC----EVEGKHIVAPSAM Thermophilic- Geo VEATGVTPQGRISERDLGIWSDDHIAGLRELVGLVKEHGAAIGIQLAHAGRKS----QVPGE-IIAPSAV YqjM VEASAVNPQGRITDQDLGIWSDEHIEGFAKLTEQVKEQGSKIGIQLAHAGRKA----ELEGD-IFAPSAI like OYEs ----> <--->αB <------>α2 β3<-----> <--->αC <>βC 150 160 170 180 190 200 210 | | | | | | | OYE1 DN----VFMDAEQEAKAKKANNPQHSLTKDEIKQYIKEYVQAAKNSIAAGADGVEIHSANGYLLNQFLDP PETNR NAN-TRTSLRDENGNAIRVDTTTPRALELDEIPGIVNDFRQAVANAREAGFDLVELHSAHGYLLHQFLSP Classic OYEs MR KAEGAECFVEFEDGTAGLHPTSTPRALETDEIPGIVEDYRQAAQRAKRAGFDMVEVHAANACLPNQFLAT OPR1 G-----LTPQIRSNGIDIAHFTRPRRLTTDEIPQIVNEFRVAARNAIEAGFDGVEIHGAHGYLIDQFMKD TOYE ------KAGDRYKLPRELSVEEIKSIVKAFGEAAKRANLAGYDVVEIHAAHGYLIHEFLSP nemA ------HWSDSYPVPEELTMAEIQKIVQAFAEAGKRAVKAGFDALEIHAAHGYLIHEFLSP Thermophilic- Geo ------PFDDSSPTPKEMTKADIEETVQAFQNGARRAKEAGFDVIEIHAAHGYLINEFLSP YqjM ------AFDEQSATPVEMSAEKVKETVQEFKQAAARAKEAGFDVIEIHAAHGYLIHEFLSP like OYEs βD<> <------α3------> <---->β4 <------>αD 220 230 240 250 260 270 280 | | | | | | | OYE1 HSNTRTDEYGGSIENRARFTLEVVDALVEAIGHEK-VGLRLSPYGVFNS-MSGGAETGIVAQYAYVAGEL PETNR SSNQRTDQYGGSVENRARLVLEVVDAVCNEWSADR-IGIRVSPIGTFQN---VDNGPNEEADALYLIEEL Classic OYEs MR GTNRRTDQYGGSIENRARFPLEVVDAVAEVFGPER-VGIRLTPFLELFG----LTDDEPEAMAFYLAGEL OPR1 QVNDRSDKYGGSLENRCRFALEIVEAVANEIGSDR-VGIRISPFAHYNE----AGDTNPTALGLYMVESL TOYE LSNKRKDEYGNSIENRARFLIEVIDEVRKNWPENKPIFVRVSADDYMEGGINIDMMVEYINMIKDKVDLI nemA LSNKRTDKYGGGIENRVRFLREVLEAVRSNVPEDMPIFMRVSATDYVEGGLDIDETIAIVNMVKDLVDVV Thermophilic- Geo LSNRRQDEYGGSPENRYRFLGEVIDAVREVW--DGPLFVRISASDYHPDGLTAKDYVPYAKRMKEQGVDL YqjM LSNHRTDEYGGSPENRYRFLREIIDEVKQVW-—DGPLFVRVSASDYTDKGLDIADHIGFAKWMKEQGVDL like OYEs <------α4------ <--->β5 <----α5------> < 290 300 310 320 330 340 350 | | | | | | | OYE1 EKRAKAGKRLAFVHLVEPRVTNPFLTEGEGEYEGGSNDFVYSIWKGPVIRAGNFALHPEVVREEVKDKRT PETNR AKRG-----IAYLHMSETDL------AGGKPYSEAFRQKVRERFHGVIIGAGAYTA-EKAEDLIGKGLID Classic OYEs MR DRRG-----LAYLHFNEPDW-----IGGDITYPEGFREQMRQRFKGGLIYCGNYDAG-RAQARLDDNTAD OPR1 NKYD-----LAYCHVVEPRMK----TAWEKIECTESLVPMRKAYKGTFIVAGGYDR-EDGNRALIEDRAD TOYE D------VSSGGLLNVDI------NLYPGYQVKYAETIKKRCNIKTSAVGLITTQELAEEILSNERAD nemA D------CSSGGLLSPRI------DLYPGYQIGFAEAVKRETGVATAAVGLITSPEMAEEIIGNQRAD Thermophilic- Geo VD------VSSGAIVPARM------NVYPGYQVPFAELIRREADIPTGAVGLITSGWQAEEILQNGRAD YqjM ID------CSSGALVHADI------NVFPGYQVSFAEKIREQADMATGAVGMITDGSMAEEILQNGRAD like OYEs --β6------> <----α6-----> <-->β7 <---α7----> 360 370 380 390 400 | | | | | OYE1 LIGYGRFFISNPDLVDRLEKGLPLNKYDRDTFYQMSAH-GYIDYPTYEEALKLGWDKK PETNR AVAFGRDYIANPDLVARLQKKAELNPQRPESFYGGGAE-GYTDYPSL------Classic OYEs MR AVAFGRPFIANPDLPERFRLGAALNEPDPSTFY-GGAEVGYTDYPFLDNGHDRLG--- OPR1 LVAYGRLFISNPDLPKRFELNAPLNKYNRDTFYTSDPIVGYTDYPFLETMT------TOYE LVALGRELLRNPYWVLHT---YTSKEDWPKQYERAFKK------nemA LVLLGRVLLRQPYWPLYAAHFLKADVGYPKQYERGRYQ------Thermophilic- Geo LVFLGRELLRNPYWPYAAARELGAKISAPVQYERGWRF------YqjM LIFIGRELLRDPFFARTAAKQLNTEIPAPVQYERGW------like OYEs β8<--><-αE--> <---α8---> <-->αF

Figure 1.6. Amino acid sequence comparison of selected Old Yellow Enzyme family members. Numbering is based on the sequence of OYE1. Highlights in red and blue show the sequence conservation of active site residues T26, Y28, W102, H181, H184, Y186, Y351 (PETNR numbering) and R333 (TOYE numbering) for classical OYEs and thermostable-like OYEs, respectively. Dark highlight shows residues conserved over the whole enzyme family. Enzymes and accession numbers: TOYE from T. pseudethanolicus (27) (B0KAH1), nemA from Coprothermobacter proteolyticus (28) (accession number B5Y655), Geo from Geobacillus kaustophilus (29) (Q5KXG9), YqjM from B. subtilis (30) (P54550), PETNR from E. cloacae PB2 (31) (U68759), MR from P. putida (32) (AAC43569), OYE1 from S. carlsbergensis (33) (Q02899), and OPR1 from Solanum lycopersicum (34) (accession number Q9XG54). Secondary structure is noted for YqjM (24).The alignment was generated using ClustalW and accession numbers were from UniProt (http://www.uniprot.org/). 27

)

, , )

, , ) ) )

31 39

, , ,

33 36 37 32

( ( (

, ,

16 34

(

, ,

References

(

His His

194

Asn Asn Asn Asn

Residue Residue

E: Old Yellow Enzyme. MR: Morphinone Yellow Old MR: E: Enzyme.

His His His His His His

191

Residue Residue

Lycopersicon esculentum

NADH

NADPH NADPH NADPH NADPH NADPH

preference

Coenzyme Coenzyme

family of enzymes. OY family enzymes. of

[b]

Formerly as known

[c]

Dimer Dimer Dimer Dimer

hasbeen shownin to small exist portion dimer, as the while functional form a is monomer

structure

Monomer

Quaternary Quaternary

Monomer

PETN

[b]

. .

(Da)

45,000 44,900 44,800 40,000 41,100 42,400

Subunitmass

[c]

[a]

M10

oxophytodienoate reductases. The residue numbering OYE1. on based is residue oxophytodienoateThe reductases.

cerevisiae

cloacae

Solanum Solanum

Saccharomycescarlsberginsis

Organism

putida

Enterobacter Enterobacter

S. S.

Pseudomonas Pseudomonas

Saccharomyces Saccharomyces

lycopersicum

Comparison of some members of the Old Yellow of Enzyme Old the members Comparisonof some

OPR1

OYE1 OYE2 OYE3

PETNR

Enzyme

Formerly known as

(http://onlinelibrary.wiley.com/doi/10.1002/cbic.201000527/suppinfo). [a]

Table 1.1. 1.1. Table OPR1: reductase, 28

O O H H H H C N N O R S 3 R1 NH2 NH R2 H3C N H N OYEox O NADPH

reduced oxidative oxidative reductive substrate half- half- reaction reaction

H O O - S H3C N N O R 1 NH2 NH + + H3C N N NADP R2 H O α,β-unsaturated OYEred substrate

Figure 1.7. The catalytic cycle of Old Yellow Enzymes (42).

The reaction cycle has been shown to have a ping-pong mechanism, where both the oxidative and reductive substrates share overlapping binding sites (13-15, 40). The reaction cycle begins with NADPH binding, resulting in a long wavelength absorbing charge- transfer complex, followed by hydride transfer from NADPH to the oxidised FMN N5 atom (Figure 1.8a) (35). PETNR binds to NADPH and the NADPH-enzyme intermediate is formed before the hydride transfer from the coenzyme to FMN, forming dihydroquinone form of the enzyme-bound FMN (26, 41). NADPH is bound so the nicotinamide C4 atom, the hydride donor, is positioned above the flavin N5 atom (Figure 1.8). Following NADP+ release, the enzyme is capable of reducing a variety of oxidising substrates (Figure 1.9). Along with alkene reduction, OYEs can also perform nitro reduction (e.g. reduction of PETN) and reduction of TNT proceeds by a combination of alkene reduction and nitro reduction (26).

R2 FMN R2 - H3C N O H3C N O

NH NH H3C N H3C N H O O

NH H NH Hs HR 2 (s) 2

O O

+ N N NAD(P)H NAD(P)+ R1 R1

NADH4 His184

His181

H-

FMN

Figure 1.8 Hydride transfer and FMN reduction in the reductive half-reaction of OYEs. A) Reaction scheme of the stereo-selective oxidation of NADH where the C4 R-hydrogen (Hp) is transferred to N5 of FMN. B) Crystal structure view of 1,4,5,6-tetrahydro NADH inhibitor bound in the active site of PETNR (accession code 3KFT). The arrow and H- refer to the relative position of where the hydride transferred from NADH to

FMN would be expected. PETNR residues, FMN and NADH4 are shown as atom coloured sticks with green, yellow and cyan coloured carbons, respectively.

O N O2N 2 A: O O

NO NO2 2 O O O O O N O2N 2 NO O 2 HO NO 2

CH3 CH3 CH3 O N NO O N NO O N NO B: 2 2 2 2 2 2 - CH C H H NO2 NO2 NO2

C: O O

O O HO HO O CH3 O CH3 OH OH

CH D: 3 CH3

O O

Figure 1.9. Example of reactions catalysed by PETNR. A: Reduction of PETN. B: Reduction of TNT. Possible hydride-Meisenheimer complex intermediate shown in parathesis. C: Reduction of 2-cyclohexenone and D: Reduction of prednisone.

Normally, the oxidative half-reaction is rate-limiting during steady-state turnover (14, 40). The hydride transfer from NAD(P)H to FMN is essentially irreversible (26). Hydride transfer is connected to FMN reduction and can be measured by following the rate of the FMN bleaching at around 465 nm (25, 40). This reduction is a monoexponential process in wild type enzymes. Stopped-flow spectrometers have been used to elucidate the rate constants in the reductive half-reaction. This must be done anaerobically or by using an oxygen depletion system (glucose oxidase/glucose oxygen scavenging system) as the reduced FMN of OYEs can be rapidly reoxidised by oxygen (25).

To complete the catalytic turnover (Figure 1.10), a number of oxidising substrates, such as α/β-unsaturated aldehydes, ketones and oxygen, can act as electron acceptors (35). When binding phenolic ligands, OYEs form long wavelength charge-transfer (CT) complexes (500-800 nm) (35). Their phenolic ligands stack their ring almost parallel to and above the FMN ring, while His191 and Asn194 (OYE1) form hydrogen bonds to the oxygen of 2- cyclohexenone (Figure 1.11) (13). The substrate acts as the electron donor, while the flavin is the electron acceptor (43). The CT complex has a characteristic absorbance band around 555 nm and forms before the reduction of the flavin at around 460 nm (40). The CT complex decay and flavin reduction are kinetically linked, with the flavin reduction proceeds concomitantly with CT decay (25, 26). The reductive half-reaction in OYEs proceeds in three steps:

( ) → [ ( ) ] → ( ) → ( )

R - H C N O 3 FMN NH H3C N H O

H A O Åsn194 2-cyclohexenone O H2N H + N His191 N H

Figure 1.10. The oxidative half-reaction with 2-cyclohexenone, a common OYE substrate. H-A is a proton donor from either a conserved tyrosine or water. Numbering of the residues is based on OYE1 (46).

OYE binds tightly to 2-cyclohexenone, and competes with NADPH binding to the oxidised enzyme due to overlapping binding sites (35). Even though some substrates used in the study of OYEs are not thought to be physiological, such as 2-cyclohexenone, they have spectroscopic properties that make their reactions easy to study. Hydride transfer from a reducing coenzyme to a flavin cofactor are, for example, performed by cytochrome P450 reductase, ferredoxin reductase, nitric oxide synthase and the OYE family. The multi-step mechanism means that individual steps in hydride transfer can be studied.

Reduced OYEs can be oxidized by many classes of substrates where an activating or electron-withdrawing group is conjugated to the double bond that is reduced (Figure 1.11). This activating group binds to the His/His (His/Asn) substrate binding couple (Section 1.3.3). The reduction of oxidative substrates often takes place in two concerted steps; hydride transfer and proton uptake (42). When comparing five OYE-family members, no substrate was found to be preferred by all enzymes (44). In most cases a C=C double bond is reduced but in the oxidative half-reaction with PETNR, the two-electron reduced enzyme reduces substrates such as nitro-ester explosives (PETN and glycerol trinitrate, nitro reduction), nitroaromatic explosives (2,4,6-trinitrotoluene (TNT) and picric acid, combination of nitro reduction and alkene reduction) and α,β-unsaturated carbonyl compounds (2-cyclohexenone) (26, 41) (Figure 1.11). Both PETNR and Escherichia coli NEM reductase have broad specificity, with activity against nitrate esters, α,β-unsaturated carbonyls. In addition, both produce an orange coloured 2H—TNT as one of their TNT products, which is due to the formation of a hydride-Meisenheimer complex (Figure 1.9 B), where the hydride is transferred from the reduced FMN to the electron deficient aromatic ring (45). OYE1 and MR on the other hand have a reduced activity towards nitrate esters (44).

Mutagenesis studies of the His/His (His/Asn) pairs on the OYE-family combined with the X-ray crystal structures of substrate-bound OYEs confirms their role in substrate binding (35, 47). Though engineering and mutagenesis, random and designed, will become more popular in the future, care must be taken with flavoproteins as amino acids around the active site not only influence substrate specificity but also the redox potential (the driving force of the reaction) and binding of the cofactor. The importance of the histidine pair in PETNR in substrate binding and their effect on substrate specificity has been demonstrated by site specific saturation mutagenesis, where a single selected amino acid is mutated into 33

all other possible amino acids (47). In this study, many mutants showed an increase in the nitroreduction, instead of the usual C=C reduction. Mutagenesis studies on PETNR and YqjM show that single mutations can change the enantioselectivity of an enzyme (48, 49).

In this thesis, four enzymes within the OYE family will be described and used for comparison; OYE1 (the archetypical enzyme of the family), PETNR (which has been extensively studied with regard to biocatalysis, as well as OYE1 and YqjM), MR (of which the hydride transfer has been greatly studied) and YqjM (a member of the thermophilic- like subgroup of OYEs).

1.3.3 Structural features of the OYE family The OYE-family members typically have structures based on an eight stranded α/β barrel structure (13, 15, 16) with a β-hairpin that forms the bottom of the barrel (Figure 1.11). They contain subunits that are commonly around 40 - 45 kDa in molecular weight (Table 1.1) and vary in their oligomeric state (from monomers to tetramers).

A) B)

C-terminal

Figure 1.11. OYE monomer structures. A) Top view of PETNR. B) Comparison between PETNR (classical OYE) and YqjM (thermophilic-like OYE). PETNR and YqjM are shown as green and turquoise cartoons respectively, with FMN shown as an atom coloured sticks with red carbon. Circled are the two largest structural distinct features of each subclass, C-terminal and loop extension next to the active site. Accession codes for PETNR and YqjM are 2aba and 1Z41.

At the bottom of the barrel is a β-hairpin structure covering its base, whilst the FMN cofactor is bound at the top of the barrel forming the bottom of the active site. The cofactor is tightly bound by hydrogen bonding, with the isoalloxazine ring perpendicular to the barrel. The FMN re-face is towards the carboxy-terminal of the barrel, buried away from the solvent, while the si-face is exposed to the active site (13) .

The active site is highly conserved throughout the family (Figure 1.12), but each enzyme has a slightly different substrate binding region that affects the enzymes specificity and cofactor preference (13, 19). Within the family, there are two amino acids that take part in substrate binding, namely His191 and His/Asn194 (OYE1 numbering). For example, in pentaerythritol tetranitrate reductase (PETNR) both residues are histidines, while OYE and morphinone reductase (MR) contain a histidine and asparagine pair (44). OYE1 and PETNR prefer nicotinamide adenine dinucleotide phosphate (NADPH) as their coenzyme while MR has more affinity for NADH (44).

Tyr116

Asn/His194

His191

Thr37 FMN

Figure 1.12. Comparison of active site residues of OYE1, PETNR and MR. FMN is shown as atom coloured sticks with green carbons. Numbering is based on OYE1 and are OYE1, PETNR and MR shown as atom coloured lines with green, turquoise and red carbons, respectively. The pdb accession codes for the structures used for OYE1, PETNR and MR were 1oya, 2aba and 1gwj, respectively.

Substrates of the OYE-family are shielded from solvent in the active site (17). Around the substrate binding site are number of amino acids that take part in the reaction catalysis. Thr37 is conserved throughout the classical OYE subfamily, and is located close to the flavin cofactor. The enzyme-bound FMN in OYE1 has a lower redox potential than free flavin (50) and forms a hydrogen bond between its C4 oxygen atom and Thr37 OG (13). It is thought that Thr37 OG stabilizes the negative charge of the flavin when in the reduced state and therefore increases the redox potential of the enzyme (51). Residue Thr37 has been replaced by Cys in thermophilic-like OYEs which may take part in the stabilisation in that subclass.

Tyr196 is highly conserved within the OYE-family and has been shown to be a proton donor in the oxidative half-reaction of OYE with 2-cyclohexenone (42). While the Y196F mutation has little effect on the reductive half-reaction, the oxidative half-reaction rate is dramatically decreased (42). This suggests that the oxidative substrate can be reduced without the need for Tyr196 as a proton donor as water can potentially be a proton donor. Residue Tyr186 of PETNR was thought to act as a proton donor in the reduction of olefinic bonds (26) as in OYE, but stopped flow studies on Y186F with TNT indicate that the Tyr186 does not have a major influence on either hydride transfer or nitroreductase pathways (38). The crystal structure of this mutant was virtually identical to that of the wild-type indicating the mutation had not caused any major structural alterations to the enzyme.

Both residues His191 and Asp194 are highly conserved within the OYE-family and interact with the activation groups of a variety of substrates and inhibitors such as oxygen O3 of prednisone (15). Mutants His191Asp/Asp194His and His191Asp of OYE1 show similar reactivity with O2, but the reduction of 2-cyclohexenone is greatly decreased even though their crystal structures are highly similar to the wild type enzyme (35). The oxidative half-reaction of the double mutant with 2-cyclohexenone was too slow to be measured by stopped flow techniques, while the FMN reduction step was rapid. These mutations also affected the FMN spectra, shifting the peaks to higher wavelengths with an increase in the flavin potential.

The active site of PETNR contains many aromatic residues that contribute to its hydrophobic nature (15). Small anions, such as acetate, chloride and thiocyanate and other compounds such as glycerol act as inhibitors of PETN as they bind tightly to residues His181 and His184 (15). Steroids can either be substrates or inhibitors of PETNR, for example prednisone and progesterone, respectively (Figure 1.13) (15). A number of crystal structures of the enzyme show ligands (e.g. nitroaromatic and steroid substrates and inhibitors) bind above the si-face of the FMN (15, 38, 48, 52, 53). The carbonyl oxygen of progesterone (O3) and prednisone (O1) form 2 hydrogen bonds with His181 and His184 (15). The binding of progesterone and prednisone, an inhibitor and a substrate respectively, have similar effects on the position of nearby side-chains and overall enzyme geometry, except that progesterone is bound slightly closer to the flavin (3.1 Å) than is prednisone (3.3 Å) (15).

Steroids containing unsaturated double bonds between the C4 and C5 carbons (Figure 1.13) are known to be reduced by the OYE family members. In addition, OYEs are inhibited by 3-keto steroids with unsaturated bonds between the C1 and the C2 carbons, such as 19-nortestosterone, progesterone and 19-hydroxyandrost-4-ene-3,17-dione. The nitroaromatic substrates have their C5 carbon close to the flavin N5, an optimal position for hydride transfer (26).

When PETNR degrades TNT (45) and PETN (16) nitrates are liberated. The enzyme does not degrade PETN further than pentaerythritol dinitrate and 2,4-DNP is not a substrate but an inhibitor. This is because of a resonance stabilisation in the 2,4-DNP structure which does not favour are further reaction (26).

The role of the conserved active site histidine and aspargine/histidine pair in ligand binding has been studied by site directed mutagenesis in OYEs such as PETNR and OYE1 (15, 35). Mutants H181A and H184A show the two histidines are not essential for enzyme catalysis, as reductive and oxidative half-reaction activities were retained in some cases (38). However, they played a role in ligand binding as the dissociation constants increased, for both substrates and inhibitors. Residues H181 and H184 are known to interact with the carbonyl group of 2-cyclohexenone and the hydroxyl groups of picric acid and 2,4- dinitrophenol (2,4-DNP) as seen in co-crystal structures (26). While these residues do not

OH H C O 3 O CH3 CH3 C5 O OH C5

CH CH3 C4 3 C4

O O Prednisone Progesterone

C5 C4 His184 Prednisone

His181 FMN N5 Figure 1.13 A) Structures of prednisone and progesterone. Circled is the C-C bond which is positioned above the N5 of enzyme bound FMN. B) Crystal structure of prednisone in the active site of PETNR and positioned of the the FMN. PETNR residues, FMN and prednisone are shown as atom coloured sticks with green, yellow and turquoise coloured carbons. Accession code: 1H61.

interact with the methyl group of TNT, this substrate is still bound in a similar way as picric acid and 2,4-DNP in the active site.

Residue Trp102 has been shown to occupy at least two crystallographic conformational states in PETNR with picrate (41). This causes nearby amino acids, Thr104, Gly67 and Ser132, also to have two conformational states. Interestingly, crystal structures of mutants W102F and W102Y do not have these multiple conformations in the active site. These mutations resulted in tighter binding of picric acid, without any other significant alterations to the enzyme and with little effect on the FMN reduction by NADPH (41). The W102F

and W102Y mutants have similar affinity for 2,4,-DNP, as wild-type in agreement with the crystal structures which indicates a steric interaction between Trp102 and a nitro group of picric acid not seen with 2,4,-DNP. Mutants W102F/Y did not affect the dissociation constant for the progesterone-enzyme complex (41), in agreement with the crystal structure of the complex, where Trp102 does not interact with progesterone (15).

1.3.3.1 Structural features of classical OYE The classical OYEs are generally longer than the thermophilic-like subclass (Figure 1.6), for example OYE1, PETNR and MR with 400, 365 and 377 amino acids, respectively, compared to 338 of YqjM. Although closely related, PETNR and MR have certain unique features setting them apart from OYE1.

PETNR and MR were initially isolated due to the ability of their hosts to degrade toxic materials. PETNR is from Enterobacter cloacae PB2 which was isolated from explosive- contaminated soil and can grow in media containing PETN (16) or TNT (45) as the sole nitrogen source. Morphinone reductase (MR), from Pseudomonas putida M10, stands out in the OYE-family for its reactivity with the opiate precursors codeinone and morphinone (32). This enzyme saturates the carbon-carbon double bonds producing the semi-synthetic drugs hydrocodone and hydromorphone, respectively (14).

While OYEs have similar subunit structures and are dimeric in solution, the majority of PETNR is monomeric in solution (15, 16). For MR, the dimer interface is between the N- terminal β-strands and helices 2 and 8 of the barrel (14) in contrast to OYE1 where the subunits interact around helices 4, 5 and 6 (Figure 1.6) (13).

Even though the active site is highly conserved, MR utilises NADH unlike OYE and PETNR which prefer NADPH (14). MR also forms a charge-transfer intermediate with the coenzyme, NADH, similar to PETNR. It has been determined by stopped-flow and steady state studies that the proton for the oxidative half-reaction of MR comes from the solvent (46). In addition, conserved residue Tyr196 in OYE1 (and Tyr186 in PETNR) is Cys191 in MR which explains its lack of proton donation in the oxidative half-reaction (14).

As in OYE1, the suggested role of Thr32 in MR is to affect the reduction potential of the FMN to drive the hydride transfer from the nicotinamide coenzyme to FMN (46). For the

T32A mutant, flavin reduction is the rate-limiting step of the oxidative half reaction.

Mutants Y72F and Y356F affected the kcat, and were ruled out as proton donors of the oxidative half-reaction. Both mutants have relatively high reactivity towards codeinone (46).

1.3.3.2 Structural features of the thermophilic-like OYEs The thermophilic-like OYEs are generally shorter than the classical OYEs, for example YqjM, TOYE, Geo and nemA are 338, 337, 340 and 340 amino acids, respectively. Shorter amino acid chains are a common feature of thermophilic enzymes (see section 1.7.1). The C-termini are different as they take part in dimer formation and surface loops are shorter than in classical OYEs. As YqjM is a mesophilic enzyme the subclass is referred to as ‘thermophilic-like’.

Of the thermophilic-like OYEs, YqjM is the most heavily studied enzyme. YqjM is from Bacillus subtilis and has 33% identity and 50% similarity to OYE1 (30). Though it is not as closely related to OYE as PETNR and MR it still shares many biochemical properties and structural characteristics of the enzyme family. The expression of YqjM has been linked to up-regulation during oxidative stress from hydrogen peroxide and trinitrotoluene, suggesting that the enzyme plays a role in detoxification and oxidative stress response (30). This enzyme was also the first in the new thermophilic-like subclass of the OYE family to be described (54). While the OYE active site is mostly conserved in both subgroups there are few key differences, including the wide access channel into the active site in thermophilic-like. When purified, YqjM is not fully flavinated and requires the addition of FMN to be reconstituted suggesting a weaker binding of the cofactor (30).

Like other OYEs, the YqjM monomer consists of a α/β-barrel with a non-covalently bound FMN (Figure 1.12) (54). YqjM stands out from the OYE family as its oligomeric state is tetrameric, the smallest unit in solution, and has the molecular mass of 147 kDa (30). The YqjM tetramer is a dimer of functional dimers, where two monomers take part in forming the active site of their neighbours (54). The access tunnel to the active site of YqjM is larger than for other OYEs as two loops, L4 and L8, around the active site are shorter than in other OYEs. It was shown that using affinity tags on YqjM can affect the catalytic properties of the enzyme and even render it inactive (55). Tagging also affected the

oligomeric state of the enzyme, suggesting that the tetramer conformation is important to the enzyme catalysis.

Within this subclass, Thr37 (OYE1 numbering) has been substituted for cysteine, in a location which has been shown to influence the FMN redox potential by stabilising the reduced flavin negative charge (46, 51). The most notable difference in the YqjM active site are Cys26 and Tyr28 (which replace Thr37 and Tyr375 in OYE1, respectively) and Arg336. The Cys26 is located in a similar position to Thr in classical OYEs but Tyr28 is not at the C-terminal end like the Tyr in classical OYEs. The C-terminal of YqjM and residue Arg336 takes part in substrate binding of the adjacent monomer by extending into the active site (54). Classical OYE active sites are composed of one monomer and do not have a residue located in the same way as the Arg336, in YqjM, and is thus a unique feature of the thermophilic-like OYEs.

1.4 Biocatalysis Biocatalysis is the use of living organisms or their enzymes in the synthesis of compounds. One of the oldest use of biocatalysis is found in the brewing industry and early biochemistry discoveries were made from the yeast enzymes used in brewing. Biocatalysis has many advantages over normal synthetic catalysis; enzymes often have very high enantioselectivity (produce one enantiomer in excess of the other and/or react with one enantiomeric substrate) and regioselectivity (selective towards the location of the chemical groups on the substrate), reactions proceed at mild conditions, the solvent is often water and toxic catalyists are not required (56). Enantioselectivity is especially important for the pharmaceutical industry as chirality can affect the biological activity of chemicals. Biocatalysis does have limits though; enzymes often have low specific activity towards non-physiological substrates, sometimes extreme temperatures, pH levels and/or solvents may be needed for substrates to be soluble, and it requires a complex optimisation process to get a new reaction usable.

Enzyme optimisation by mutagenesis for biocatalysis is a growing field due to advances in genomics, directed evolution, genome shuffling using high-throughput screening and bioinformatics (57). Whole cell reactions are often used when either the enzymes are unstable at the reaction condition or the enzyme requires an expensive coenzyme which needs to be regenerated (58). Therefore whole cell cofactor regeneration is often easier and 41

less expensive than in vitro reactions. Cofactors can be recycled using enzyme recycling systems, by another enzyme, electrochemically or light-driven, for example the use of a NADP+/glucose-6-phosphate dehydrogenase cofactor regeneration system to recycle NADPH as shown in the scheme below (59).

glucose-6-phosphate dehydrogenase phosphate-6-glucose + NADP+ 6-phosphogluconate + NADPH

Biocatalysis is usually performed in bulk batches and the product purification which follows is filtration or centrifugation of the biomass (58). As the products are often hydrophobic this requires a phase separation and liquid extraction or distillation. An industrial example of biocatalysis is the hydration of acrylonitrile into acrylamide using nitrile hydratase (60).

1.4.1 Biocatalysis potential of the OYE-family OYEs are catalysts for stereoselective reduction of activated alkenes (Figure 1.15) (18, 19).

The reaction of OYEs is stereospecific as the flavin transfers the hydride to the Cβ of the substrate while a proton is added on the Cα from the opposite side, either by water or active site amino acid. The reaction is therefore a trans-hydrogen addition. Up to two new chiral centres can be formed when a C=C double bond is reduced (Figure 1.14). OYEs are chemoselective and the stereoselectivity (ees) can reach >99%, although side reactions are possible (such as nitro reduction of nitroalkenes) (61). The enzyme family reduces a variety of substrates (Table 1.2), which usually contain carbonyl groups (aldehyde, ketone, carboxylic acid, ester, nitrogroup, lactone or imide) as the electron-withdrawing or ‘activating’ substituent (EWG) conjugated to the C=C (18).

Nitroalkanes are versatile synthons as they can be used for addition of nitro or amino groups. They can also be useful for coupling two molecules (for example in the synthesis of bucindolo, a beta blocker (62)). OYE-catalysed biocatalysis could therefore provide a greener alternative to some currently used synthetic routes. An example is the reduction of phenyl-2-nitropropene by PETNR, which reduces the (E)- and (Z)-enantiomers to the (S)- product with high yield and enantiopurity (Table 1.2) (59).

FMN - [H ] H R1 R3 R1 R3 OYEs * C C * R R 2 EWG 2 H EWG + [H ]

solvent

Figure 1.14. Asymmetric reduction of activated alkenes by OYEs. *: potentially new chiral centre and EWG: electron-withdrawing group.

Morphine and derivatives are complex molecules, and modifying them chemically is difficult. This makes an enzymatic approach for morphone synthesis an attractive option (63). The soil bacterium P. putida is known to catalyse the formation of the analgesics hydromorphone and hydrocodone, from morphine and codeine, respectively (64). Two enzymes catalyse this process, namely morphine dehydrogenase (MD, which is not a member of the OYE family) and MR (Figure 1.15). Both MD and MR have been successfully utilised in E. coli to produce hydromorphone and hydrocodone in the new host (65).

Whole cell systems are often used to avoid time consuming and the costly enzyme purification and external NAD(P)H recycling system (61, 66, 67). However site reactions often occur due to the reaction of additional constitutive oxidoreductases that yield alcohol. (18, 68).

A cloned cofactor recycling system, like the use of a pyridine nucleotide transhydrogenase from Pseudomonas fluorescens, where hydride is transferred from NADPH to NADH (69) or NAD+/NADH recycling using monoxygenase (70), can be used in a whole cell system to avoid cofactor depletion within the host. Among the prospects of OYEs in biocatalysis is the use of novel continuous recirculating anaerobic bioreactors that utilise two phases (organic and aqueous) to increase substrate solubility and be able to extract product from the system without stopping the reaction (71).

Table 1.2. Examples of the substrate diversity for the OYE family. OYE/ Substrate Product %yield %ee Reference Whole cell H C CH H3C CH3 3 3 PETNR >99 93 (59) COH (S)- COH O O H3C H3C CH3 CH3 H C H3C 3 PETNR 99 21 (59)

O (R)- O O O CH CH 3 3 PETNR 99 94 (59)

(S)- H3C H3C

PETNR 99 99 (59) O O O O N N H (R)- H NO2

NO 96 70 CH3 2 (E)- CH PETNR (59) 3 CH3 (S)- >99 87 NO2 (Z)- CH CH2 3 Baker’s CN CN 64 - (72) yeast (R)- O

OH XenA - - (73)

O CH3 OH Cl Cl Cl Cl O O Baker’s 84-92 97-98 (74) OCH3 OCH3 yeast Cl (R)- Cl CH3 YqjM >99 >99 (75) (E/Z)- (R)-HOOC COOH

RO + RO NADH + RO NADP NADPH NAD

O O O MD NCH3 NCH3 MR NCH3

HO O O

R = H: morphine R = H: morphinone R = H: hydromorphone

R = CH3: codeine R = CH3: codeinone R = CH3: hydrocondone

Figure 1.15. Reactions catalysed by morphine dehydrogenase (MD) and morphinone reductase (MR). Hydromorphone and hydrocodone produced from morphine and codeine, respectively (64).

Although the reaction mechanism is the same between OYEs, the stereo selectivity can be remarkably different, which is attributed to subtle differences within each active site. A good example of different stereoselectivity between closely related enzyme is the reduction of 1-nitro-2-phenylpropene by OPR1 and OPR3, yielding the (R)-enantiomer and (S)- enantiomeric product, respectively (Figure 1.16) (61).

PETNR has been shown to possess potentials outside the chemical industry. As it can reduce nitroaromatic explosives, which is are environmental pollutant (26), it has been studied for bioremediation (76). The bacterial gene was cloned into tobacco plants to allow growth in the presence of TNT or glycerol trinitrate (GTN) and the subsequent degradation of these chemicals to remove explosives from contaminated soils.

OPR1 OPR3

NAD(P)H NAD(P)H

H3C H3C H3C

NO2 NO2 NO2 (R) (S)

Figure 1.16. Isoenzyme stereocontrolled catalysis using OPR1 and OPR3. 45

Among industrially important enones that are substrates is ketoisophorone as the reduced product, levodione, is a precursor to optically active carotinoids (Figure 1.17) (77, 78). OYEs achieve this reaction with high conversion and enantiopurity.

O O OYE

NAD(P)H O O

Ketoisophorone (R)-Levodione

Figure 1.17. Asymmetric biocatalysis of (R)-Levodione by enoate reductases.

1.5 Quantum mechanical tunnelling Many enzyme reactions involve transfer of hydrogen and have they been studied in detail (79, 80). Theoretical and experimental work regarding hydrogen transfer has been facilitated by the fact that hydrogen isotopes are stable and relatively less complex than other molecules. The relatively small size of hydrogen can in some cases allow for a mechanism based on less conventional transfer mode, quantum mechanical tunnelling. This transfer mode is dependent on the energy barrier of the reaction and has been used to study the role of motions within enzymes during catalysis.

1.5.1 Transition state theory Enzymes are effective catalysts that enhance the rates of chemical reactions. They bind substrates in well-defined active sites, often with acidic, basic and nucleophilic groups. Over the years, transition state theory (TST) has been used to describe enzyme catalysis.

Many of the current mechanisms of enzyme catalysis are based on TST, a theory which focuses on the structure-activity relationship of enzymes. TST describes the free potential energy of the ground state reagents (X) and the unstable transition state (X‡) along a reaction coordinate pathway, where the energy of the species involved are plotted with the progress of the reaction (Figure 1.18). (81) The transition state (TS) is described as a high energy state that resembles both reactant and product states. During the TS chemical bonds are broken and formed. More stable intermediates with stable bonds can form during the reaction.

X‡

‡ ΔG soln Uncatalysed

‡ ΔΔG cat (the reduction in ΔG‡ by the catalyst)

‡ ΔG enz Catalysed

A + B

P + Q

Reaction Coordinates

Figure 1.18. The effect of a catalyst on the transition state diagram of a reaction. Curves represent a two- dimensional potential energy surface for the reaction A + B ⇌ P + Q. The transition state (X‡) is at the top of ‡ ‡ each curve and ΔG is the energy required to reach the TS from the ground state. ΔG soln is the energy needed ‡ for the reaction in solution, uncatalysed (blue line), while ΔG enz is the energy required in an enzyme ‡ ‡ ‡ catalysed reaction (red line). Here ΔΔG = ΔG soln – ΔG enz. Substrates or substrate and enzyme before the reaction are labelled A and B and products or product and enzyme after the reaction are labelled P and Q.

The ground states and TS are considered to be in thermodynamic equilibrium, and the concentration of TS can be calculated from the energy difference between the two states. The Arrhenius equation (Equation 1) is used to describe the temperature dependence of rate constants,

( ) ( ) Eq. 1

Where k is the rate constant, Ea the activation energy, R the gas constant, T the temperature (in Kelvin) and ln A the constant of integration. Equation 1 can be rearranged as shown in Equation 2: 47

( ) Eq. 2

where A represents the frequency of collisions of reacting species and is called the Arrhenius pre-exponential factor. This indicates that a reaction will not commence unless the energy of the reactants is greater than Ea, allowing the TS to form. The concentration of TS [X‡] can be found for the bimolecular reaction of the enzyme and substrate according to Equation 3:

[ ] [ ][ ] Eq. 3 where [A] and [B] are the concentrations for the two molecules reacting and K‡ is the equilibrium constant for the formation of TS. The energy required to overcome the energy barrier is the activation energy (or Gibbs-energy, ΔG‡). In thermodynamics, the relationship between an equilibrium constant of a reaction and the Gibbs-energy change is described as follows (Equation 4):

Eq. 4 and the relationship between the Gibbs-energy change enthalpy and entropy is described in Equation 5:

Eq. 5 where ΔG‡ is the Gibbs-energy, ΔH‡ the enthalpy and ΔS‡ the entropy of activation. Lowering the energy barrier between the two energy minima will lead to a rate enhancement (Figure 1.19). In TST, this is achieved by stabilising (or lowering the energy) the TS of the reaction. As the reaction barrier is lowered for both the forward and backward reactions, the equilibrium not affected by enzyme catalysis.

1.5.2 Theory of Quantum Mechanical Tunnelling Quantum mechanics came forward in the middle of the 20th century and the field progressed rapidly in the following decades. In 1966, it was suggested that electron tunnelling takes place between cytochrome c and bacteriochlorophyll (82). A short overview of the quantum mechanical tunnelling follows.

1.5.2.1 The wave/particle duality The wave/particle duality proposed by the French Nobel laureate Louis de Broglie suggests that matter possesses the attributes of both a wave and particle simultaneously. Wave quanta can be described by two equations (Equations 6 and 7):

Eq. 6

Eq. 7 where E is energy of the wave, h is the Planck’s constant, p the momentum of the particle, v is the mean speed of the particle and c is the speed of light. From these two equations the de Broglie relation described that all matter has wave-like characteristics (Equation 8):

Eq. 8 where h is the Planck’s constant, λ is the wavelength of a particle and µ the reduced mass of the particle with mean speed of v. In equation 9, the Heisenberg’s uncertainty principle shows the wave-like character is connected to its particle-like behaviour.

Eq. 9 where Δq and Δp are the uncertainty or probability of position and momentum of a particle and ħ is the reduced Planck’s constant (h/2π). This principle states that in a system, a particle cannot have a definite value for both the location and momentum at the same time. This means that the more accurately the location of a particle is known, the less is known about its momentum. Conversely, the more that is known about the momentum of the particle, the less is known about its location. This means that a particle has a certain probability of existence at any particular point and this distribution of probability is said to be the wave function of the particle (ψ 2).

This means that a particle is never in any fixed location and thus the kinetic energy of the particle cannot be zero. Zero point energy (ZPE) is the lowest energy that a particle can possibly have according to the uncertainty principle (Figure 1.19). The ZPE is dependent on the mass of a particle, and mass can be measured more accurately for larger particles. This is in agreement with the de Broglie relation (Equation 8), that the smaller the particle

Energy

ΔED ΔEH

0 EH

0 ED

Nuclear distance

A-H/D A H/D A H/D A H/D

Partial overlap Slight overlap (not a stable Separated atoms (stable bond) covalent bond)

Large overlap (repulsion between nuclei)

Figure 1.19. Schematic illustration of a potential energy curve of a bond between chemical A and hydrogen/deuterium (H/D). The potential well describes the energy associated with the diatomic bond between A and either H or D. The effect of difference in zero point energy between isotopes means that A-D requires more energy to break the bond than A-H. the larger its wavelength. Small particles like isotopes hydrogen and deuterium, with wavelengths of ~0.63 and ~0.45 Å respectively, have a wave-function that is close to the width of an energy barrier of a proton-transfer (83). This means that a wave-function can reach through an energy barrier and allow a particle to pass through, without obtaining the energy needed for the transition state according to classical models. The term quantum tunnelling (or just tunnelling) is used to describe these circumstances.

1.5.3 Hydrogen tunnelling Atoms have the same number of electrons and protons. Isotopes are atoms that have the same number of protons but vary in the number of neutrons. The mass of nucleus (and the atom) varies with different isotopes as the number of neutrons varies. As the electron (and proton) content is not changed, the effect on the chemical properties is negligible. The effect of the mass change is most pronounced for hydrogen as it has only a single proton in its nucleus. Deuterium (2H) has an added neutron and has therefore twice the mass of hydrogen (1H, protium). For other atoms the difference is less, for example, the difference between 12C and 13C is ~8%. Molecules that contain different isotopes are called isotopologues.

The discovery of isotopes in the early twentieth century, followed by their increased use in chemistry and biological sciences, has allowed detailed analysis of mechanisms and rates of chemical reactions at quantum levels. As these methods have become more accepted they have allowed a deeper understanding of reaction mechanism beyond the simplification of the transition state theory (84). At first a tunnel-correction model was made to accommodate tunnelling within TST (79), but as large numbers of enzyme reactions involve a hydrogen transfer, over the last decade an agreement has been reached that these reactions likely proceed via quantum mechanical tunnelling (85-88).

1.5.4 Comparison of transition state and tunnelling As described in section 1.5.1, the classical over-the-barrier transition state theory is traditionally shown as an energy barrier that has to be surmounted for the reaction to move from reactants to products. The higher the energy barrier, the more energy is required so the slower the reaction is. In some cases, this description of catalysis is imperfect.

In TST, matter is solely considered as particles but, as described in section 1.5.2, matter also has a wavelength in accordance to wave-particle duality theory (the smaller the particle, the larger the wavelength). This wave-like property of matter means that it can potentially pass through regions which particles could not, like visible light through clear glass. The pathway of the reactants may not need to pass over the energy barrier but could pass through it, often referred to as tunnelling (Figure 1.20). The energy required for the

X‡ Over the barrier

Higher energy of a molecule increases wave function across the barrier which increases the chance of tunnelling.

Through the barrier

Potentialenergy A-H/D + B Reactants

A + H/D-B Products

Reaction coordinates

Figure 1.20. Reaction coordinate diagram comparison between transition state theory and tunnelling of hydrogen and deuterium. According to TST (green), for the reaction (A-H + B → A + H-B) to proceed, reactants need to pass over the energy barrier and through X‡ (transition state) to the product side. Quantum tunnelling predicts that relatively light atoms can tunnel through energy barriers without reaching the energy needed for X‡. Lighter atoms, here hydrogen (blue), have a larger wave function (described by a coloured line) than heavier atoms, here deuterium (red), and require less energy to tunnel through the barrier. In this case, the hydrogen transfer would be faster than deuterium due to this difference. reaction can this way be decreased and therefore increase the reaction rate in enzyme catalysed reactions, especially for small molecules (like electrons (89) and hydrogen (90)). This method of catalysis would be very advantageous for enzymes with large activation energy of a hydrogen transfer. Early theoretical models (Bell correction model (79)) interpreted the transfer reactions as hybrid of ‘over’ and ‘through’ the barrier (TST and quantum-tunnelling, respectively).

This model is not applicable in cases where H-transfer proceeds only through quantum tunnelling, as in the case of methylamine dehydrogenase (MADH) (91). In this example

the tunnelling takes place in the ground state, without proceeding up the energy barrier. Despite proceeding fully through tunnelling, the reaction rate is temperature dependent as with TST as the enzyme dynamics are temperature-dependant and affect the barrier width between reactants and products (Section 1.6).

1.5.3.2 Kinetic isotope effect Kinetic isotope effects (KIEs) on reactions are investigated by studying the differences in the kinetics of reactions between enzymes and isotopologue substrates. The difference in the reaction rate with isotopologue substrates is referred to the kinetic isotope effect (KIE = kH/kD), with kH and kD referring to the rate with protiated and deuterated substrates, respectively. Kinetic isotope effects have been used successfully to explain non-classical TST behaviour in many enzyme catalysed H-transfer reactions.

H-tunnelling can be detected and KIEs studied because of the difference in mass between hydrogen and deuterium. This effect can be described in a simple way with Equation 1.10, where the tunnelling probability, P, is related to the mass of the tunnelling atom:

[( √ ) ] Eq. 10 where ℓ is the width and V is the height of a rectangular potential energy barrier and is the reduced Plank constant. Given that electrons can be transferred up to distances of around 25 Å during biological catalysis and protium is about 1840 times heavier than an electron, the protium tunnelling distances reach to around 0.58 Å, which is similar to the distance between reaction coordinates (92). On the same principle, deuterium can tunnel distances of 0.41 Å. This difference in possible tunnelling distance allows the studies of H- tunnelling by KIE studies.

Different kinds of KIE can be measured. Primary (1°) KIE is the rate difference between substrate with isotopically labelled and non-isotopically labelled transferred atom. Secondary (2°) KIE is the effect of isotope labelling of the proximal atom to the transferred atom (α-2° KIE) or other atom than the proximal atom (β-2° KIE). Secondary KIEs can be monitored as the mass difference between isotopes changes the centre of gravity (reduced mass) and thus the chemical bond frequency between the isotope and the rest of the isotopologue. The vibrational frequency of a diatomic molecules is defined in Equation 11:

√ Eq. 11

where vH-X is the frequency of the chemical bond, fH-X is the H-X bond force constant and

µH-X is the reduced mass. This changes the ground state ZPE, the stability of the bond and the reaction rate. The expected 1° KIE, if tunnelling is not considered, can be calculated from Equation 12.

( ) Eq. 12

And from the definition that KIE = kH/kD it can be rearranged as in Equation 13:

( ) ( ) Eq. 13

where kH and kD are the reaction rates with protiated and deuterated substrates, AH and AD are the Arrhenius prefactors, vH and vD are the bond frequency for hydrogen and deuterium, respectively, R is the gas constant and T the temperature (K).

At 300 K the 1° KIE is calculated to have an upper limit of ~ 7 for reactions obeying a classical TST mechanism. Enzyme-catalysed reactions can have KIEs below this limit due to the complex nature of enzyme-catalysed reactions (i.e. multi-step process and steps which do not include breaking of a C-H bond (92)). Inflated KIEs (>7) suggest the reaction does not proceed by a semi-classical system and the reaction likely proceeds via tunnelling. Examples of a large 1° KIE are methylamine dehydrogenase and soybean lipoxygenase, with a KIEs of 16.8 (91) and ~80 (93), respectively.

The 2° KIEs have more complex origin than 1° KIE. For C-H and C-D, the electronic energy surface is essentially the same while the bond length is longer for C-H (94, 95). This leads to the theory that the secondary isotope effect is derived from the different energies in the nuclear motion, the vibrational frequency. This different comes from the mass differences between isotopes. The increase in 2° KIE can be thought as a change in bond force constant which affects the rehybridisation during a reaction (95, 96). Computational methods have been used to predict KIEs, complementing experimental work on hydrogen nuclear tunnelling and enzyme dynamics (85, 97).

1.6 Temperature studies on tunnelling and enzyme dynamics The link between quantum mechanical tunnelling and motion within the protein active site (protein and/or substrate motion) during enzyme catalysis has been the subject of heated debate over the last two decades (98-100). The role of enzyme dynamics in catalysis is a complex subject. Protein dynamics has been detected by a variety of techniques, and ranges from domain motions to bond vibrations, in the time frame of seconds to femtoseconds, respectively (101). Thermally equilibrated motions within protein structures tend to be stochastic and lead to faster enzyme catalysed reactions. Vibrational modes of the enzyme and/or substrate could possibly increase H-tunnelling rates, by shortening the interatomic distance, but one of the limitations in the study of promoting vibrations is the lack of methods to directly measure them experimentally (86, 100, 102). The function of these dynamics in enzyme reactions has been analysed by kinetic isotope effects and the temperature dependent reaction rate changes between substrate isotologues (103).

The relationship between temperature and reaction rates is often shown in a temperature dependence plot (Arrhenius plot1), shown in Figure 1.21. The plot consists of four regimes (105). In regime I a classical TST behaviour takes place and ΔH‡ values are large and A’H : A’D is ~ 1. Quantum tunnelling effects on temperature dependence are visible and increase from regime II to IV. Over regime II protium has more possibility of tunnelling than deuterium, resulting in inflated KIEs, >7, and A’H : A’D is < 1. More tunnelling effect is visible between protium and deuterium in regime III. In regime IV the tunnelling proceeds only through ground state tunnelling, with no temperature dependence and A’H : A’D is equal to the KIE. This temperature dependence of the reaction rate can be used to detect non-classical reactions in enzymes, when A’H : A’D ≠ 1 and the ΔΔH‡ > 5.4 kJ mol-1 (the difference in the zero point vibrational energy of C-H and C-D).

Temperature dependence plots of KIEs (KIE vs 1/T) are also used to study tunnelling properties and link motions of protein and substrates to H-tunnelling. As thermally induced vibrations in protein can drive H-tunnelling (106) a theoretical model was made by Kuznetsov & Ulstrup (98) and adapted by Knapp & Klinman (107) as Equation 14:

1 Arrhenius plot is shown as ln (k / T) vs 1 / T plot with the Eyring equation (104. Gladstone, S., Laidler, K. J., and Eyring, H. (1951) Theory of rate processes, McGraw-Hill, New York.). ln (k / T) = lnA’ – ‡ ‡ ‡ ΔH /RT, where ln A’ = ln (kB / h) + ΔS / R, where the slope is Ea (Ea = ΔH + RT), and ln A’ is analogous to the Arrhenius ln A term.). 55

I II III IV

H H

)

/ /

/ / k k

k ln ( ln ( ln D

1/T 1/T

Figure 1.21. Temperature effect on tunnelling reactions. A) H-tunnelling regimes as described in text. Regime I is a classical TST behaviour, regimes II and III require some thermal energy to proceed through tunnelling while in regime IV ground state tunnelling takes place. B) Typical temperature dependence plot of a reaction partially proceeding through quantum tunnelling. Rates of hydrogen (H) and deuterium (D) are shown as blue and red lines, respectively.

( ) ( ) [ { }]

( ) ( ) Eq. 14 where ktunnel is the tunnelling rate constant, const. is an independent term of electron coupling, ΔG° is the driving force of the reaction, λ is the reorganisation energy, R is the gas constant and T is temperature, F.C. Term is the Frank-Condon nuclear overlap over the hydrogen coordinate (HC) which is connected to the overlap of the initial and final nuclear wave function of the hydrogen, Active Dynamics Term is thought as a compression of the potential energy barrier.

The model suggests that KIEs can be temperature dependent when the active dynamics term (gating) is dominating as the transfer distance is different between isotopes (107), where A’H : A’D < 1. When the F.C Term is dominating (the energy required for gating is greater than the thermal energy) the KIE is temperature independent, though occupation of excited vibrational levels might make the KIE temperature dependent. At 298 K the majority of tunnelling of hydrogen will be at ground state.

With H-tunnelling where the KIE is temperature independent, there are no (detectable) gating motions (motion effecting the HC). In contrast, H-tunnelling where the KIE is temperature dependent, gating motion is involved, and enzyme motions involved in tunnelling have been considered as ‘passive dynamics’. This results in nuclear configurations being in locations where tunnelling is possible, or as ‘active dynamics’, which enhance the tunnelling probability after the right configuration has been reached (90, 98, 107). This does not adequately describe the cases of temperature dependent KIEs where the reaction partially goes through tunnelling (40, 108) as both the ‘over vs through the barrier’ and/or active gating dominance would be in agreement with the temperature dependent KIE. Another fact that needs considering is that the accessible experimental temperature range is narrow (5 – 45 °C) and it has been shown computationally that a ‘false’ temperature independent KIEs can be observed (109).

The importance of short-range conformational changes in the active site and coupling with conformational changes further away on H-tunnelling reaction rates has been debated (86, 110-114). Short-range promoting vibrations have been shown to promote tunnelling by affecting the proton-acceptor distance from the substrate in aromatic amine dehydrogenase (AADH) (86, 102).

The motions affecting H-tunnelling may reach further than residues lining the active site. Both computational studies (97, 113) and experimental work (115, 116) show that hydride transfer during the reduction of dihydrofolate reductase by NADPH involves long-range motions that reach throughout the enzyme. These relatively slow motions form an equilibrium-coupled network of motions which facilitate conformations for H-tunnelling (97, 113). Computational calculations on soybean lipoxygenase suggest that the motion of proton donor and acceptor (protein cofactor and substrate) and affect the magnitude and temperature dependence of the KIE (117).

Enzyme reactions that proceed through H-tunnelling are well suited to study the effects of dynamics on reactions as the hydride transfer rate is dependent on the shape and width of the reaction barrier (118). Energy barrier compression from promoting vibrations has been shown to enhance the rates of enzyme reactions. A decrease in barrier width increases the probability of tunnelling (99, 118), and this has been supported by pressure-dependence studies on the 1° (119) and secondary (2°) KIEs (118) of the reduction of MR by NADH.

Hydride-transfer KIEs can either be independent or dependent on temperature. When the KIE is temperature dependent it can be viewed as resulting from gated motions that would decrease the tunnelling distance after the alignment of the substrate and enzyme. A temperature independent KIE is in agreement with H-tunnelling taking place without detectable gating motion. The behaviour of KIEs has been a great tool when investigating H-tunnelling reactions. Protein motion and structure affects both the magnitude and temperature dependence of the KIE, in majority of cases, by changing the donor-acceptor distance (117). Sub-picosecond promoting vibrations have also been shown to be important in the oxidative deamination catalysed by AADH, even though the observed protium/deuterium KIE does not show measurable temperature dependence (102).

The three-dimensional directions of vibrations within an enzyme are important to the reaction as promoting vibrations need to be symmetrical to the reaction coordinates to be effective and reduce the reaction barrier (106, 120). A long-range network of promoting vibrations has been suggested to play a role in tunnelling in dihydrofolate reductase (DHFR) (97, 121) and in liver alcohol dehydrogenase (LADH) (122-124). This is not universal for tunnelling reactions as similar network has not been detected in AADH (102).

Both NMR (125) and X-ray crystallographic structures of 5 different intermediates over the catalytic cycle (126) have shown dynamics areas and conformational changes within DHFR. The dynamics form a network across the enzyme and mutations relatively far from the active site can significantly affect the hydride transfer by disrupting a network of couple motions and decreasing the probability of configuration which facilitates hydride transfer (127, 128). This far reaching effect can be linked to residues which are conserved in 36 different DHFR homologues (121), from humans to E. coli.

The temperature dependence and its magnitude of KIEs has been used to examine the role of enzyme dynamics during catalysis (40, 91, 107, 108, 129). Temperature dependence has been suggested to be connected to thermally activated promoting vibrations of the enzyme- substrate complex at the subpicosecond time scale and enhances tunnelling rate by compressing the reaction barrier (99, 107, 119). Direct mutagenesis around the active site of MR has been shown to influence the donor-acceptor distance, by less than 1 Å, of the reaction complex during H-transfer (130). These changes were visible spectroscopically as the intensity of the charge transfer complex is affected by the donor-acceptor distance.

Increase of the force constant for the promoting vibration was calculated to correlate with decreasing donor-acceptor distance. Mutations around active sites can change the 1° KIE of H-transfer (111, 130-132) and can affect the donor-acceptor distance and the force constant of promoting vibrations through changes in active site orientation.

1.7 Extremophilic Enzymes Extremophiles are organisms that grow in extreme conditions and are on the outer boundaries of life. This adaptation often makes these organisms dependent on these harsh conditions. The extreme condition can be referred to as physical extremes (temperature, pressure and radiation) or geochemical extremes (pH and salinity) (133). Though heavily studied, a full understanding of the molecular basis for the adaptation of extremophiles is not clear. Extremophiles (Table 1.3) can survive at either high temperatures (thermophiles and hyperthermophiles), low temperatures (psychrophiles), high salinity (halophiles), low pH (acidophiles), high pH (alkalophiles), high pressure (piezophile) and/or high radiation level (radiophile). Extremophiles can adapt to more than one external force, for example the case of Thermococcus profundus which is a hyperthermophilic piezophile which lives near deep sea hydrothermal vents (134). The outlying life forms are interesting; Picrophilus oshimae can live both at high temperatures (60°C) and extremely low pH (0.7) (135), Trichococcus patagoniensis can grow at temperatures as low as -5 °C (136), Alkaliphilus transvaalensis can grow in pH as high as 12.5 and the deep sea organism designated as Strain 121 can live at temperatures up to 121 °C (137).

Table 1.3 Types of extremophilic microorganisms Microorganism phenotype Habitat Hyperthermophilic 80-113 °C Thermophilic 55-80 °C Psychrophilic -5-20 °C Halophilic 2-5 M NaCl Acidophilic pH < 4 Alkalophilic pH > 9 Piezophile High pressure Radiophile High radiation levels

When it comes to adaptation to temperatures, living organisms can be divided into four groups; psychrophiles, mesophiles, thermophiles and hyperthermophiles, which have optimal temperatures at ≤15 °C, 15 – 50 °C, 50 – 70 °C and above 70 °C. Temperature is a crucial factor for life; the lower it gets the slower enzymes catalyse their reactions due to increased rigidity, and higher temperatures increase enzyme flexibility leading to protein unfolding (138). Due to effects on membrane fluidity, extremophiles retain their optimum fluidity with different lipid compositions (139, 140), for example psychrophiles contain a higher ratio of long and flexible unsaturated lipids in their membranes.

The aim of adaptation is often homeostasis, such as the preservation of the structural integrity of macromolecules and their interactions or metabolism of the organism. This means that the chemistry and reaction rates of the enzymes need to be near equivalent to comparable mesophilic enzymes at extreme temperatures, in order to maintain structural integrity and catalytic activity. By comparing the sequence and structural differences in enzymes between psychrophiles, mesophiles and thermophiles, some understanding has been gained on how enzymes retain activity at extreme temperatures.

Cold adapted (psychrophilic) enzymes tend to have fewer nonpolar and small amino acids, but an increased number of polar amino acids than the corresponding mesophilic proteins (141). Their surface also contains more short-chained amino acids. Thermophilic enzymes tend to have more small and polar amino acids than mesophilic enzymes, and fewer long and polar amino acids. This makes the enzyme core more tightly packed (141, 142). The surface contains more large and charged amino acids than mesophiles. In addition, thermophiles tend to have shorter surface loops than mesophiles and psychrophiles, which reduces the flexibility of the protein (143). Thus, organisms employ slight structural changes to their overall structures to deal with their environment but while strictly conserving their active site and chemistry (144), a balance between enzyme flexibility for catalysis and structural integrity. An example is the nitroreductase family where the overall structural theme has been conserved in organisms living at high and low temperatures (Figure 1.22), yet amino acid changes have allowed the enzymes to maintain structural integrity at their optimum temperatures.

Figure 1.22. Structural similarities of nitroreductases adapted to different temperatures. Overall structures are highly similar between organisms that have adapted to very different environments and areas with visible structural differences are circled. Red: thermophilic nitroreductase from Thermus thermophilus (pdb code: 1NOX). Orange: mesophilic nitroreductase from Streptococcus pneumoniae (pdb code: 2B67). Green: mesophilic nitroreductase from E. coli (pdb code: 1ICR). Blue: psychrophilic nitroreductase from Exiguobacterium sibiricum (pdb code: 3GE6). All structures are shown as cartoons.

1.7.1 Thermo- and hyperthermophiles Hyperthermophiles can withstand high temperatures, up to 140°C, yet remain active (145). They are heavily studied for biotechnological and biocatalytical use as they are can also be more solvent stable than their mesophilic homologues (146, 147). The best known use for a thermostable enzyme is DNA polymerase in the polymerase chain reaction (PCR). This thermostability may be useful in the purification of thermophilic enzymes cloned into mesophilic hosts, such as E. coli, as the incorporation of a heat treatment step precipitates 61

the majority of the contaminating proteins. This has been used successfully in many enzyme preparations such as in the isolation of the thermostable aldolase from Sulfolobus solfataricus (148).

To withstand high temperatures, thermophilic enzymes have structural alterations from their mesophilic homologues but the overall structural changes are very few. At mesophilic temperatures (below 50 °C), thermophilic enzymes are more rigid than their mesophilic homologues due to their increased packing and smaller flexible loop regions (142, 149). Their heat stability and rigidity comes at a price with often lower specific activities at mesophilic temperatures, as opposed to cold-adapted enzymes. However, they are often more stable in organic solvents and against denaturants, than their mesophilic counterparts at ambient temperatures. Alkaline α-amylase, for example, from the thermophilic Bacillus sp. A3-15 is highly resistant to sodium dodecyl sulphate (SDS) (150). Neutron scattering has been used to measure global flexibility and enzyme rigidity of macromolecules inside cells and this technique showed that macromolecules in hyperthermophiles and thermophiles in vivo were shown to have less flexibility than mesophiles, while psychrophilic macromolecules had the most flexibility (151, 152). This makes thermophiles and hyperthermophiles interesting targets for investigating the importance of enzyme dynamics during catalysis.

1.8 Thermophilic Old Yellow Enzyme: Thermoanaerobacter pseudethanolicus was the first strain in a new genus (153). It was isolated from both alkaline and acidic hot springs in Yellowstone National Park (USA). The natural habitat of T. pseudethanolicus has the temperature range 37-78 °C and the pH range is 4.4 – 9.8, at ambient pressure. Its name derives from the high production of ethanol and CO2 via fermentation of hexoses, which classifies it as chemoorganotrophic anaerobe. It is a facultative anaerobe with an optimal growth temperature and pH range of 69 °C and 5.8 – 8.5, respectively (153).

A gene from T. pseudethanolicus with high sequence similarity to the OYE-family member YqjM, here termed TOYE for ‘Thermophilic Old Yellow Enzyme’, was chosen for the work in this thesis. Sequence alignment (Figure 1.11) shows that TOYE is a member of the thermophilic-like subclass of OYEs but previously only the mesophilic 62

YqjM had been characterised within the subclass. TOYE was the first thermophilic OYE fully characterised kinetically and structurally (23). Since then two other thermophilic OYEs been described, CrS (154) and Geo (155), supporting the thermophilic-like subclass.

In the work for this thesis, the basic characteristics of TOYE were examined. Thermal stability of the thermophilic enzyme was verified by CD and fluorescence studies and the preference towards reductive coenzyme by stopped-flow studies. Structural studies were conducted using X-ray crystallography, electron microscopy and sedimentation velocity studies. The potentials for TOYE as biocatalyst were explored by steady-state reaction and biotransformation studies. Lastly, the temperature dependence of the hydride transfer of TOYE and KIEs were studied as increased thermostability allowed for an increased temperature range compared to previously studied mesophilic homologues.

1.9 Aims and Objectives In this thesis, the basic characteristics of TOYE will be examined as well as structural adaptation to increased physiological temperature to enable future application as an industrial catalyst. The thermal stability of the thermophilic enzyme will be measured using spectroscopy, kinetic methods and structural studies, such as X-ray crystallography, electron microscopy and sedimentation velocity studies. The effects of these structural changes on substrate specificity and proton tunnelling will be compared to other members of the OYE-family by stopped-flow and steady state kinetic studies. As thermophilic enzymes are important in industrial application, the potential for TOYE as a biocatalyst will be explored by steady-state reaction and biotransformation studies. The increased temperature stability should allow for a larger temperature range compared to previously studied mesophilic homologues when studying the hydride transfer of TOYE and KIEs. This thesis makes a considerable contribution to the knowledge base of the OYE-family.

2. Materials and Methods

Table of Contents 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 64

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

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

2.1 Materials All chemicals were of analytical grade or better and the water used was double distilled unless mentioned otherwise. Chemicals and enzymes used in this thesis are listed in Table 2.1.

Table 2.1. Chemical list. Chemicals and solutions used in this thesis, listed with their sources or manufacturer. Chemical Source Acetone Sigma-Aldrich Acetonitrile Sigma-Aldrich Ampicillin Melford Calcium chloride Fischer Scientific Carbenicillin Formedium (5R)-Carvone Alfa Aeser (5S)-Carvone Alfa Aeser trans-Cinnamaldehyde Sigma-Aldrich Citral Alfa Aeser 1-Cyclohexene-1-carboxylic acid Sigma-Aldrich 2-Cyclohexen-1-one Acros Organics Deoxyribonuclease I Sigma-Aldrich Dimethyl formamide (DMF) Fischer Scientific Dipotassium hydrogen orthophosphate Fischer Scientific (K2HPO4) Ethanol Fischer Scientific 2 1-[ H6]-Ethanol Cambridge Isotope Laboratories Ethylene glycol Fischer Scientific

Flavin mononucleotide (FMN) Sigma-Aldrich

Gentamicin Formedium 1-[2H]-Glucose Cambridge Isotope Laboratories Glycerol Fischer Scientific Hydrochloric acid (HCl) Melford

Imidazole Melford Isopropanol Fischer Scientific

Isopropyl-β-D-1-thiogalactopyranoside (IPTG) Melford

Potassium chloride (KCl) Fischer Scientific Ketoisophorone Alfa Aeser Lysozyme Sigma-Aldrich Magnesium formate Fischer Scientific 2-Mercaptoethanol Sigma-Aldrich Methanol Fischer Scientific 3-Methyl cyclohexenone Alfa Aeser 2-Methyl cyclopentenone Sigma-Aldrich 3-Methyl cyclopentenone Sigma-Aldrich 2-Methyl maleimide * 2-Methyl pentenal Sigma-Aldrich Sodium chloride (NaCl) Fischer Scientific Sodium hydroxide (NaOH) Fischer Scientific

Nickel Sulfate (NiSO4·(H2O)6) Fischer Scientific

Nicotinamide adenine dinucleotide (NADH) Melford

Nicotinamide adenine dinucleotide phosphate Melford (NADPH) Ni-NTA Agarose QIAGEN Palladium-activated charcoal Sigma-Aldrich 3-Phenyl cyclohexenone * n-Phenyl-2-methyl maleimide * 2-Phenyl nitropropane * Propanol Fischer Scientific Protease inhibitors Roche Q-Sepharose GE Healthcare Sodium acetate Fischer Scientific

Sodium ethylenediaminetetraacetic acid Fischer Scientific (NaEDTA) Superdex-200 GE Healthcare Tetrahydrofuran Fischer Scientific Tris base – Tris(hydroxymethyl)aminomethane Melford *: Made in house by Dr. Anna Fryszkowska and/or Dr. David Mansell

2.1.1 Media and solutions SOC medium was used during transformations as a recovery medium for the heat-treated E. coli cells. The medium was prepared by dissolving tryptone, yeast extract, NaCl and

KCl into ddHO2, followed by pH adjustment to 7.0 using HCl/NaOH and the volume was made up to 975 ml. The medium was autoclaved, followed by the addition of 5 ml of filter sterilised 2M MgSO4 solution and 20 ml of filter sterilised 1M glucose solution was added.

SOC (1 L): Tryptone 2% (w/v) (20 g)

Yeast extract 0.5% (w/v) (5 g)

NaCl 8.6 mM (0.58 g)

KCl 2.5 mM (0.19 g)

MgSO4 20 mM

Glucose 20 mM

Luria-Bertani (LB) medium (156) was used for growing E. coli host strains. Premixed LB powder (25 g) (Formedium; Tryptone 10 g/L, Yeast Extract 5 g/L and NaCl 0.5 g/L) was dissolved in 1 L of ddH2O. The pH of the medium was adjusted to 7.0 with HCl/NaOH followed by autoclaving.

Ampicillin, gentamycin and IPTG stock solutions were made to 1000x concentration, 50 -1 -1 -1 mg ml , 20 mg ml and 24 mg ml respectively, in ddH20. The stock solutions were filter sterilised through 0.2 µm filters (Appleton Woods).

2.2 Synthesis and Cloning of TOYE The expression vector for TOYE was synthesised by Entelechon (Regensburg, Germany) and the gene was incorporated into the expression vector pET21b via NdeI (5’ end) and

XhoI (3’ end) restriction sites, including a C-terminal His6-tag on the enzyme. The pET21b encodes an ampicillin resistance gene. Agar plates with ampicillin were used to screen for transformed cells. The full gene sequence, vector map and synthesis report can be found in Appendix A. The gene codons were optimised for expression in E. coli hosts while maintaining the correct amino acid sequence. The construct was transformed into the following E. coli strains: XL10-Gold (Stratagene) and ArcticExpressTM (DE3) (Stratagene) for plasmid maintenance and enzyme expression, respectively. The genotypes for TM – – – + r ArcticExpress (DE3) and XL10-Gold are E. coli B F ompT hsdS(rB mB ) dcm Tet gal λ(DE3) endA Hte [cpn10 cpn60 Gentr] and E. coli TetrΔ(mcrA)183 Δ(mcrCB-hsdSMR- mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac Hte [F´ proAB lacIqZΔM15 Tn10 (Tetr) Amy Camr], respectively.

2.2.1 Transformation of TOYE into expression strains Chemically competent cells (100 µl) were pre-treated by the addition of sterile 2- mercaptoethanol to a final concentration of 0.03%. The cells were incubated for 10 minutes on ice followed by the addition of 0.5 µl of pET21b containing the TOYE gene (expression construct). The solution was gently mixed, followed by incubation on ice for 30 minutes. This transformation mixture was heat-treated at 42 °C for 30 seconds then cooled on ice for 2 minutes. SOC media (900 µl) was added to the mixture, followed by one hour incubation at 37°C with 180 rpm shaking. The cell slurry was centrifuged at 16,000 g for 1 minute (Thermo MicroCL 17) and 900 µl of the supernatant was removed. The remaining 100 µl and cell pellet were resuspended and plated onto LB agar plates, containing 100 µgml-1 ampicillin (plus 20 µgml-1 gentamycin for ArcticExpress cells), and incubated overnight at 37 °C. Glycerol stocks were made by adding 500 µl of sterile cryoprotection buffer (80% phosphate buffered saline containing 20% glycerol) to 500 µl of cell culture followed by freezing on dry ice and storage at -84 °C.

2.2.2 Plasmid synthesis and purification Plasmids were purified using a QIAprep Miniprep kit (QIAGEN) for PCR, DNA sequencing and storage. Culture (5 ml) was allowed to grow for 16 hours at 37 °C and 220 rpm shaking. The cells were harvested by centrifuging at 16,000 g for 3 minutes (Thermo MicroCL 17) at room temperature. The cell pellet was resuspended in 250 µl of resuspension buffer P1, which includes RNAse A and LyseBlue. Lysis buffer P2 (250 µl) was added and solutions gently mixed by inverting few times until a uniform blue colour is obtained. Neutralisation buffer N3 (350 µl) was added and solutions were mixed by inverting the tube. The solution turns colourless and a white precipitate formed indicating that SDS had been successfully precipitated. This is followed by centrifugation for 10 minutes at 16,000 g at room temperature. The supernatant is removed by pipetting, leaving a white pellet, and moved to a QIAprep spin column. The spin column is centrifuged for 1 minute at 16,000 g at room temperature and the flow through is discarded. The spin columns were then washed with 750 µl of buffer PE and centrifuged again for 1 minute. The flow through was discarded and the column dried by centrifuging for 60 seconds. The QIAprep column was then placed in a 1.5 ml Eppendorf tube and 25 µl of water was added on top of the column. The Eppendorf tube was allowed to stand for 1 minute before centrifuging for 1 minute. The eluted DNA was stored at -84 °C.

2.2.3 Removal of the His6-tag from TOYE-His6 A non-His6-tagged version of TOYE was made due to the observation that the presence of a His6-tag dramatically reduced the activity of the closely related OYE YqjM (55). A stop codon was incorporated before the His6-tag to remove the tag from the enzyme using the PCR Quickchange mutagenesis strategy (Stratagene) before the XhoI restriction site. This method is designed for site specific mutation on double-stranded plasmids. Primers, that are complementary to the opposing vector strands and include the mutation, are extended by Pfu DNA polymerase during a temperature cycling. This produces a mutated plasmid.

PCR primers were designed and synthesised by Eurofins MWG Operon (Germany) to incorporate a stop codon (CTC → TGA, Figure 2.1) before the 3’ XhoI restriction site to eliminate the His6-tag from the enzyme.

5’-GAACGTGCCTTCAAAAAATGAGAGCACCACCACCACCAC-3’

5’-GTGGTGGTGGTGGTGCTCATTTTTTGAAGGCACGTTC-3’

Figure 2.1. Primers used for removing the His6-tag. The mutated codon is underlined. The native gene and

His6-tag corresponding bases are shown red and green, respectively.

The PCR reaction (50 µl) was composed of the following:

1. 1 µL of TOYE-His6 in pET21b vector (5-10 ng/µl). 2. 1 µL of each primer (5µM). 3. 1 µL of dNTP mix (dATG, dTTG, dCTG and dGTG; 5 µM each). 4. 5 µL of Pfu polymerase reaction buffer (10x; Fermentas). 5. 40 µL of sterile water. 6. 1 µL of Pfu polymerase (2.5 U; Fermentas).

The following PCR protocol was used in the mutagenensis:

1. 95 °C for 30 seconds. 2. 95 °C for 30 seconds. 3. 52 °C for 1 minute. 4. 68 °C for 7 minutes. 5. Steps 2 through 4 were repeated 25 times. 6. 72 °C for 7 seconds. 7. Dpn I (10 U) was added to product and incubated for 1 hour at 37°C 8. Storage at -20 °C.

To remove the parental DNA template, the product was treated with Dpn I as E. coli produces methylated DNA (target sequence: 5’-Gm6ATC-3’). As DpnI cleaves methylated DNA, like the template from E. coli, this removes the unmutated DNA template before transformation. The PCR product was transformed into XL10-Gold cells for plasmid production and storage (Section 2.2.2). The presence of the new stop codon was verified by gene sequencing (Eurofins MWG Operon, Ebersberg Germany; Appendix B) and the

plasmid was transformed into the expression host ArcticExpressTM (DE3) (Section 2.2.1). Ligation was not required before transformation as E. coli ligates

2.3 Expression trials of the recombinant TOYE-His6 Small cultures were grown to examine the expression levels of the synthesised gene. The

E. coli ArcticExpress expression cultures containing the TOYE-His6 construct were grown in LB broth containing ampicillin (50 µg ml-1) and gentamycin (20 µg ml-1). A 250 ml culture, inoculated from a glycerol stock was grown at 37 °C (220 rpm) until the OD600 reached 0.5. The cultures were split into 10 ml aliquots and induced with different final concentrations of IPTG (0 – 1 mM) followed by overnight growth at 25 °C (220 rpm). ArcticExpressTM (DE3) cells which did not contain the enzyme construct were grown without ampicillin and used as the negative control. Expression of proteins was analysed by SDS polyacrylamide gel electrophoresis as described in Section 2.5.

Overnight culture samples (1 ml) were centrifuged at 16,000 g for 2 minutes (Thermo MicroCL 17). The supernatant was discarded and the cells pellet was resuspended in a lysis buffer (phosphate buffer pH 7.0) with DNAse (0.1 mg/ml) and lysozyme (0.1 mg/ml).

2.4 Large scale production and purification of TOYE

2.4.1 Pretreatment of dialysis membranes Dialysis membranes used in this work had molecular weight cut-off 12 – 14 kDa (Medicell International Ltd.). Heavy metals and preservatives are incorporated into the dialysis tubes during the manufacturing process. The tubes need to be pre-treated to remove those unwanted chemicals. The tubes are cut to suitable length boiled first in 50 mM NaHCO3, then in 1 mM NaEDTA and finally in distilled water for 5 min each time and washed in between with distilled water. Dialysis tubes were stored in 20% EtOH at 4 °C.

2.4.2 Large scale of production of TOYE

Both native TOYE and TOYE-His6 starter cultures were made by taking a small sample from an ArcticExpressTM (DE3) glycerol stock with a sterile pipette tip to inoculate 10 ml of LB media containing ampicillin (50 µg/ml) and gentamycin (20 µg/ml). This starter culture was allowed to grow overnight at 37 °C, shaking at 180 rpm. The starter culture was transferred into a 250 ml of fresh medium including the same concentrations of antibiotics as before and the culture was grown overnight at 37 °C and 180 rpm. An 72

inoculum (5 ml) of the second culture was transferred into 0.5 L of LB, containing ampicillin (50 µg/ml) and gentamycin (20 µg/ml) and grown at 37 °C as before. The cultures were grown until the OD600 measured 0.5 – 0.7, followed by IPTG induction (0.5 mM) and grown overnight growth at 25 °C, 220 rpm.

The cultures were harvested by centrifugation at 6,000 g for 30 minutes at 4 °C (Avanti J- 26 XP centrifuge; Beckman Coulter). The supernatant was removed and the cell pellets were freeze-thawed twice on dry ice. The pellets were resuspended in 50 ml of 50 mM dipotassium phosphate buffer, pH 8.0, containing small amount of deoxyribonuclease (Sigma), lysozyme (Sigma) and 1 tablet of EDTA-free Complete protease inhibitors (Roche). The extract slurry was stirred at 4 °C for 30 minutes, followed by the addition of an excess amount of FMN when highly flavinated TOYE was required. The cells were further lysed by running them through a precooled French Press (Thermo Electron Corporation) four times. The resulting extract was then centrifuged at 40,000 g for 90 minutes at 4 °C (Avanti J-26 XP centrifuge; Beckman Coulter). The supernatant was dialysed at 4 °C in 10 L of 50 mM potassium phosphate buffer, pH 8.0, for 30 minutes. This was followed by a second dialysis in 15 L of the same buffer overnight at 4 °C.

2.4.3 Purification of TOYE-His6 The dialysed supernatant was loaded onto a Ni-NTA nickel-column (50 ml) preequilibrated in 50 mM potassium phosphate, pH 8.0, containing 40 mM imidazole and 0.3 M NaCl. Aliquots of extract (10 – 30 ml) were loaded onto the column followed by a 250 ml wash of the equilibrating buffer. TOYE was eluted with a single step of 50 mM potassium phosphate, pH 8.0, containing 250 mM imidazole and 0.3 M NaCl. The yellow fractions were pooled and dialysed twice in 10 L of either 10 mM Tris pH 7.0, for crystallographic studies, or 50 mM potassium phosphate buffer pH 7.0, for other studies. Freshly purified enzyme was concentrated through centrifugation using Vivaspin columns (molecular weight cutoff: 30,000).

2.4.4 Purification of native TOYE

For the non His6-tagged TOYE, the dialysate was loaded onto 100 ml Q-Sepharose column (GE Healthcare) equilibrated with 50 mM Tris/HCl pH 8.0. The column was washed with

4 column volumes of equilibrium buffer followed by protein elution using a 500 ml gradient of equilibration buffer containing 10 – 500 mM NaCl. Yellow fractions (20 ml) collected, pooled and dialysed in 3 aliquots of 15 L of 50 mM Tris/HCl pH 8.0, for 30 minutes each. The procedure produced ~75% pure samples of TOYE and the above Q- Sepharose procedure was repeated to improve the protein purification. This was followed by dialysis and concentrated as above using Vivaspin columns (molecular weight cutoff: 30,000).

2.5 Protein purity and molecular mass determination

2.5.1 Polyacrylamide gel electrophoresis The degree of protein expression and protein purity was assessed using precast 10% polyacrylamide gel (Gene Bio-Application Ltd (GeBA), Israel). The running buffer used was 40 mM glycine, 50 mM Tris, 1% SDS and 14 mM 2-mercaptoethanol in ddH2O. Samples (20 µl) were mixed with 5 µl of 5x SDS sample buffer and boiled for approximately 10 minutes followed by centrifugation at 16,000 g for 1 minute (Thermo MicroCL 17). The samples (20 µl) and a pre-stained protein ladder (Fermentas) were loaded onto the GeBA gels and run as above. Gels were run on constant current (60 mA, 180V) until the sample dye had reached the end of the gel.

Native polyacrylamide gel electrophoresis was performed as above, excluding SDS and β- mercaptoethanol from buffer and sample buffer. Samples were not boiled before electrophoresis either.

2.5.2 Staining and destaining of polyacrylamide gels Protein staining dye was made by dissolving 250 mg of Coomassie Blue in 90 ml of 50% methanol and 10 ml of glacial acetic acid and the solution was filtered through Whatman #1 filter paper. Gels were covered in the Coomassie solution and microwaved for 1 minute. Destaining was performed by pouring away the Coomassie solution and soaking the gel in water and microwaving (850 W) for 4 minutes. The water was replaced and microwaved again until the gel was destained.

2.6 Determination of enzyme concentration The UV/Vis spectrum between 200 – 800 nm (Figure 2.2) of purified native TOYE and

TOYE-His6 showed peaks at ~456 and ~380 nm (corresponding to the bound FMN) and a peak at 280 nm (corresponding to aromatic amino acids in the enzyme). Due to partial flavination of TOYE, verified by partial occupancy in the crystal structures, it was not possible to determine accurate extinction coefficient (ε) for the enzyme bound FMN. Concentration of the flavinated enzyme was determined using the commonly used extinction coefficient of 11,300 M-1 cm-1 for bound FMN in PETNR and MR (15, 157) according to the Beer-Lambert law,

Equation 2.1

Where Abs is the absorbance, c is the molar concentration and l is the path length in cm. This formula gives the monomeric concentration of TOYE. While this enzyme is a homotetramer (dimer of dimers), each tetramer contains four complete active sites. FMN reduction at 456 nm was monitored directly in reductive half reaction studies.

1.0

0.8

0.6

0.4 Absorbance

0.2

0.0 300 350 400 450 500 550 600 Wavelength (nm)

Figure 2.2 Sample UV/VIS spectrum of TOYE around the FMN absorption. FMN absorption peaks are at 456 nm and around 362 nm.

2.7 Re-flavination of TOYE

The degree of flavination of TOYE-His6 and native TOYE preparations was determined due to the relatively low flavin absorbance of the purified enzymes suggestive of a low level of flavination. To determine this degree of flavination, the total protein concentration of purified TOYE was determined by both the Bradford assay (158) and the 205/280 nm absorbance methods (158) (Equation 2.2). Bovine serum albumin (BSA) (New England Biolabs) was used as the protein standard. The absorbance peak at 456 nm was used to determine the concentration of flavinated TOYE using the extinction coefficient for bound FMN (11.300 M-1 cm-1). The ratio of flavination was determined using Equation 2.3.

A P (mg / ml)  205 Equation 2.2  A280  27.0 120   A205 

( ) Equation 2.3 ( )

Re-flavination of TOYE was achieved by adding an excessive amount of free FMN to enzyme solutions and incubating at both ambient temperature and 42 °C for 5 – 15 minutes. Free flavin was removed by desalting the solution (10-DG BioRad desalting column) into 10 mM Tris pH 8.0 (159). The degree of flavination and % increase in flavination was determined as above.

2.8 Temperature stability studies on TOYE-His6

2.8.1 Circular dichroism studies on TOYE-His6 The thermostability of TOYE-His6 was determined by monitoring the changes in the secondary structure with increased temperature using circular dichroism (CD; Chirascan circular dichroism spectrometer; Applied Biophysics). TOYE was desalted into 10 mM potassium phosphate buffer, pH 7.0, using a 10-DG BioRad desalting column. The same buffer was used to measure the baseline for the experiment. TOYE-His6 (300 µl of a 5.3

mg/ml solution) was placed into a 0.5 mm cuvette (Starna Scientific) and the CD spectrum was measured every 5 °C, from 10 °C up to 90 °C with the temperature ramp of 1 °C per minute. The absorbance changes at 222 nm were also monitored over time between 70 and 80 °C. Data was collected and processed in the Pro-Data software of Chirascan. The absorbance at 222 nm was plotted to assess the degree of α-helical content with increasing temperature, which is indicative of the level of folded protein.

2.8.2 Fluorescence spectroscopy The changes in fluorescence were monitored with increasing temperature to determine the melting temperature (Tm) of TOYE-His6. These studies were performed by Dr. Thomas A. Jowitt at the Biomolecular Analysis Facility of the University of Manchester, Faculty of

Life Sciences. TOYE-His6 samples (5 µM) were heated from 20 °C to 80 °C and incubated for 20 minutes at 10 °C intervals in 50 mM potassium phosphate buffer pH 7.0. Tryptophan fluorescence was monitored using 295 nm excitation, and emission was monitored between 310 and 400 nm (Jasco FP570 spectrofluorimeter). Flavin fluorescence emission was monitored between 460 and 600 nm after excitation at 430 nm. One enzyme sample was scanned for each temperature.

2.9 Crystallisation of TOYE

2.9.1 Screening for crystallisation conditions of TOYE All crystallisation trials were performed using the sitting drop vapour diffusion. The initial crystallisation screening kits (Qiagen) used were; Classics, Classics Lite, Anions, Cations, PEGs, PACT, MPDs, Procomplex, MBClassII, pH Clear and pH Clear II. A Mosquito robot (TTP-Labtech) was used to make 200 nl drops in MRC 96 well trays (Molecular Dimensions). In each drop, 100 nl of screening kit crystallisation solution (mother liquor) was combined with 100 nl of the enzyme solution with 80 µl of crystallisation solution from the reservoir using a Mosquito liquid handling robot (TTP). The concentrations of

TOYE-His6 used in these trials ranged from 1 to 80 mg/ml and both native and reflavinated enzyme samples were trialed. Crystal trays were sealed with Crystal Clear tape (Jena Biosciences) and were incubated at 20 °C for 3 days before first inspection. The conditions that most consistently resulted in crystals were selected for further crystallisation studies

(Tables 4.1). Higher quality crystals were usually obtained using TOYE that had not undergone reflavination (~25% flavination).

2.9.2 Crystallisation optimisation trials of TOYE-His6 and TOYE-NADH4 Conditions similar to those that gave promising crystals in screening were used for larger crystallisation trials (Table 4.2). Enzyme samples (2 µl) were mixed with an equal amount of mother liquor and allowed to equilibrate against 1 ml of mother liquor in the reservoir. Crystals were first soaked for about 30 seconds in mother liquor containing FMN followed by soaking in a second drop of mother liquor containing 15% PEG 200 as a cryoprotectant.

To determine the NADH4-TOYE co-crystal structure, the soaks were performed as above, except NADH4 was added to the second soak. Crystals were flash frozen in liquid nitrogen. Professor David Leys and Dr Helen Toogood froze the crystals and collected crystallographic data for the TOYE-His6 and TOYE-NADH4 structures, respectively, at the European Synchrotron Radiation Facility (Grenoble, France) on Station ID 14.4 (wavelength 0.97 Å; 100 K) by using ADSC CCD detector, and Dr Toogood processed and refined both data sets.

2.9.3 X-ray diffraction data collection and processing All data processing and structure refinement was performed by Dr. Helen Toogood (University of Manchester). Data was processed and scaled using the programs MOSFLM (160) and SCALA (161). The structure was solved via molecular replacement using the coordinates for a Swiss-MODEL-generated pdb file of TOYE (http://swissmodel.expasy.org). This model was generated based on the known three dimensional structure for the YqjM from B. subtilis (PDB accession code 1Z41) (30), a closely related OYE (Section 1.3.1). Model rebuilding and water addition was performed automatically using REFMAC combined with ARP/wARP (162). Positional and isotropic B-factor refinement was performed using REFMAC5 (163), with alternate rounds of manual rebuilding of the model in COOT (163). Structures were validated and deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutger University, New Brunswick, NJ (http://www.rcsb.org/). The model coordinates for TOYE-

His6 and TOYE-NADH4 were deposited with the codes 3KRU and 3KRZ, respectively. Crystallographic images were generated using Pymol (164). 78

2.10 Sedimentation velocity and bead modelling of TOYE Experiments and data interpretation was performed by Dr Thomas A. Jowitt. A Beckman XL-A ultracentrifuge with an An60Ti four-hole rotor with a two-sector epon-filled centrepieces with quartz glass windows. TOYE-His6 was used in 50 mM potassium phosphate buffer, pH 7.0. Prior to sedimentation the samples were purified with gel filtration. The rotor speed used was 129024 g at 20 °C and sedimenting boundary was monitored at 230 nm every 90 seconds until full sedimentation was reached. Sedfit software (165) was used to interpret the data, which was corrected for standard water conditions at 20 °C ( ̅ = 0.714) from the amino acid composition calculated by Sednterp

(166). Bead models of the quaternary structure of TOYE-His6 were generated using the

PDB structure file for TOYE-His6 and the SOMO solution bead modelling program (167). Experimental results were compared to the hydrodynamic parameters for the models until a good fit was obtained.

2.11 Transmission Electron Microscopy of TOYE Sample preparation and data processing were performed by Dr. Richard Collins (Manchester Interdisciplinary Biocentre, University of Manchester). Drops (10 µl) of -1 TOYE-His6 (10 µgml ) in 50 mM potassium phosphate buffer, pH 7.0, were absorbed onto glow discharged carbon-coated copper transmission electron microscopy (TEM) grids. Uranyl acetate (4% w/v) drops (10 µl) were then added for negative staining. Data was recorded digitally using a Joel 1220 transmission electron microscope coupled to a Gatan Orius charge-coupled device camera and subsequent reference free projection averaging of observed single particles was performed using EMAN (http://blake.bcm.tmc.edu/eman/).

2.12 Multi angle laser light scattering studies on TOYE

The solution molecular mass and oligomerisation state(s) of TOYE-His6 were assessed using multi angle laser light scattering (MALLS) by Dr. Thomas A. Jowitt (Biomolecular Analysis Facility of the University of Manchester, Faculty of Life Sciences). Soluble

TOYE-His6 was purified from any aggregates on a Superdex-200 24/30 gel filtration column (GE Healthcare) preequilibrated in 50 mM potassium phosphate buffer pH 7.0, on a Dionex BioLC HPLC at 0.71 ml/min. TOYE-His6 was run through a Wyatt EOS 18- angle laser photometer with a Wyatt QELS detector in place of the 13th detector for

simultaneous measurement of the hydrodynamic radius. This was connected to a Wyatt Optilab rEX refractive index detector. Hydrodynamic radius, molecular weight moments and concentration data was determined using Astra 5.2 software.

2.13 Isotopologues for kinetic studies and crystallisation

2.13.1 Isotopologue synthesis (R)-[4-2H]-NADH was prepared by the stereospecific reduction of NAD+ (300 mg) with 2 [ H6]-ethanol (500 µl) using equine liver alcohol dehydrogenase (50 U) and aldehyde dehydrogenase (90 U) 20 mM TAPS, pH 9.0 dissolved in D2O, at room temperature. The reaction was monitored by UV/VIS spectra and the reaction was stopped, by storing the solution at -80°C until purified, when the Abs340 had stopped increasing and the

Abs260:Abs340 < 3.0. The reaction mix was freeze dried and resuspended in 5 ml of same buffer.

(R)-[4-2H]-NADPH was prepared by the stereospecific reduction of NADP+ (300 mg) with 2 1-[ H6]-isopropanol (1 ml µl) using thermophilic alchohol dehydrogenase (50 U) from

Thermoanaerobacter brokii in 100 ml D2O containing 10 mM NH4HCO3, pH 9.0, at 42

°C. The reaction was monitored by UV/VIS spectra and quenched when the Abs340 had stopped increasing and the Abs260:Abs340 < 3.0. The reaction mix was freeze dried and resuspended in 5 ml of same buffer.

The inhibitors, 1,4,5,6 tetrahydro-NADH (NADH4) and 1,4,5,6 tetrahydro-NADPH + (NADPH4), were prepared by adding NAD(P) (300 mg) to palladium-activated charcoal (30 mg) in 5 ml of 10 mM Tris/Cl, pH 8.0. The reaction was kept under ~1.2 bar pressure of hydrogen (> 99%) while stirring on ice. The reaction was stopped, by storing the solution at -80 °C until purified, when no absorption was observed at 340 nm. The reaction mix was freeze dried and resuspended in 5 ml of same buffer.

2.13.2 Isotopologue purification Purification of isotopologues was achieved by using Äkta FPLC and Q-Sepharose column

(20 ml) that had be equilibrated with 2 column volumes of 10 mM NH4HCO3, pH 9.0.

Isotopologues were eluted using a 10 – 500 mM NH4HCO3 gradient. Abs260:Abs288 ≤ 1.1 2 and Abs260:Abs340 < 2.3 were considered sufficiently pure for NAD(P)H4 and (R)-[4- H]- NAD(P)H, respectively (40).

Isotopologue solutions were made fresh, and their concentrations were determined by absorbance measurements at 340 nm, ε = 6.22 mM-1cm-1 (NADH), or at 288 nm, ε = 9.2 -1 -1 mM cm (NADH4). The isotopologues were purified by anion-exchange chromatography and stored freeze-dried at -80 °C. Coenzyme purity was typically better than 95% determined by 1H NMR (Bruker 400 MHz spectrometer, 10 mM potassium phosphate buffered D2O, pD 7.1, 10 °C) by assessing relative peak height of the pro-R and pro-S protons. KIE values were not corrected for this impurity due to the typically high purity achieved.

2.14 Preparation of anaerobic solutions To prevent any unproductive flavin reoxidation of reduced FMN by oxygen, kinetic studies of TOYE were carried out under anaerobic conditions (168). These conditions were maintained by using a Belle Technology glove box containing a positive pressure atmosphere of oxygen-free nitrogen. In addition, residual oxygen was removed by continuous gas recirculation through an oxygen scavenging catalyst (BASF R3-11). Oxygen meter (Type 02M-1, Belle Technology) was used to monitor that the oxygen level was below 5 parts per million (ppm).

Anaerobic buffer solutions were made by sparging continuously with oxygen-free nitrogen at ~5 psi for 1 hour while continuously stirred. The buffers were then moved into the anaerobic box and allowed to equilibrate with the oxygen-free atmosphere overnight. Enzyme samples were made anaerobic by passing them through a 10-DG Biorad desalting column preequilibrated in anaerobic buffer inside the anaerobic glove box. Anaerobic substrate and coenzyme solutions were prepared by dissolving the compounds in anaerobic buffers within the anaerobic glove box.

2.15 Potentiometric titrations of TOYE Anaerobic redox potentiometry was performed in an anaerobic Belle Technology glove box (oxygen maintained at <5 ppm) under positive nitrogen atmosphere at between 5 – 30 °C in 50 mM potassium phosphate buffer pH 7.0. The titration was performed in a 10 ml water-jacketed beaker attached to a thermostated water bath. The electrochemical potential was measured using a DC 740 pH meter (Mettle Toledo) that was coupled to a Russell Pt/calomel electrode and absorption spectra were measured using a Cary 50 Probe UV-

visible scanning spectrophotometer. The electrode was calibrated using the Fe(II)/Fe(III)- EDTA couple (108 mV) as a standard. Enzyme stock solutions were brought into the glove box and deoxygenated by passage through a 10-DG BioRad desalting column equilibrated with anaerobic 50 mM phosphate buffer pH 7.0. TOYE-His6 (typically between 40 – 50 μM) was titrated with sodium dithionite (reductant) to systematically reduce the flavin. Good electrical communication between the enzyme and electrode over +100 mV to -480 mV was facilitated by the use of redox mediators (7 μM 2-hydroxy-1,4-napthaquinone, 2 μM phenazine methosulfate, 0.3 μM methyl viologen and 1 μM benzyl viologen). After each addition of reductant, the solution was allowed to equilibrate for 10 – 15 minutes before the absorbance spectrum was measured between 280 – 800 nm and the corresponding redox potential was noted. To obtain a potential value relative to the standard hydrogen electrode (SHE) +244 mM was added to the observed values recorded with the Pt/calomel electrode. Titration curves consisted of ~30 different potential measurements (from -50 mV to -400 mV). The flavin absorbance of TOYE at 456 nm

(λmax for oxidised flavin) was plotted against the redox potential (Figure 2.3) and fitted to an extension of the Nernst equation and the Beer-Lambert law (Equation 2.4) (169).

A) B)

1.0 100

0.8 80

0.6 60

0.4 Absorbance

PercentageOxidised 20 0.2

0.0 300 400 500 600 -400 -300 -200 -100 Wavelength (nm) Potential versus hydrogen eletrode (mV)

Figure 2.3 Sample redox potentiometric titration of TOYE. A) Spectral changes during reductive titration of TOYE. B) Percentage of oxidised TOYE as a function of observed potential for redox potentiometric titrations of TOYE. Conditions: 50 mM potassium phosphate buffer, pH 7.0, at 25 °C. Mediators used: 7 μM 2-hydroxy-1,4-napthaquinone, 2 μM phenazine methosulfate, 0.3 μM methyl viologen and 1 μM benzyl viologen.

( ( ) ) Equation 2.4 ( )

Where Aobs is the absorbance value at the oxidised flavin peak at 456 nm, the electron potential E, and a, b are the absorbance values of the fully oxidised and reduced enzyme, respectively. E12 is the midpoint potential for the two-electron reduction of the flavin. Analysis was performed using Origin, version 7.0 (OriginLab, Northampton, USA).

The temperature dependence of the midpoint potential with sodium dithionite was used to calculate the thermodynamic parameters for the reduction of TOYE-His6. Equation 2.5 is used to calculate standard free energy change (ΔG°’) of the reaction when the standard reduction potential ( ) is measured and the number of electrons (n) involved in the redox reaction are known.

Equation 2.5

Where ΔG°’ is the standard free energy change of the reaction, n is the number of electrons taking part in the reaction, F is the Faraday constant and is the standard reduction potential. The equilibrium constant (K’eq) for the redox process and the direction of the reaction can be calculated when ΔG°’ is known and inserted into Equation 2.6.

Equation 2.6

Where R is the gas constant and T is temperature in Kelvin (K). Measuring the flavin midpoint potential of TOYE at different temperatures allows calculations of the standard entropy change of the reaction (ΔS°’) can be calculated using Equation 2.7.

( ) Equation 2.7

From this the standard enthalpy change in the reaction (ΔH°’) can be calculated by using Equation 2.8 after rearranging Equation 2.6.

Equation 2.8

2.16 Enzyme kinetics studies

2.16.1 Steady-state kinetic measurements of TOYE-His6 Anaerobic steady-state kinetic experiments were carried out using either a Cary 50 Bio spectrophotometer (Varian), with a temperature controlled cuvette holder, or BioTek Synergy HT microtiter plate reader with a 0.84 cm path length. Reactions (0.3 – 1.0 mL) were performed in 50 mM potassium phosphate buffer pH 7.0, containing oxidising substrates (0.01 – 1 mM; diluted from stock solutions in 100% ethanol), NADPH (100

µM) and TOYE-His6 (0.1 – 2 µM). The final ethanol concentration was kept constant at 5%. The initial velocity of the reaction was calculated by continuously monitoring the oxidation of the coenzyme NAD(P)H at 340 nm for 1 minute at 25 °C. Reactions with the substrate trans-cinnamaldehyde were monitored at 365 nm due to overlap in the spectra between NAD(P)H and the substrate (ε365 = 3256) (59). The rate of the reaction (δAbs/δt) was calculated using the known extinction cefficient of NAD(P)H at 340 nm (6220 M-1cm- 1). The data was fitted to the Michaelis-Menten equation (170),

[ ] Equation 2.9 [ ]

Where kmm is the initial rate of reaction, Vmax is the theoretical maximal value of kmm, Km is the concentration at half-maximal kmm and [S] is the substrate concentration. For each experimental condition, an average of 3 – 4 measurements were analysed. The specificity constants (kcat / Km) were determined from the slope of the initial rates in cases of substrate concentrations considerably lower than the estimated Km (5 – 20 µM). The kinetics parameters were determined by fitting the data using the Origin 7 software (MicroCal). 84

Solvent stability comparison of TOYE and PETNR (a kind gift from Dr. Helen Toogood) by steady-state measurements was performed in 0.3 ml reactions using a microtiter plate reader in 50 mM potassium phosphate buffer pH 7.0, at 25 °C, over 2 minutes, containing

100 µM NADPH, 1 mM 2-cyclohexenone and 200/400 nM TOYE-His6/PETNR. The effect of ethanol concentration on enzyme activity at 25 °C, was determined (2 minutes reaction; no preincubation) at ethanol concentrations of 5 – 50%. To investigate the reversibility of the organic solvent effects on activity, TOYE (4 µM) and PETNR (8 µM) were pre-incubated in ethanol solutions (0 – 90% in 50 mM potassium phosphate buffer, pH 7.0) followed by activity determination. The final ethanol concentration in each reaction was 5% and final enzyme and substrate concentrations were as before.

2.16.2 Stopped-flow kinetic studies of the reductive half-reaction of TOYE. To prevent oxidase activity of TOYE, all kinetic studies were performed under strict anaerobic conditions (<5 ppm O2) within a glove box environment (Belle Technology). An Applied Photophysics SX.18MV-R stopped-flow spectrophotometer was used for all stopped flow studies and was wholly contained within the glove box. Spectral changes accompanying flavin reduction were monitored at 456 nm over a log timebase (3 decades). Typically 3 – 5 measurements were taken for each reaction condition. Reaction transients were fit to Equation 2.10:

∑ ( ) Equation 2.10

Where Ai is the amplitude and ki is the rate constant of the ith exponential component, obtained from the stopped-flow trace, ΔA is the total absorbance change and b is a stretch function which describes how well each exponential component fits to a true exponential relationship. Values of b = 1 ± 0.2 are considered to give an accurate fit to a single exponential component (130). The term b is allowed to vary only to assess the quality of fitting and is fixed to b = 1.0 for final extraction of kinetic data. The number of exponential components were chosen based on the number which gave b = 1 ± 0.2 for the major phase and where additional exponentials did not significantly alter the extracted rate, k1, of the first (fastest) kinetic phase. Individual reaction absorption traces were fit using n = 2

(NADH and oxidative substrates) or n = 3 (NADPH) using Origin 7 software (MicroCal). For temperature dependence studies of hydride transfer, saturating concentrations of coenzyme (5 mM) were used to confer pseudo-first-order reaction kinetics. All measurements were made in 50 mM potassium phosphate buffer pH 7.0.

2.17 Biotransformations of TOYE-His6 Reactions were done in 50 mM potassium phosphate buffer pH 7.0 (1.0 ml) containing alkene substrates (5 mM) from a stock solution in DMF (final concentration of DMF was 2%), NAD(P)H (6 mM) and TOYE (10 µM) at 30 or 50 °C and shaken at 130 rpm for 24 hours. Addition reactions were performed using different organic solvents (dimethyl formamide, tetrahydrofuran, ethanol, methanol, propanol acetone and acetonitrile) in 2 – 30% v/v concentration over 2 hours at 30 °C.

Products were extracted and analysed by Dr. Anna Fryszkowska and Dr. David Mansell (Manchester Interdisciplinary Biocentre, University of Manchester). The reactions were halted by extraction with ethyl acetate (0.9 ml) with limonene (5% v/v) as the internal standard. The organic phase was dried by passage through a small column of anhydrous

MgSO4. The substrate and product(s) were analysed by GC or HPLC to determine % yield, % conversion and enantiomeric excess (% ee) and products were identified with authentic reference material as comparison. Conversion and yields were analysed by GC on a 30 m DB-Wax (0.32 mm, 0.25 mm) column, for 2-methylmaleimide % yield and % conversion were determined using a Chiraldex B-TA column (40 m, 0.25 mm). Enantiomeric excess (% ee) was determined by HPLC using a Chiralcel OD column (N-phenyl-2- methylsuccinimide), Chiraldex B-TA column (2-methylsuccinimide, 3- methylcyclopentanone, 3-methylcyclohexanone, dihydrocarvones), CP-Chirasil-DEX-CB column (levodion, nitroalkanes), Hydrodex-β-TBDAc column (2-methylpentanal, citronellal).

2.18 Temperature-dependence of observed kinetics Temperature-dependence experiments were performed with 5 °C intervals from 5 – 50 °C. Enzyme concentrations and methods are the same as described in Section 2.15.1 and 2.15.2 for steady-state and stopped-flow measurements, respectively. Before starting reactions at each temperature the samples were allowed to equilibrate for 10 minutes. The Eyring

equation (Gladstone et al. 1951) characterises the temperature-dependence of the rate constant.

( ) Equation 2.11

Where,

( ) Equation 2.12

and k is the rate constant, T is the temperature, A’ is the Eyring prefactor, ΔH‡ is the enthalpy of activation, R is the Universal gas constant, kB is the Boltzmann constant, h is the Planck’s constant and ΔS‡ is the entropy of activation. A plot of ln(k/T) vs 1/T of the ‡ ‡ temperature-dependence reveals the Ea (as the slope), ΔH (from Ea = ΔH + RT) and ln A’.

Where a breakpoint was visible the following equations, made by Dr. Sam Hay, was used to determine the Eyring parameters and the temperature of the breakpoint:

( ) ( )

(( ) √(( ) ( ))) ( ) Equation 2.13

Where ln A’ is the natural log of the prefactor, R is the gas constant, BP is the breakpoint in the fit and ΔH1 and ΔH2 are the enthalpy of the high and low temperature components, respectively.

2.19 Error propagation of observed rates and KIEs H D Kinetic isotope effects (KIEs) were defined as KIEobs = k obs – k obs. Observed rates obtained from stopped-flow were extracted from traces, the mean rate constant observed is reported as kobs and the error is one standard deviation (SD) of the rate constants at each temperature. For KIEs, the standard error (SE) was calculated as,

√(( ) ( ) ) Equation 2.14

3. Enzyme production and general properties

Table of Contents 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

3.1 Plasmid synthesis and transformations During gene synthesis the gene codons for TOYE were optimised for expression in E. coli to avoid potential problems during overexpression. A total of 247 bases (out of 1014 bases) in the original gene sequence were changed whilst maintaining the original amino acid sequence (silent mutations; Appendix A). Amino acids are encoded by three nucleotide codons; with 61 codons (plus 3 stop codons) for 20 amino acids (171). There are 2 to 6 codons per amino acid, and the frequency of each codon is similar to the expression of corresponding tRNAs. This ratio is not the same between organisms and translational problems can occur during overexpression of non-native proteins, especially when expressing extremophilic proteins in E. coli (172, 173).

The expression vector for TOYE was pET21b, which incorporated a C-terminal His6-tag

(TOYE-His6) and expression was controlled indirectly by IPTG. IPTG induces synthesis of a viral T7 RNA polymerase (DE3 phenotype of E. coli), which produces the mRNA transcript of TOYE under the control of the viral T7 promoter in pET21b.

The closely related OYE, YqjM, (Table 3.1; Figure 3.1) was inactive when a His6-tag was incorporated at the C-terminal end of the protein as it inhibited tetramer formation (55). To ensure that TOYE-His6 was active and comparable to the natural enzyme, another clone was made of TOYE without the His6-tag. This was done by incorporating a stop codon using the QuikChange mutagenesis strategy. The first amino acid base codon for the His6- tag was changed into a stop codon by mutating CTC (Leu) into TGA (STOP) and the mutation was verified by sequencing (Appendix B). The untagged and His6-tagged versions of TOYE were similar in initial kinetic studies (see Chapter 3.4).

Table 3.1. Amino acid sequence identity and similarities within the Old Yellow Enzyme family of enzymes. Sequence similarities are shown in the lower left half and sequence identity is shown in grey and top right of the table. Numbers directly relating to TOYE are bold. Enzymes and accession numbers: TOYE from T. pseudethanolicus (YP_001664021); YqjM from B. subtilis (30) (P54550); PETNR from E. cloacae PB2 (31) (U68759); MR from P. putida (32) (AAC43569); OYE1 from S. pastorianus (formerly S. carlsbergensis) (33) (Q02899). Accession numbers from UniProt (http://www.uniprot.org/). Monomer Enzyme TOYE PETNR MR OYE1 YqjM mass (kDa) TOYE 30% 29.9% 26.9% 51.6% 38.2 PETNR 51% 51.4% 36.5% 32.8% 40 MR 49.6% 67.4% 36.9% 29.8% 41.1 OYE1 45.5% 52.8% 55% 26.4% 45 YqjM 71.9% 52.9% 50.1% 45.3% 37.6 89

Figure 3.1. Amino acid sequence alignment between TOYE and YqjM. Enzymes and accession numbers: TOYE from T. pseudethanolicus (27) (B0KAH1) and YqjM from B. subtilis (30) (P54550). Colouring is based on residue polarity and the numbering within each protein is above the corresponding amino acids. Helices and β-sheets are shown as red barrels and blue arrows, respectively. Secondary structures shown are based on the crystal structures for TOYE, from chapter 4, (PDB code 3KRU) (23) and YqjM (PDB code

1Z41) (54). Numbering of the helices and β-sheets taking part in the (α/β)8–barrel formation is based on YqjM and are numbered by red and blue numbers within the barrel and arrows, respectively (54). The alignment was generated using Geneious 5.4 (174) and accession numbers were from UniProt (http://www.uniprot.org/). 90

3.2 Induction trials for TOYE TM Induction trials of TOYE-His6 in E. coli Arctic Express (DE3) grown in LB at 25 °C showed a high level of over-expression as seen by a thick protein band around 40 kDa on

SDS PAGE (Figure 3.2 B). The predicted monomer size of TOYE-His6 was 38 kDa according to the amino acid sequence. Over-expression was visible at all IPTG concentrations. For larger scale protein expression 0.5 mM IPTG was chosen, which was the lowest concentration of IPTG to give the maximal protein expression level (Figure 3.2 B). Arctic ExpressTM (DE3) strain was chosen as an expression host as it contains additional chaperones Cpn60 and Cpn10 from Oleispira Antarctica RB8. These extra chaperones assist with protein folding, and minimise the formation of insoluble aggregates of the target protein (175). This over-expression was accompanied by a visible detection of a large 60 kDa protein band on SDS PAGE in all cases.

A) B)

Cpn60 72 72 Cpn60 55 55 43 43 TOYE 34 34

L 0 0.01 0.05 0.1 0.5 1 L L 0 0.01 0.05 0.1 0.5 1 L

TM Figure 3.2. SDS PAGE analysis of TOYE-His6 induction trials in E. coli ArcticExpress (DE3) expression strain. Cultures were grown overnight at 25°C and 180 rpm in LB media. The protein ladder band sizes are shown on the left of each panel. Below each panel the IPTG concentration is shown in mM and protein ladder is marked as L. A) Soluble protein control of untransformed E. coli ArcticExpressTM (DE3). B) TM Soluble proteins after TOYE-His6 expression in E. coli ArcticExpress (DE3).

A) L A B C D E L B) T L C) LN TN

130 170 480 95 130 242 72

72 55 55 146

43 43

34 66 34

Figure 3.3. SDS PAGE analysis of TOYE-His6 purification. A) Purification of TOYE-His6, after a single step through a Nickel-NTA column. A: cell lysate, B, C and D: flow through. E: final purified TOYE-His6 sample. B) TOYE-His6 SDS-PAGE gel was analysed by mass spectrometry. The bands analysed are circled.

T: TOYE-His6. C) Native PAGE analysis of TOYE-His6. Lane TN: TOYE-His6. In all parts, L or LN: molecular mass markers, whose size are shown to the left or right of each panel.

3.3 Production and purification of TOYE

The TOYE-His6 enzyme was purified in a single step, with typically 75 – 100 mg of purified TOYE and TOYE-His6 isolated per litre of culture. The purity of the enzyme was assessed by 10% SDS PAGE and native PAGE analysis. The majority of TOYE was visible around 40 kDa (Figure 3.3). Both analysis methods showed multiple bands of TOYE in final purified samples and their sizes were often multiplications of the expected monomeric molecular weight of TOYE (eg. 40, 80, 120 kDa). The intensity of the bands varied with the enzyme concentration in the SDS-PAGE samples and heat treatment time during sample preparation. Nine individual and prominent bands on a SDS PAGE gel were subjected to mass spectrometry protein identification analysis (23). The analysis was performed by Emma Keevil at the in house Biomolecular Analysis Facility2. Mass spectroscopy verified that each of the higher molecular mass protein bands on SDS PAGE were higher oligomeric states of TOYE (Figure 3.3 B).

2 University of Manchester Biomolecular Analysis Facility: http://www.ls.manchester.ac.uk/research/facilities/biomolecularanalysis/ 92

Both native TOYE and TOYE-His6 were on average around 25% flavinated (mole enzyme-bound FMN : mole enzyme) when purified initially. Reflavination was achieved by adding excess amount of FMN to the enzyme, followed by removal of excess FMN by dialysis, leading to around 70 – 80% flavination. The same level of flavination was achieved by adding the FMN during the initial purification stage.

3.4 Initial transient kinetics of the reductive half-reaction of TOYE The cofactor preference of TOYE was studied by monitoring the reductive half reaction by stopped-flow kinetics with NADPH and NADH (Table 3.2 and Figure 3.4). The kinetic parameters (klim and Ks) of the reduction of the TOYE-bound FMN are comparable to other OYEs, such as MR, PETNR and XenA. NADPH is oxidised at higher rate than NADH at -1 -1 25 °C, with a klim of 36 s and 2.8 s , respectively. The reduction of TOYE by NADH is three times as fast as for OYE1 (0.9 s-1) and twice as fast as for XenA (1.5 s-1) with the same coenzyme, both of which show preferences for NADPH. MR stands out of the classical OYEs as it shows preference towards NADH. MR oxidises NADH 20 times faster than does TOYE (56 s-1 and 2.87 s-1, respectively).

The reduction of TOYE-His6 by NADH was also investigated at 50 °C closer to the physiological temperature of 69 °C. Reactions could not be performed at higher temperature due to temperature limitations of the stopped-flow instrument. The klim had increased more than six fold from 2.8 to 18.4 s-1. However, this rate is still slower than the rate for NADPH at 25 °C.

When reduced by NADH, both the native TOYE and TOYE-His6 had virtually the same -1 -1 klim values (2.82 ± 0.1 s and 2.87 ± 0.12 s , respectively) and similar Ks values (35.1 ± 7.0 µM and 62.6 ± 12.6 µM, respectively). This suggests that the structure had not been compromised by the addition of the His6-tag, unlike with YqjM where the addition of a

His6-tag resulted in an inactive enzyme (55), presumably due to compromising the formation of active dimers. This suggests that further kinetic studies and characterisation can be performed on the TOYE-His6 enzyme, which is easier to purify in larger quantities.

A) B)

1.0 1.0

0.8 0.8

0.6 0.6

0.4 0.4

0.2 0.2

RelativeAbsorbance RelativeAbsorbance

0.0 0.0

1E-3 0.01 0.1 1 10 100 1E-3 0.01 0.1 1 10 100 Time (s) Time (s)

0.01 0.01

0.00 0.00

-0.01 -0.01

Residuals Residuals 1E-3 0.01 0.1 1 10 100 1E-3 0.01 0.1 1 10 100 Time (s) Time (s)

C) D)

4.0 40 3.5 35 3.0 30

2.5 25

) )

-1 20

-1 2.0 (s

(s 1.5 15

obs

obs k k 1.0 10 0.5 5 0.0 0 -0.5 -5 0 1000 2000 3000 4000 5000 0 1000 2000 3000 4000 5000 [NADH] (µM) [NADPH] (µM)

Figure 3.4. Representative stopped-flow traces and concentration dependences for FMN reduction of 20 µM TOYE at 25 °C. A) Stopped-flow trace for FMN reduction by NADH (5 mM). Red line describes the fit from Equation 2.10 for a double exponential component and the difference between the fit and stopped-flow trace is shown below. B) Stopped-flow trace for FMN reduction by NADH (5 mM). Red line describes the fit from Equation 2.10 for a double exponential component and the difference between the fit and stopped-flow trace is shown below. C) Dependence of the observed rate constant for FMN reduction on the concentration of NADH. Red line shows the fit to Equation 2.9. D) Dependence of the observed rate constant for FMN reduction on the concentration of NADPH. Red line shows the fit to Equation 2.9.

Table 3.2. Stopped-flow kinetics of the reductive half reaction of TOYE with NADH and NADPH. Performed at 25 °C unless stated otherwise. a (40) b (176) c (35). -1 Protein/Cofactor klim (s ) Ks (µM) TOYE/NADH 2.8 ± 0.1 35.1 ± 7.0 TOYE-His6/NADH 2.8 ± 0.1 62.6 ± 12.6 TOYE-His6/NADH 50°C 18.4 ± 0.2 186.1 ± 8.1 TOYE/NADPH 31.7 ± 2.1 58.0 ± 19.4 TOYE-His6/NADPH 36.1 ± 0.5 33.1 ± 3.9

MR/NADH a 56 ± 0.6 101 ± 7 PETNR/NADPH a 34 ± 0.4 73 ± 0.4 XenA/NADH b 1.5 ± 0.2 176 ± 14 XenA/NADPH b 35.7 ± 0.6 256 ± 12 OYE1/NADH c 0.9 ± 0.1 <10 OYE1/NADPH c 5.1 ± 0.1 100

3.5 Thermal stability of TOYE The thermal stability of TOYE-His6 was investigated using CD, DSC and fluorescence methods. The CD spectropolarimeter was used to monitor changes in the degree of α- helical content in the secondary structure of TOYE-His6 at 222 nm during increased temperature and prolonged exposure to high temperatures. TOYE-His6 was shown to be stable at 72 °C for at least 60 minutes (Figure 3.5). The enzyme starts to unfold above that temperature and white aggregates were visible to the naked eye in the sample cuvette.

By monitoring at different wavelengths it was possible to distinguish between changes in tryptophan and FMN fluorescence (310-400 and 460-600 nm, respectively). Fluorescence studies for tryptophan residues showed a linear decrease in fluorescence with increased temperature up to 65 °C. This suggests an increased flexibility of tryptophan residues. The fluorescence of FMN shows a linear decrease up to 70 °C, the signal decreases at 80 °C (Figure 3.4 B, open squares). This decrease can be explained by quenching caused by aggregation in the sample during the unfolding process above around 80 °C (177). Differential scanning calorimetry (DSC) showed that enzyme unfolding begins at 79 °C and aggregates start forming at 86.5 °C. The melting temperature (Tm) and enthalpy (ΔH) of the unfolding event could not be determined due to this aggregation.

A) B)

0 1.0 70°C 0.9 -10 72°C 74°C 0.8 76°C (mdeg) 78°C -20 222 0.7 79°C 79.5°C

Molar ellipticity (mdeg) 80°C

-30 0.6 RelativeA

0.5 -40 200 220 240 260 280 0 10 20 30 40 50 60 Wavelength (nm) Time (min)

C) D) -20

C) o -40

Cp (kcal/mole/ Cp

-60 30 40 50 60 70 80 90 100 o Temperature ( C)

Figure 3.5. Thermal secondary structure unfolding of TOYE. A) CD spectra of thermal unfolding of TOYE after 0, 7.5 and 45 min at 80 °C (solid, dotted and dashed lines, respectively). TOYE (5 – 10 µM in 50 mM potassium phosphate buffer, pH 7.0) was monitored by molar ellipticity at 222 nm over time (1 h) between 70 – 80 °C. B) Thermal secondary structure unfolding of TOYE was determined by CD showing TOYE stable up to 72 °C. The second phase of the unfolding event could be attributed to aggregation formation. TOYE (5 – 10 µM in 50 mM potassium phosphate buffer, pH 7.0) was monitored by absorbance change at 222 nm over time (1 h) between 70 – 80 °C. C) DSC analysis of TOYE from 20 °C to 100 °C. TOYE (0.5 mg/mL) was denatured with a scan rate of 60 °C/h from 20 °C to 100 °C. Unfolding is visible around 75 °C (with the increase of signal) and protein aggregation around 85 °C (sharp decrease in signal). D) Fluorescence studies of TOYE. Tryptophan fluorescence was studied with an excitation of 295 nm (filled squares) and scanning between 310 and 400 nm. Flavin fluorescence was studied using an excitation of 430 nm (open squares) and scanning emissions between 460 and 600 nm. All TOYE samples (5 µM) were in 50 mM potassium phosphate buffer, pH 7.0.

The thermal stability of OYE family members has not been extensively studied. The mesophilic xenobiotic reductase A (XenA) has a Tm of 50.4 °C (178) and closely related YqjM starts unfolding at around 50°C (155). The thermostable GkOYE shows a similar thermal stability to TOYE as it is stable to up to 75 °C (155). Geobacillus kaustophilus HTA426 has anoptimal growth temperature of 60 °C (179) similar to the host of TOYE Thermoanaerobacter pseudethanolicus (69 °C) (153). This thermal stability of TOYE makes it an excellent candidate for biocatalysis studies as thermostable enzymes are frequently more solvent resistant than their mesophilic homologues (180).

3.6 Redox potentiometry of electron transfer in TOYE Potentiometric redox titrations were carried out using the reductant sodium dithionite and at temperatures over the range of 6 – 30 °C at pH 7.0. During the titrations no turbidity was observed and no evidence for flavin semiquinone accumulation was visible during the reduction of the oxidised flavin to dihydroflavin (typical titrations are shown in figure 3.6, all titrations can be found in Appendix C). The formation of either a red anionic or a blue neutral semiquinone during reduction is normally detected by an increased absorbance at around 350 and 600 nm, respectively (181). The lack of accumulation of a semiquinone species is unlike OYE, which proceeds through anionic red semiquinone (182), but is similar to both PETNR (26) and MR (183). Like other OYEs, there is a residual absorbance at 456 nm in the presence of excess reductant which indicates that the reduction of the flavin was not complete (~8% oxidised enzyme at the end of reduction)

+ - 2H , 2e and the reaction is reversible, TOYEox TOYEred. Starting and final absorbance values were used as 100% and 0%, respectively, for the application of the Nernst equation (Figure 3.6 B).

Data were fitted to the two-electron Nernst equation (Equation 2.4) by using least square regression giving a two-electron and RTF value (2.303RT/nF) for each temperature. The RTF value corresponded to the theoretical value of 29.5 mV for an enzyme-bound two- electron reduction of FMN at 25°C. Measurements of midpoint potentials at different temperatures were used to determine that TOYE-bound FMN showed a linear temperature dependence over the observed range (6.0 °C to 30 °C: Figure 3.7 B)). Temperatures above 30 °C could not be observed due to equipment limitations and increase experimental inaccuracy. 97

A) B)

1.0 100

0.8 80

0.6 60

0.4 Absorbance

0.2 PercentageOxidised

0 0.0 300 400 500 600 -400 -300 -200 -100 Wavelength (nm) Potential versus hydrogen eletrode (mV)

Figure 3.6. Redox potentiometric titration of TOYE. A) Spectral changes during reductive titration of TOYE with sodium dithionite at 20 °C in 50 mM potassium phosphate buffer, pH 7.0. B) Absorbance changes at 456 nm as a function of observed potential for redox potentiometric titrations of TOYE with sodium dithionite at 20 °C. Starting and final absorbance values were used as 100% and 0%, respectively, for the application of the Nernst equation. Mediators used: 7 μM 2-hydroxy-1,4-napthaquinone, 2 μM phenazine methosulfate, 0.3 μM methyl viologen and 1 μM benzyl viologen.

100 -170

-175 80

-180

60 -185

-190 40

-195

PercentageOxidised 20

Midpointpotential(mV) -200

-205 0

-210 -300 -250 -200 -150 -100 5 10 15 20 25 30 Potential versus hydrogen electrode (mV) Temperature (°C)

Figure 3.7. Temperature dependence of Em during redox potentiometric titration of TOYE. Panel A, overlay of absorbance changes as % oxidised TOYE against titration potential at different temperatures. ▼: 6 °C, ▲:

10 °C, ◄: 15 °C, ►: 20 °C, ●: 25 °C and ■: 30 °C. Panel B, plot showing Em against temperature showing a gradient of -1.4 ± 0.2 mV/°C in a linear fit (R2 = 0.92). The midpoint potential at physiological temperature (65 °C for T. pseudethanolicus) can be extrapolated to -255 ± 13 mV if no unforeseen changes take place outside the observable range.

The temperature dependence of the midpoint potential was calculated to be -1.4 ± 0.2 mV/°C. A midpoint potential Em65°C = -255 ± 13 mV at physiological temperatures (65 °C for T. pseudethanolicus) can be extrapolated from the plot assuming that no changes in the linear trend outside the accessible temperature of the experiment occurred. The standard free energy change3 (ΔG°’) value was calculated from Equation 2.5 for the reaction: -1 ΔG°’298 = -39.22 ± 0.1 kJ·mol at 25 °C. The free energy change was used to calculate the 4 -7 equilibrium constant (K’eq) of 1.34 * 10 for the reaction (Equation 2.6). The standard entropy change of the reaction5 (ΔS°’) and the standard enthalpy change of the reaction6 (ΔH°’) were derived from Equations 2.7 and 2.8, respectively, giving ΔS°’ = -270 ± 38 J·mol-1·K-1 and ΔH°’ = -80.47 kJ·mol-1. These parameters are similar to the L-proline -1 - dehydrogenase from Pyrococcus furiosus (ΔG°’298= -41.1 kJ·mol , ΔS°’ = -290.4 J·mol 1K-1 and ΔH°’ = -127.6 kJ·mol-1) (184). This is not surprising as temperature dependence is instrinsic to the oxidation-reduction midpoint potential, ⁄ ⁄( ) (185). The reduction spectrum showed a two-electron reduction without the accumulation of a semiquinone species with a midpoint potential similar to other OYEs at 25 °C (Table 3.3), both the classical OYEs and other thermophilic-like OYEs (YqjM and GkOYE).

Table 3.3 Thermodynamic parameters associated with the two electron reduction of TOYE and selected OYEs. a(26) b(183) c(50) d(155). NS: not specified in source.

Temperature (K) Em (mV) TOYE 338 -255 ± 13 TOYE 298 -203 ± 0.5 PETNR 298 -193 ± 5a b MR 298 -237 ± 6 OYE1 298 -230c d GkOYE NS -208 ± 8 d YqjM NS -207 ± 2 c Free FMN 298 -210

3 -1 Standard free energy change (ΔG°f ; kJ mol ) for any given reaction is defined as ∑ ( ) ∑ ( ). . 4 Equilibrium constant, Keq, is a description for a dynamic equilibrium. Equilibrium constant can be derived [ ] [ ] from standard free energy or the substrate and product concentration at equilibrium: [ ] [ ] ⁄ . 5 Standard entropy change is the change in entropy (or disorder) in a system during a reaction. 6 Standard enthalpy change is the change in enthalpy, total thermodynamic energy of a system, in a system during a reaction. 99

3.7 Discussion A novel thermostable old yellow enzyme from T. pseudethanolicus E39 (TOYE) was cloned, overexpressed, purified and characterised. TOYE was successfully isolated in both native and His6-tagged forms. A high yield of purified protein was obtained, but multiple bands were visible on SDS-PAGE gels. These multiple bands were identified as being TOYE by mass spectroscopy. This suggests that TOYE has a high oligomeric state, similar to the tetramer of the highly related YqjM, or aggregates heavily during unfolding. Native gels showed that the lowest band had a molecular weight around 160 kDa (Figure 3.2 C). Some of the bands may be a combination of oligomeric states as an aggregate or an incomplete oligomeric disassembly during sample handling. This supports the idea that the lowest oligomeric state of TOYE is a tetramer. This needs to be investigated further by crystallography and other techniques.

TOYE is thermostable as it is stable for at least 1 hour at 70 °C and starts unfolding around temperatures of 74 °C. This thermostability is comparable to GkOYE, which starts unfolding in around 76 °C. Unfolding parameters, like Tm, were not obtainable as TOYE aggregates heavily during heating. Some of the additional TOYE bands seen on SDS- PAGE might arise from incomplete oligomer disassembly and possibly aggregation.

Transient kinetic studies show that TOYE has preference towards NADPH over NADH as a reductive coenzyme. At 25 °C, which is 44 °C lower than the optimal growth temperature of the original host organism, TOYE is reduced at similar rates as mesophilic OYEs (31.4 -1 s for TOYE-His6 in reaction with NADPH). The addition of a His6-tag did not affect the reductive half-reaction of TOYE as was the case with YqjM. This allows the more readily purified TOYE-His6 to be used in further kinetic studies.

100

4. Structural studies of TOYE

Table of Contents 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 4.5 Sedimentation velocity and hydrodynamic bead model of TOYE ...... 127 4.6 Discussion ...... 129

101

TOYE is a member of the thermophilic-like subclass of OYEs and YqjM, the first member characterised, was shown to have a tetrameric structure (24). TOYE shows unusual SDS PAGE results that suggest higher oligomeric states such as octamer and dodecamer, which had previously not been reported for OYEs. Multi-angle laser light scattering (MALLS) and sedimentation velocity techniques were employed to investigate whether these higher oligomeric solutions exist in solution. TEM was used to determine the low resolution structure of oligomeric state(s) of TOYE. High resolution three-dimensional crystal structures of TOYE were determined and compared to YqjM and other OYEs to observe any thermostable determinants and aid in the understanding of substrate specificity (Chapter 5).

4.1 Transmission Electron Microscopy of TOYE In order to study the oligomeric state and overall solution structure of TOYE further,

TOYE-His6 was examined using TEM with negative staining. The image showed a homogeneous population of squared particles with a 10 – 11 nm diameter in random orientations (Figure 4.1). Image analysis calculations from 1450 particles predicted a structure with C4 rotational symmetry with an axis of pseudo mirror symmetry, consistent with a tetrameric particle that forms a two-layered stack with a possible D-symmetrical arrangement. The tetramer is formed by four monomers in a square with a central opening (Figure 4.1, B)). Potential octamer formation is consistent with two tetramers stacking on top of each other (Figure 4.1 C)). There was no evidence for the larger oligomeric states suggested by MALLS and SDS-page other than aggregations (Sections 3.2 (SDS-PAGE), 4.4 (MALLS) and 4.5 (Sedimentation velocity)). This might be due to the transient nature of the larger oligomers, or that the TEM sample preparation is not favourable for them.

4.2 Generation of the crystal structure of TOYE

4.2.1 Crystallisation of TOYE Crystallisation screens were set up with TOYE to assess the optimal crystallisation conditions at 20 °C. Out of 1056 screening conditions, 17 produced crystals (Figure 4.2 and Table 4.1). High concentrations (80 and 40 mg/ml) of TOYE-His6 were used in this screening. The most promising conditions were: i) 200 mM magnesium formate; ii) 200 mM CaCl in 0.1 M sodium acetate pH 4.6 containing 20% isopropanol; iii) 12.5% ethylene

102

B) C)

Figure 4.1. Electron microscopy of negatively stained TOYE. A) Overview showing individual stable single particles highlighted in square boxes and atypical non-selected aggregations shown by an asterisk. The black scale bar is 50 nm. Inset boxes show selected reference free projection averages calculated with likely B) top- view and C) side-view projections. The white scale bar for the inset is 10 nm.

glycol; iv) 10% isopropanol in 0.1 M HEPES pH 7.0; v) 10% isopropanol in 0.1 M Tris pH 8.0 and vi) 30% isopropanol in 0.1 M MES pH 6.0. These conditions were chosen for further crystallisation optimisation studies (Table 4.2). In these latter studies, different enzyme concentrations were used ranging from 5 mg/ml to 80 mg/ml. Typically, crystals were visible after 48 hours and diffraction quality crystals were obtained within a week. Crystals grew best from protein that had not been reflavinated during the purification process (25% flavination). Prior to flash freezing in liquid nitrogen, crystals were soaked in FMN-saturated mother liquor for 30 seconds to more fully flavinate the enzyme (~70%). Similarly, in attempts to gain a structure in the presence of an inhibitor, crystals were soaked in NAD(P)H4 saturated mother liquor before freezing, after being soaked in the

FMN solution. The NAD(P)H4 soaks lasted from 30 s to 3 minutes dependent on the stability of the crystal matrix in the soaking conditions. In some cases the crystals redissolved during soaking in the presence of the inhibitor, likely due to conformational changes in TOYE disrupting the crystal lattice upon NAD(P)H4-binding. 103

4.2.2 Crystallographic data collection

High quality X-ray crystallography data for both TOYE-His6 and the inhibitor bound native TOYE-NADH4 were collected. Crystals were soaked in mother liquor containing the cryoprotectant polyethylene glycol 200 (15% PEG200) and flash frozen in liquid nitrogen. Each dataset was collected from a single crystal under a nitrogen cryo-stream (100 K). Ice rings were visible on crystallographic images due to initial difficulties in finding optimal cryoprotectant conditions, leading to the formation of opaque ice around the enzyme crystals (Figure 4.3).

Table 4.1. Crystallisation screening conditions yielding crystals for TOYE. In each drop 100 nl of screening kit mother liquor was combined with 100 nl of the enzyme solution, and 80 µl of the mother liquor was located in the reservoir. PEG: polyethylene glycol, MPD: 2-methyl-2,4-pentadiol. The enzyme concentration was 40 mg/ml and 2 drops were made of each condition. Kit Buffer pH Salt Precipitant (QIAGEN) Classics - - - 0.2 M magnesium formate 0.1M sodium acetate pH 4.6 0.2M calcium chloride 20% isopropanol Classics L - - - 12.5% ethylene glycol - - - 0.1M magnesium formate pHClear II 0.1M HEPES pH 7.0 - 10% isopropanol 0.1 TRIS pH 8.0 - 10% isopropanol 0.1 M MES pH 6.0 - 30% isopropanol pHClear 0.1 M MES pH 6.0 5% PEG 6000 - 0.1 M HEPES pH 7.0 5% PEG 6000 - 0.1 M citric acid pH 4.0 10% PEG 6000 - 0.1 M MES pH 6.0 1.6 M ammonium sulphate - 0.1 M MES pH 6.0 2.4 M ammonium sulphate - 0.1 M sodium acetate pH 5.0 10% MPD - 0.1 M MES pH 6.0 10% MPD - 0.1 M HEPES pH 7.0 10% MPD - 0.1 M TRIS pH 8.0 10% MPD - 0.1 M sodium acetate pH 5.0 20% MPD - MB Class II 0.1 M sodium acetate pH 4.6 - 1.0 M magnesium sulphate 0.1 M HEPES sodium pH 7.5 0.1 M ammonium sulphate 12% PEG 4000

104

Table 4.2. Crystallisation optimisation screen of TOYE-His6 and native TOYE. *: chemical of which the concentration was varied as per corresponding table column; magnesium formate (50 – 300 mM), CaCl (50 – 300 mM), ethylene glycol (10% - 14%), isopropanol (8% - 32%). Concentrations from the successful screening wells are shown in bold. The enzyme concentration was 10 mg/ml and 4 drops were made of each condition.

sodium HEPES 0.1 M TRIS 0.1 M MES 0.1 M *magnesium acetate 0.1 *ethylene Condition pH 7.0 pH 8.0 pH 6.0 formate M glycol *isopropanol *isopropanol *isopropanol *CaCl

Line\Column 1 2 3 4 5 6

A 50 mM 50 mM 10 % 8 % 8 % 26 %

B 100 mM 100 mM 11 % 10 % 10 % 28 %

C 200 mM 200 mM 12.5 % 12 % 12 % 30 %

D 300 mM 300 mM 14 % 14 % 14 % 32 %

A) B)

C) D)

Figure 4.2. Selected TOYE-His6 crystals from the screening trials. Conditions A): 0.2 M magnesium formate B): 0.1 M magnesium formate C): 0.1 M HEPES and 10% isopropanol at pH 7.0 with added FMN in the mother liquor and D): as-purified crystals (25% flavinated) grown in 0.1 M HEPES and 10% isopropanol at pH 7.0. 105

Figure 4.3. Selected diffraction image of TOYE-His6 crystal from which a dataset was collected. The final resolution was 1.6 Å. The dark concentric circles are due to the presence of opaque ice in the frozen crystals due to sub-optimal cryoprotectant conditions. The white bold line is the beam stopper, which protects the detector from direct X-ray beams.

4.2.3 Data processing and refinement

The TOYE crystals belonged to the space group P21 with unit cell dimensions of a = 86.93/87.88 Å, b = 97.38/98.56 Å, c = 94.39/95.13 Å and α = 90°/90°, 92.34°/92.40° and γ

= 90°/90° for TOYE-His6 and TOYE-NADH4, respectively. This unit cell is sufficiently large to accommodate 4 TOYE monomers. Further crystallographic statistics can be found in Table 4.3.

The crystal structures were solved via molecular replacement using a TOYE Swiss- MODEL-generated pdb file (http://swissmodel.expasy.org) to facilitate initial atom placement. This model was based on the structure of the closely related OYE YqjM from B. subtilis (PDB accession code, 1Z41) (30). In both structures, electron densities of a few of the C-terminal amino acids and the His6-tag (in TOYE-His6) were not visible in the

2|F|o-|F|c and |F|o-|F|c maps, and were therefore not modelled. The final models for TOYE-

His6 and native-TOYE-NADH4 were refined to 1.60 Å and 1.80 Å with Rfactor/Rfree values of 15.2/17.9 and 15.5/19.4, respectively. The final structures were deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/) and received the PDB accession codes 3KRU

(TOYE-His6) and 3KRZ (TOYE-NADH4).

106

Table 4.3 X-ray crystallographic statistics for data collection, processing and refinement of TOYE-His6 a and TOYE-NADH4. The highest resolution shell is shown in parentheses. Ramachandran statistics were determined by MolProbity, http://molprobity.biochem.duke.edu/ (186)

Parameters TOYE-His6 TOYE-NADH4 PDB code 3KRU 3KRZ

Space group P21 P21

Resolution (Å) 64.8-1.6 (1.64-1.60) 68.4-1.8 (1.85-1.80)

Unit cell a,b,c (Å) 86.93, 97.38, 94.39 87.38, 98.56, 95.13

α,β,γ (°) 90.00, 92.34, 90.00 90.00, 92.40, 90.00

Redundancy7 2.8 (3.0) 3.3 (3.4)

Reflections

Total observed 560259 482529

Total unique (hkl) 189805 141968

Completeness (%) 98.9 (95.0) 98.9 (99.4)

8 Rmerge (%) 6.4 (35.1) 10.9 (30.2)

11.7000 10.6000 Solvent molecules 1831 1690

9 Rwork(%)/Rfree(%) 15.2/17.9 (22.5/25.6) 15.5/19.4 (18.5/21.8)

Mean B-factor (Å2) 18.43 15.30

Ramachandran plots (%)a

Favourable 97.45 97.44 Allowed 99.9 99.8 Outliners 0.15 0.15 Buried surface area (Å2) 7280 7320 Surface Area (Å2) 46150 46660

7 Redundancy is the total number of reflections diveded by total number of unique reflections. 8 Where ∑ ∑ | ( ) [ ( )]|⁄∑ ∑ ( ), where li(hkl) is the intensity of the ith observation of unique reflection hkl. 9 ∑ || | | ||⁄∑| |, where Fobs and Fcalc are observed and model factors, respectively. Rfree was calculated by using a randomly selected set (5%) of reflections. 107

4.3 Structural analysis of TOYE

4.3.1 Overall structure TOYE was the first published structure of a thermophilic OYE (23), and since then structures of other thermostable OYEs CrS (187) and Gio (155) have been described (see structural comparison in Chapter 4.3.6). These latter two enzymes also belong to the thermophilic-like subclass of OYEs. A superimposition of the structures of TOYE-His6 and native-TOYE-NADH4 showed they had very similar overall structures (Figure 4.4), and were similar to the structure of YqjM (24).

TOYE is a dimer of functional dimers, with each monomer composed of the typical OYE fold, an (α/β)8-barrel (TIM barrel) (13). The tetramer arrangement of TOYE is similar to related thermostable-like OYEs such as YqjM (54) and CrS (187). In the middle of the tetramer is a water filled central cavity accessible by the active site (Figure 4.5).

Some residues were not modelled into the structures, as the electron density was absent due to high flexibility. These residues included some at both C- and N-termini. In the

TOYE-His6 structure Lys336 was missing in subunit C and both Lys337 and the whole C- terminal-tag are absent in all four subunits. The C-terminal-tag consists of Leu338, Glu339 and His340-345. Inhibitor bound TOYE-NADH4 structure lacked Lys337 in all subunits and Ser2 was absent in subunits B and C.

4.3.2 Structural analysis of the crystal structures of TOYE-His6 and TOYE-NADH4 An analysis of the secondary structure of TOYE-His6, TOYE-NADH4 and YqjM was performed using the programs PyMol (164) and SwissPDB viewer ((188); http://www.expasy.org/spdbv/). The number and boundaries of each α-helix and β-sheet differed between the programs, the analysis in this thesis is based on the analysis from Pymol as it was closer to the published secondary structure analysis of YqjM (24).

The structure is based on an (α/β)8-barrel, formed by an alternating series of α-helices and β-sheets and begins with β1 (Figure 1.6). Additional small structural elements have been reported for YqjM: two 310-helices (αA and αF), four α-helices αB-αE), two short antiparallel β-strands (βA-β-B) and two β-sheets (Figure 3.1). Analysis of the two TOYE structures showed that most of these features were present (Figure 4.6).

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Helix α6

Helix α7

N-terminus

Helix α8

Helix αFa C-terminus Helix αFb

Figure 4.4. Overall structures of TOYE-His6 and native-TOYE. The two structures were superimposed based on the structure backbones by Pymol. TOYE-His6 and native-TOYE are shown as red and green cartoons, respectively. The N- and C-termini are labelled and the functional and non-functional interacting areas are indicated with a circle and line, respectively. The location of Arg333 at the C-terminus, which takes part in substrate binding of the adjacent monomer, is indicated with a box. Accession pdb codes for TOYE-His6 and native-TOYE: 3KRU and 3KRZ, respectively.

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A) Functional dimer

Figure 4.5. X-ray crystal structure of holo-TOYE tetramer. A) Top view and B) side view of the tetramer of TOYE. Residues are shown as rainbow cartoons from N- (blue) to C-terminus (red) in each monomer. Each FMN molecule is shown as atom coloured sticks with magenta carbons. Two monomers, forming a functional dimer are shown in a box. Accession pdb code used: 3KRU.

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Overall, the a-helices and b-sheets of TOYE tend to be shorter by 1-2 amino acids compared to its mesophilic homologue YqjM. Missing secondary structural elements were

αA 310-helix and αC-helix. The analysis by PyMol showed an absence of the -sheets C, D and 7, however these elements were present in the analysis by SwissPDB viewer.

Other minor deviations were seen in α3 and α6 and one additional 310-helix (αFa) was located between the α8 and αF helices (αFb in TOYE) due to different orientation of the C- terminus in TOYE. There was only one minor difference in the overall secondary structure of TOYE-His6 and TOYE-NADH4, a difference in the α-helical shape of the last two residues on α4-helix.

βA βB β1 α1 β2 TOYE MS-ILHMPLKIKDITIKNRIMMSPMCMYSAS-TDGMPNDWHIVHYATRAIGGVGLIMQEA YqjM MARKLFTPITIKDMTLKNRIVMSPMCMYSSHEKDGKLTPFHMAHYISRAIGQVGLIIVEA αA* βA βB β1 α1 β2

αB α2 β3 TOYE TAVESRGRITDHDLGIWNDEQVKELKKIVDICKANGAVMGIQLAHAGRKCNISYEDVVGP YqjM SAVNPQGRITDQDLGIWSDEHIEGFAKLTEQVKEQGSKIGIQLAHAGRKAELEG-DIFAP αB α2 β3 αC βC

α3 β4 αD TOYE SPIKAGDRYKLPRELSVEEIKSIVKAFGEAAKRANLAGYDVVEIHAAHGYLIHEFLSPLS YqjM SAIAFDEQSATPVEMSAEKVKETVQEFKQAAARAKEAGFDVIEIHAAHGYLIHEFLSPLS βD α3 β4 αD

α4 β5 α5 TOYE NKRKDEYGNSIENRARFLIEVIDEVRKNWPENKPIFVRVSADDYMEGGINIDMMVEYINM YqjM NHRTDEYGGSPENRYRFLREIIDEVKQVWDG--PLFVRVSASDYTDKGLDIADHIGFAKW α4 β5 α5

β6 α6 β7 α7 TOYE IKDK-VDLIDVSSGGLLNVDINLYPGYQVKYAETIKKRCNIKTSAVGLITTQELAEEILS YqjM MKEQGVDLIDCSSGALVHADINVFPGYQVSFAEKIREQADMATGAVGMITDGSMAEEILQ β6 α6 β7 α7

β8 αE α8 αFa αFb TOYE NERADLVALGRELLRNPYWV---LHTYTSKEDWPKQYERAFKK YqjM NGRADLIFIGRELLRDPFFARTAAKQLNTEIPAPVQYERGW-- β8 αE α8 αF*

Figure 4.6. Sequence alignment of TOYE and YqjM. Helices and sheets are shown with green and turquoise highlight, respectively. Secondary structures were determined using parameters from Pymol (164). * marks

310-helix and underlined amino acids in α6 and β7 show some secondary structure contacts.

111

4.3.2 Functional dimer interactions Functional dimers occur when interactions between two monomers are required to form a functional active site. In TOYE, the monomers in the functional dimer take part in the formation of the active site of each other. The interaction surface, between the functional dimers, covers 1054 Å2 with a complexation significance score (CSS) (189) of 1.0 indicating a functional interaction between those monomers. These two monomers interact via hydrogen bonds between the equivalent residues of the two monomers (Figure 4.7, Table 4.4).

TOYE is arranged into a dimer of functional dimers where the C-terminus takes part in forming the active site of the adjacent monomer (Figure 4.7 A-E). The α8 and αF (a and b) helices form extensions that interact with the active site area of the neighbouring monomer to form a functional dimer. In YqjM the C-terminal arm is extended and interacts with the adjacent monomer as well, reinforcing the connection between the dimers (54). This arm contains Arg333 (Arg336 in YqjM), which extends into the active site of an adjacent monomer (Figure 4.7). This arm is conserved within the thermophilic-like OYEs but not in the classical OYEs. This residue is important for substrate binding and the embedding of this residue into the active site of an adjacent monomer reinforces the idea that TOYE is a dimer of functional dimers. In the holoenzyme of TOYE, the Arg333 has a partial occupancy in a position that clashes with the FMN. This is possible as the structure is only ~70% flavinated and allows the Arg333 to occupy the space when the enzyme lacks the FMN. The majority of the interactions in the formation of functional dimers are between αFb (Gln330, Tyr331 and Ala334) of one monomer and α1 (His42, Arg46), αE (Arg312) and a loop between β1 and α1 (Ser28) of the other. Other interactions within the functional dimer are between Thr45 (α1) of each subunit, and between Arg46 (α1) and Try315.

4.3.3 Interaction between non-functional dimers The functional dimers interact to form a tetrameric structure through interactions between helix 7 (residues 287-300) and helix 6 (Tyr261/Lys274) of adjacent subunits (Table 4.5). The electron density of Arg300 shows two possible conformations for the residue of each subunit (Figure 4.8 D). The position of these two conformations of Arg300 for each monomer within the non-functional dimeric contact is visible as a diamond pattern. In both monomers, Glu270 is located near Arg300 and also displays dual occupancy. The dimers

112

A) Helices 8, αFa and αFb

Helix α2 Helix α1 B) C) Ala334 FMN Thr45

Tyr315 Tyr331 Arg46 Gln330 Arg312

Thr45 Tyr331

Arg46 Ser28 Tyr315 His42 D) E) Gln330

Ser28 Arg333

FMN FMN Tyr331 Ala334

Arg312 His42

Figure 4.7. Functional interaction between TOYE monomers. A) Interaction between two monomers of TOYE in the functional dimer, with monomer 1 and 2 shown as cyan and green cartoons. FMN is shown as atom coloured sticks with yellow carbons. B)-D) Closer view of the functional dimer interactions. Residues are atom coloured sticks with green and cyan coloured carbons to indicate which monomer they belong to. B) Interactions between monomer 1 Tyr315, Tyr331 and Gln330 with residues from the adjacent monomer. C) Interactions between monomer 1 Ala334, Thr45 and Arg46 and residues from the adjacent monomer. D) Interactions between monomer 1 Ser28, His42 and Arg312 and residues from the adjacent monomer. E) Interaction between Arg333 of monomer 2 and nearby water molecules by the active site of monomer 1.

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connect through hydrophobic interactions (Tyr261, Pro262, Tyr264 and Leu291), hydrogen bonds (Tyr261, Gly263, Lys267, Thr287, Thr288, Glu290 and Asn298) and salt bridges (involving Lys267, Glu270 and Arg300). As these interactions do not involve the active sites it is referred to as a ‘non-functional’ interaction. The non-functional interaction surface is 776 Å2, less than for the functional dimer interaction, and has a CSS value of 0.325, indicating a less stable connection than the functional dimer.

Table 4.4. Interactions between two TOYE-His6 subunits that form a functional dimer and surrounding solvent. The structure was refined to 1.6 Å and the interactions are based on the position in chains A and B. Water molecules are shown as HOH. Subunit Atom Distance Atom Subunit Subunit Atom Distance Atom Subunit A B B A Ser28 OG 2.92 NE2 Gln330 Ser28 OG 2.95 NE2 Gln330 His42 ND1 2.71 OH Tyr331 His42 ND1 2.66 OH Tyr331 NE2 2.79 HOH NE2 2.81 HOH Thr45 OG1 2.93 HOH Thr45 OG1 2.85 HOH 3.02 OG1 Thr45 3.02 OG1 Thr45 Arg46 NH1 2.98 OH Tyr331 Arg46 NH1 3.05 OH Tyr331 NH2 3.18 OH Tyr315 NH2 3.19 OH Tyr315 Arg312 NH1 2.96 O Ala334 Arg312 NE 2.95 O Ala334 NH2 2.82 HOH NH1 3.12 HOH NH2 2.97 O Ala334 3.06 HOH 3.08 HOH Tyr315 OH 2.77 HOH Tyr315 OH 2.72 HOH 3.19 NH2 Arg46 3.18 NH2 Arg46 Gln330 OE1 2.73 HOH Gln330 OE1 2.67 HOH NE2 3.02 HOH NE2 2.92 OG Ser28 2.95 OG Ser28 3.05 HOH Tyr331 OH 2.84 HOH Tyr331 OH 2.79 HOH 2.66 ND1 His42 2.71 ND1 His42 3.05 NH1 Arg46 2.98 NH1 Arg46 Ala334 O 2.97 NE Arg312 Ala334 O 2.96 NH1 Arg312 2.95 NH2 Arg312

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Table 4.5. Interactions between two TOYE-His6 subunits that form a non-functional dimer and surrounding solvent. The structure was refined to 1.6 Å and the interactions are based on the position in chains A and C. When a residue has two conformations they are marked with 1 and 2. Water molecules are shown as HOH.

Chain A Chain C Chain C Chain A Amino Distance Amino Amino Distance Amino Acid Atom (Å) Atom Acid Acid Atom (Å) Atom Acid Tyr261 OH 2.62 ND2 Asn298 Tyr261 OH 2.75 HOH 2.72 OD1 Asn298 Gly263 OH 3 OD1 Asn298 Gly263 O 3.01 ND2 Asn298 Glu2701 OE1 3.11 HOH Glu2701 OE2 3.03 HOH 2.99 NH2 Arg3001 2.78 NH1 Arg3001 2.78 NH1 Arg3001 3.03 NH2 Arg3001 OE2 2.91 HOH Glu2702 OE2 2.5 HOH Thr287 O 3.04 HOH Thr287 O 3 HOH N 2.97 OE2 Glu290 N 2.97 OE2 Glu290 OG1 2.64 OE1 Glu290 OG1 2.61 OE1 Glu290 OG1 2.71 HOH 2.77 HOH Thr288 OG1 2.82 OE2 Glu290 Thr288 N 2.91 OE2 Glu290 OG1 2.81 OE2 Glu290 Glu290 OE1 2.76 HOH Glu290 OE1 2.66 HOH 2.61 OG1 Thr287 2.64 OG1 Thr287 OE2 2.97 N Thr287 OE2 2.97 N Thr287 2.91 N Thr288 2.94 N Thr288 2.81 OG1 Thr288 2.82 OG1 Thr288 Glu294 OE2 3.01 NH1 Arg3002 Glu294 OE2 2.97 NH1 Arg3002 2.83 NH2 Arg3002 2.71 NH2 Arg3002 Asn298 OD1 2.8 HOH Asn298 OD1 3 O Gly263 2.72 OH Tyr261 ND2 2.62 OH Tyr261 ND2 3.01 O Gly263 2.78 HOH Arg3001 NH1 2.78 OE2 Glu2701 Arg3001 NH1 2.78 OE1 Glu2701 NH2 3.03 OE2 Glu2701 NH2 2.99 OE1 Glu2701 Arg3002 NH1 2.97 OE2 Glu294 Arg3002 NH1 3.01 OE2 Glu294 NH2 2.71 OE2 Glu294 NH2 2.83 OE2 Glu294

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Arg300

C) D)

Gly263

Thr287 Glu270 Tyr261

Glu290 Thr288 Glu294 Asn298 Thr288 Glu290

Arg300 Thr287

Figure 4.8. Interactions within the non-functional dimer interface of TOYE. A) Interaction between two monomers of TOYE-His6 (pdb accession code: 3KRU) in the non-functional dimer, with monomer 1 and 2 shown as green and cyan cartoons. FMN is shown as atom coloured sticks with yellow carbons. B) and D) Closer view of a non-functional dimer interaction. Residues are green and cyan coloured sticks to indicate which monomer they belong to. B) Diamond formation of the two possible conformations by Arg300 of each monomer. D) Interactions between monomer 1 Arg300 and Asn298 with residues from the adjacent monomer. C) Interactions between monomer 1 Thr287, Thr288 and Glu290 and residues from the adjacent monomer.

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4.3.4 Active site of holo-TOYE-His6 TOYE is a member of the thermophilic-like subclass of the OYE-family (Section 1.3.1) that typically has an active site similar to the classical OYEs but with unique features. Compared to PETNR, TOYE contains a larger and more accessible hydrophobic active site (Figure 4.9) and might affect the substrate specificity of TOYE and allow access for larger substrates. This is mainly due to a shorter β-strand 6 which is oriented towards the turn before β-strand 7 and a longer reoriented turn between α-helix 5 and β-strand 5, which means that TOYE lacks two β-strands, C and D, on the surface compared to PETNR and MR.

β -strand C

β -strand D

Figure 4.9. Access tunnel to the active site of TOYE. TOYE is hown as a green surface and cartoon, with PETNR shown as red cartoons in the areas where the structures differ.

Residue Cys25 (TOYE numbering) is highly conserved within the thermophilic-like subclass, with the thiol group interacting with the FMN N3 atom. In classical OYEs there is a conserved Thr in this position with OG1 interacting in a similar manner. This difference might affect substrate specificity as Thr has an additional methyl group which has been shown to clash with substrates in PETNR (38, 48, 59). The conserved substrate binding histidines (His163 and His166) are positioned in a similar way to other OYEs (Figure 4.10) (35, 38, 47). One or both of these residues are known to interact with the activating group of a variety of OYE substrates and inhibitors, helping to anchor and orient the ligand. Residue Tyr351 (PETNR numbering) is highly conserved in classical OYEs, but is absent in the thermophilic-like OYEs due to the latter having a shorter C-terminus. In contrast, Tyr27 is highly conserved in the thermophilic-like OYEs, but not classical 117

OYEs. This residue in TOYE is located in the active site near where Tyr351 is found in PETNR and other classical OYEs (Figure 4.10). Residue Tyr27 of TOYE may play a similar role as Tyr351 of PETNR, as they are located in relatively similar positions in the active site. In PETNR, Try351 is known to be involved in substrate and/or inhibitor binding (13, 39).

His166 / Tyr351 FMN His184 N10

Cys25 / Thr26 His163 / Tyr27 His181

Figure 4.10. Comparison between TOYE and a classical OYE. TOYE and PETNR are shown as green and cyan coloured carbon sticks, respectively, while the FMN from is shown as atom coloured sticks with yellow carbons. Dashed line shows the N-5-N-10 axis for the butterfly bend.

The active site contains a non-covalently bound FMN molecule. As with other OYEs, the si-face of the FMN points towards the solvent. The isoalloxazine ring is not completely planar (butterfly bend), with a bend at the N5-N10 axis (Figure 4.10). This is similar to YqjM (54), Old Yellow Enzyme 1 from Shewanella oneidensis (190), and other non-OYE FMN containing enzymes such as azoreductase from E. coli (191) and YcnD from Bacillus subtilis (192). PETNR exhibits minor butterfly bending of the FMN in some structures but this may be an artefact due to X-ray beam exposure during synchrotron data collection (47). In contrast, the crystal structure of OYE1 shows the FMN contains a planar isoalloxazine ring (13). The importance of a butterfly bend in FMN has not been elucidated, but it has been suggested that a methionine is required in position 24 to facilitate this (TOYE numbering) (190) as in the case of TOYE.

The FMN interacts extensively with the enzyme and solvent (Table 4.6). The electron density suggests that the FMN was only 70-80% occupied (Figure 4.11; Table 4.6), however, the ribityl chain of the FMN is similarly positioned as in other OYEs. The phosphate is secured by multiple interactions; Arg308 (N, NE and NH2), Leu285 (N),

Gly307 (N) and 3 water molecules. In the holo-enzyme structure (TOYE-His6), the omit 118

map |F0| - |FC| electron density shows a small molecule, possibly formate or an acetate ion, bound on the si-face of the isoalloxazine ring of FMN. The electron density was in a similar position to acetate in a PETNR structure (15) (PDB accession code: 1H50) where the acetate oxygen atom interacts with His163 NE2 and His166 ND1. In a YqjM structure a sulphate ion was modelled in the same location (54).

4.3.5 NADH4 binding The |Fo| - |Fc| electron density showed clear density for the nicotinamide moiety of the inhibitor in all subunits, while there was only clear density for the adenosyl ribose moiety in subunit B of the tetramer. The C4N atom of NADH4 is positioned 3.7 Å from the FMN N5 atom (Figure 4.12). This atom is located in the position where C4N for NAD(P)H would be, from which hydride is transferred from to the FMN during the reductive half- reaction of OYEs (35). The NADH4-inhibitor interacts both with the surrounding amino acids and solvent (Table 4.7). The carbonyl oxygen of the nicotinamide moiety interacts with the highly conserved His163 and His166 (Figure 4.12). Other interactions include the N6A and N7A of the adenine base with Tyr264, Glu265 and Ser250 of TOYE and the O2’ atom of the adenosyl ribose forms an hydrogen bond with the backbone oxygen of Val256.

Figure 4.11. Active site of TOYE showing the residues/solvent involved in binding FMN. Residues/FMN are shown as atom-coloured sticks with green/yellow carbons while water is shown as red spheres. Interactions are shown as black and blue dotted lines for FMN and acetate interactions, respectively. The 2|Fo| - |Fc| electron density map of FMN is contoured at 1 σ and is shown as a blue and orange mesh for FMN and acetate, respectively. 119

Table 4.6. Interactions of the enzyme bound FMN cofactor with the surrounding protein/solvent in the TOYE-His6 crystal structure. The structure was refined to 1.6 Å and the interactions are based on the position in chain A. FMN atom TOYE residue (atom) Interaction distance (Å) N3 Glu100 (OE1) 2.83 O2 Glu100 (NE2) 2.82 O2 Arg216 (NH1) 2.78 N1 Arg216 (NH1) 3.13 N1 Pro23 (O) 3.03 N5 Cys25 (N) 2.72 O4 Ala58 (N) 3.09 O2* Prp23 (O) 2.62 O2* Ser22 (OG) 2.64 O3* Arg216 (NH1) 3.06 O3* HOH 2.66 O4* HOH 2.71 O1P Arg308 (N) 2.82 O1P Arg308 (NE) 2.91 O2P Arg308 (NH2) 2.77 O2P HOH 2.79 O2P Leu285 (N) 2.74 O3P Gly307 (N) 2.82 O3P HOH 2.71 O3P HOH 2.75

120

Table 4.7. Interactions of the enzyme bound NADH4 cofactor with the surrounding

protein/solvent in the TOYE-NADH4 crystal structure. The structure was refined to 1.8 Å and the interactions are based on the position in chain A.

NADH4 atom TOYE residue (atom) Interaction distance (Å) O7N His163 (NE2) 2.85 O7N His166 (ND1) 2.72 O4’A HOH 3.00 O4’A HOH 2.84 O2’A HOH 2.67 O2’A HOH 2.83 O3’A HOH 2.93 O1N HOH 3.02 O2N HOH 2.55 N1A Tyr264 (OH) 2.69 N6A Glu265 (NE2) 2.96 N6A Ser250 (O) 2.97 N7A HOH 2.51

As with the holo-enzyme structure, the electron density for the Arg333 side chain in the

TOYE-NADPH4 structure shows two discrete positions. This may be due to an incomplete occupancy of the inhibitor as the second position of Arg333 overlaps with the inhibitor.

This residue interacts through NH1 and NH2 with both phosphate groups of the NADH4 inhibitor (O1P and O1N). The ribose ring of the inhibitor is folded back towards the loop between Ser249 and Tyr264, instead of pointing towards the solvent-exposed central cavity as in the NADH4-bound PETNR structure (Figure 4.13 A; (52)). The low electron density for the adenosyl ribose tail suggests this part of the inhibitor is flexible, extending towards the solvent. In the case of MR, the ribose ring is oriented in an alternate position and is wedged between Tyr356 and Phe137, which form a solvent exposed groove on the surface of the protein. This groove is formed by two surface loops that are in significantly different locations in TOYE, notably the loop between β-strand 3 and α-helix 3 (which is more extended in MR and PETNR than in TOYE; Figure 4.13 B-C).

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NADH4

His166 Arg333

His163

Tyr27 FMN

Ala102 B)

His166 His184 Arg189 His163 His181 His186

NADH4 Ala102 FMN Trp102 Trp106

Tyr351 Tyr27 Tyr356

Figure 4.12. Active site of TOYE. A) The active of TOYE with a NADH4 inhibitor bound. TOYE His166,

His163, Ala102 and Tyr 27 are shown as atom coloured sticks with green carbons. FMN and NADH4 are shown as atom coloured sticks with yellow and magenta coloured carbons, respectively. The Arg333 from the adjacent monomer is shown as atom coloured sticks with cyan carbons. The 2|Fo| - |Fc| electron density map of NADPH4 is contoured at 1 σ and is shown as a blue mesh for NADPH4. B) Superimposition of the active sites of TOYE, PETNR and MR. Selected residues surrounding the active site are shown. TOYE Tyr27, Ala102, His163 and His166 are shown as atom coloured sticks with green carbons. Corresponding amino acids from PETNR (Tyr351, Trp102, His181 and His184) and MR (Tyr356, Trp106, His186 and

Arg198) are shown as atom coloured lines with cyan and red carbons, respectively. FMN and NADH4 are shown as atom coloured sticks with yellow and magenta coloured carbons, respectively. 122

A) B)

Val256

His163 Ser250

His166 His166

TOYE-NADH4 His163

MR-NADH4 Tyr264 NADH4

Tyr264 FMN

PETNR-NADH4 Arg333*

C) D)

His184 His181 His186

Asp189

NADH4

NADH4 FMN FMN

Thr26 Thr32

Tyr351

Tyr356

Figure 4.13. Structural differences in NADH4 inhibitor binding within the OYE family. A) Overview of the different adenosyl ribose tail arrangements between TOYE, PETNR and MR. TOYE is shown as green coloured carbon sticks. TOYE NADH4 is shown as atom coloured sticks with red carbons and PETNR and

MR NADH4 are shown as atom-coloured lines with orange and purple coloured carbons, respectively. B)

NADH4-binding to a TOYE monomer (atom coloured sticks with green carbons). The Arg333 from the adjacent monomer is shown as atom coloured sticks with cyan carbons. C) NADH4-binding in PETNR (atom coloured stick with cyan carbons). NADH4 is shown as atom coloured stick with orange carbons. D) NADH4- binding in MR (atom coloured sticks with blue carbons). NADH4 is shown as atom coloured sticks with purple carbons. In all parts, FMN is shown as yellow coloured carbon sticks. Crystal structures used were 3KRZ (TOYE-NADH4), 3KFT (PETNR) and 2R14 (MR). 123

4.3.6 Overall structural comparison between mesophilic and thermophilic OYEs The monomer structure of TOYE is highly similar to classical OYEs, such as PETNR. The main difference is the loop extension next to the active site (see Chapter 4.3.4) and the C- terminus, which takes part in the active dimer formation of TOYE (Figure 4.14). Along with TOYE, the other two characterised thermophilic OYEs are CrS and Geo. CrS was identified as a chromate synthase but has an OYE-like activity and structure (154) and Geo was studied for use in a novel biotransformation application (155). The thermophilic OYEs have nearly identical active site residue positions (Figure 4.15 C). The dimer interactions are similar but at the C-terminal TOYE has antiparallel α-helices (αFa-b) while YqjM, CrS and Geo only have one helix (αF).

The thermophilic-like subclass has the characteristic C-terminal arm that takes part in the formation of the active dimers (Section 4.3.3). A comparison between the thermophilic OYEs, TOYE, CrS and Geo also show high structural similarities with the exception that CrS has a loop extension around the active site, similar to the classical and mesophilic PETNR (Figure 4.15). The smallest oligomeric state of the thermophilic-like OYE enzymes (TOYE, YqjM, CrS and Geo) are homotetramers, with TOYE and CrS known to form higher oligomeric states (187). The formation of higher oligomeric states is a possible adaptation to higher temperatures by stabilising the monomers.

C-terminus

Figure 4.14. Monomer comparison of the thermophilic TOYE and the classical mesophilic OYE PETNR.

Circled is the extension of PETNR which takes part in forming the active site. PETNR and TOYE-His6 are shown as red and green cartoons, respectively. 124

B) C)

His166 His163 His167 His164 His172 His175 Ala102 Ala104 His102 Tyr27 Tyr28 Tyr27

FMN Cys25 Cys26 Cys25

Figure 4.15. Thermophilic-like OYE structural comparison. A) Comparison between the overall structures of the thermophilic enzymes TOYE, CrS and Geo. TOYE, CrS and Geo are shown as green, purple and orange coloured cartoons, respectively The extension outside the active site of CrS is circled. B) The access tunnel to the active site of TOYE and a closer view of the loop forming outside the active site of CrS, similar to PETNR (Figure 4.9). TOYE is hown as a green surface and cartoon, with CrS and Geo shown as purple and orange cartoons in the areas where the structures differ. C) Superimposition of the active sites of TOYE, CrS and Geo. Selected residues surrounding the active site are shown. TOYE residues are shown as atom coloured sticks with green carbons. Corresponding amino acids from CrS and Geo are shown as atom coloured lines with purple and orange carbons, respectively. FMN is shown atom coloured sticks with blue carbons.

125

There are very few significant structural differences observed between the thermophilic and mesophilic members of the thermophilic-like subclass, but significant differences towards the classical OYE subclass, confirming the grouping of these enzymes. These distinct features of the former subclass are typical of adaptation to thermophilic conditions (e.g. shortened loops and higher oligomeric states). It is not known why the mesophilic YqjM has the structural features of thermophilic OYEs, however, given the observation that the net free energy of stabilisation of proteins is small (4-12 kJ/mol), only subtle differences in protein structure can lead to dramatically increased or decreased protein stability (193).

4.4 Multi Angle Laser Light Scattering

To investigate further the oligomeric state of TOYE-His6 in solution, multi-angle laser light scattering (MALLS) was performed on newly purified samples of TOYE-His6. The minimal quaternary structure was shown to be tetrameric (154 kDa). Octameric (341 kDa) and possibly higher states (dodecameric) were visible in smaller quantities (Figure 4.16). No peaks were seen that could indicate dimers or monomers of TOYE. This supports the detection of multiple oligomeric states seen with SDS and native PAGE (Figure 3.3), and supports the crystallographic tetrameric structure as the minimal oligomeric state. When the tetramer peak was run again through MALLS there were no major indications for higher state oligomers (Figure 4.16, inset). This suggests that the equilibrium for octamer formation is not a fast process, but could be facilitated by high enzyme concentration during purification or the folding process.

1.0

0.8 1.0 156 kDa 0.8 0.6 0.6

341 kDa 0.4

0.4

Relative Scale Relative Relative scale 0.2

0.2 0.0

0 2 4 6 8 10 12 14 16 18 20 0.0 * mL 0 2 4 6 8 10 12 14 16 18 Volume (ml) Larger Molecular size Smaller

Figure 4.16. Multi angle laser light scattering of purified TOYE-His6. The tetramer (156 kDa) and octamer (341 kDa) are indicated by arrows. A potential dodecamer peak is labelled with an asterisk. Inset: tetramer peak rerun.

126

4.5 Sedimentation velocity and hydrodynamic bead model of TOYE

There have been numerous discussions as to whether X-ray crystal structures are a true reflection of the average overall enzyme structure. This is because they generate structures of enzyme conformations that are most likely to form a crystal lattice, and this state may not be a physiologically active state in solution. Techniques such as hydrodynamic bead modelling and molecular envelope determination by small angle X-ray scattering (SAXS) can be used to look at the low resolution structure of proteins to probe the range of conformations that can exist in in vitro (194). Sedimentation velocity analysis by analytical ultracentrifugation was used to assess the hydrated molecular mass of the enzyme oligomers present in purified TOYE samples. Small amounts of a hexamer were visible in the sedimentation velocity, which is likely due to incomplete oligomer formation. In addition, small quantities of higher oligomeric states were detected (Figure 4.17). This was also seen in SDS and native PAGE and to some degree in MALLS. Hydrodynamic bead models for TOYE-His6 were calculated to assess if the crystal structures and hydrodynamic data were complementary (Figure 4.17 B)-C); Table 4.8).

One bead model was chosen for each oligomeric state, tetrameric and octameric, and the modelled radii (RH) and sedimentation coefficients (S0) were found to be consistent with the experimental ultracentrifugation results (Table 4.8). Rejected models for the octamer, based on the experimental hydrodynamic data, can be found in Appendix D. The bead model (Figure 4.17 C) for the octameric structure shows two tetramers stacked up on each other with the central cavity solvent exposed, consistent with the observed averaged TEM structure (Figure 4.1). It cannot be determined if the same or opposite sides of the tetramers face each other as they are indistinguishable in these models, with two top and two bottom monomer sides in a cross position. This data and associated bead models are consistent with the observed averaged TEM structures of the octamer (Figure 4.1).

Table 4.8. Comparison of in vitro oligomeric state of TOYE. aDetermined by MALLS. bDetermined by ultracentrifugation. M = observed mass. S = sedimentation coefficient. RH = radius of the structure. f/f0 = estimated frictional ratios. Experimental Bead modelling a b a TOYE M (kDa) S° 20,W RH (nm) RH f/f0 S°20,W RH (nm) state Tetramer 155,900 ± 1050 8.4 4.4 4.2 1.24 8.1 4.48 Octamer 338,000 ± 5650 12.7 5.8 5.1 1.29 12.4 5.61 127

A) 3.5

3.0

2.5 Tetramer, 156 kDa

2.0

(s) 1.5

1.0

0.5 Octamer, 341 kDa

0.0

B) 5 10 15 20 Sedimentation coefficient

Figure 4.17. Sedimentation velocity analysis of TOYE-His6. A) A potential hexamer peak is indicated by an arrow. B) Crystal structure (left) and bead model (right) of the top view and side view of the tetrameric structure of TOYE. C) Crystal structure (left) and bead model (right) of the top view and side view of the octameric structure of TOYE suggested by the bead model. Bead models were generated by using the PDB file and the SOMO solution bead modelling program, and are shown as multicoloured spheres (167). The crystal structure oligomers are shown as cartoons and were generated with Pymol (164). 128

TOYE is the first OYE-family member shown to have a higher oligomeric state than a tetramer. CrS has been reported to form an octamer in crystallographic and solution studies, however, the deposited crystal structure does not show an octamer and electrophoresis gels were not provided in the publication to support this higher order state(s) (187). Both these enzymes are thermophilic and belong to the thermophilic-like OYE sub-group, and this may be a feature of their thermophilic nature. YqjM is a mesophilic member of the thermophilic-like OYE subclass, but no higher oligomeric states above the tetramer have been reported with this enzyme.

4.6 Discussion TOYE was the first published example of a thermophilic OYE (23). The crystal structure shows a tetrameric enzyme with a relatively large active site cavity compared to classical OYEs. TOYE can form higher order oligomeric states in solution (octamer and dodecamers) as shown by gel electrophoresis, MALLS and sedimentation velocity. This is the first OYE to be shown to exist in higher oligomeric states in solution. The average low resolution TEM structure was remarkably similar to the bead model derived from both sedimentation velocity analysis and the three-dimensional crystal structure. This suggests that the tetrameric crystal structure is an accurate ‘snapshot’ of the solution structure of TOYE.

The active site structure and mode of FMN binding in TOYE is similar to YqjM, while the

NADH4 inhibitor was bound in a unique conformation compared to MR and PETNR. In the case of NADH4-bound TOYE, the adenosyl ribose moiety of the inhibitor is folded back and not directly out of the active site like in PETNR. The poor electron density of the adenosyl ribose moiety of the inhibitor suggests that it may have a partial occupancy where it extends towards the central solvent-filled cavity, which was also seen in a PETNR structure. The nicotinamide moiety of the inhibitor is positioned in a similar way as in other OYEs, interacting with the highly conserved substrate-binding histidine pair (His163/His166). Other features such as a wider access tunnel to the active site might influence substrate specificity compared to the mesophilic homologues, which will be investigated in Chapter 5.

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Chapter 5 – Substrate profiling of TOYE

Table of Contents 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 143

5.3.3 Solvent and substrate concentration 143

5.3.4 Solvent type 145

5.4 Discussion ...... 147

130

The biocatalytic abilities of OYEs have been studied extensively as they are capable of reducing the double bond of a variety of useful compounds in the biotechnological and pharmaceutical industries (53, 59, 61, 195-199). These compounds also contain an ‘activating group’ (an electron-withdrawing group), such as aldehydes, carboxylic acid, acyclic and cyclic ketone, ester and nitro groups (18). Other substrate types are nitroglycerin, nitroaromatic explosives and cyclic trazines (26, 44), where the nitro group is reduced rather than an alkene group. This capacity to reduce toxic chemicals has been shown to have potentials in the bioremediation of explosive-contaminated soils by using transgenic plants expressing PETNR (76).

Thermophilic enzymes have proved to be more useful in biocatalysis as they are more stable at ambient temperature, high temperatures and towards solvents and detergents that might be used, for example β-glucoside from Pyrococcus furiosus in glucocongjugate synthesis at 95 °C (200). In this chapter the substrate profile of TOYE and potential use in biocatalysis was investigated by both steady state and biocatalytic reactions. Basic biotransformation optimisation studies were performed by altering the reaction conditions (temperature, reaction time, solvent, substrate and enzyme concentration). The concentration of NADPH used was well above the KM (100 µM). Activity was determined indirectly by monitoring the rate of NADPH oxidation where hydrogen is transported to the enzyme bound FMN, which is visible as a decrease in the NADPH absorbance at 340 nm (Section 1.3.2), in the presence of the oxidising substrate where subsequent alkene reduction is presumed to be occurring. The enantiomeric purity of the products formed is not known and so the chemical identity and enantiopurity of the products was determined under biotransformation conditions, as described in the next section.

5.1 Specific activity of substrates The OYE family of enzymes are known to catalyse C=C bond reduction of a variety of α,β-unsaturated aldehydes, ketones, nitroalkenes, carboxylic acids and their derivatives (61, 195-199, 201-203). The reaction proceeds typically via the trans-addition of a FMN- derived hydride and a proton from either a conserved tyrosine residue or the solvent (18). The biocatalytic potential of TOYE was investigated by comparing the ability to reduce the C=C bond of a number of typical OYE substrates (59, 61, 196, 199). The kinetic constants of the steady state apparent kcat/KM were determined with oxidative substrates exhibiting

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significant activity with TOYE. The reactions were performed anaerobically with NADPH as the source of the reducing equivalents as TOYE was less specific for NADH (Section 3.4).

5.1.1 2-Cyclohexanone and derivatives

The rate of reduction for 2-cyclohexenone at 1 mM substrate concentration by TOYE-His6 -1 -1 -1 was 1.18 ± 0.09 s (kcat/KM: 0.5 s mM ) (Table 5.1) and at 50 °C, which is closer to the expected optimal temperature of TOYE, it increases to 2.29 ± 0.35 s-1. The enzyme shows a preference for 2-substituted substrates such as substrates 2-methyl-cyclopentenone, (5R)- carvone and (5S)-carvone (Table 5.1), while no significant activity was seen with 3- -1 methyl-2-cyclopenten-1-one (rate < 0.01 s ). No activity was detected with TOYE-His6 towards other 3-substituted substrates (3-methyl-2-cyclohexen-1-one, 3-methyl-2- cyclopenten-1-one and 3-phenyl-2-cyclohexen-1-one). TOYE has Cys25 located in the active site towards 3-substituted group of substrates. Modelling has suggested that the 3- methyl substituent can clash with the CG2 carbon of Thr26 (PETNR numbering) in PETNR (48, 59).

5.1.2 Maleimide related substrates

TOYE-His6 shows a high activity towards maleimide substrates (Table 5.1) at 25 °C, which is 44 °C lower than the optimum growth temperature of the native host, T. pseudethanolicus, (69 °C). The activity is even higher than the mesophilic homologue PETNR towards these substrates. At 1 mM substrate concentration, TOYE reduces 2- methylmaleimide (26.85 s-1) and 2-methyl-N-phenyl-maleimide (16.90 s-1) while it displays poor activity (0.02 s-1) towards ketoisophorone, a substrate typically rapidly reduced by other OYEs, suggesting that the active site may not accommodate the 6- dimethyl substituent (59).

5.1.3 Other substrates The reduction of the C=C double bond in substrates containing ester, aldehyde and carboxylic acid activating groups by TOYE was monitored by steady-state kinetics (Table 5.2). Typically TOYE showed poor or no activity with these substrates. For example,

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Table 5.1. Steady-state kinetics of TOYE with substituted and non-substituted cyclohexenones, cyclopentenones, maleimides, ketoisophorone and thymine. 1 -1 -1 Substrate Rate (s ) kcat/KM (s mM )

2-cyclopenten-1-one O 0.74 ± 0.07 0.6

2-methyl-2-cyclopenten-1-one 0.17 ± 0.01 0.1 O

3-methyl-2-cyclopenten-1-one O < 0.01 ND

2-cyclohexen-1-one O 1.18 ± 0.09 0.5

3-methyl-2-cyclohexen-1-one O ND ND

Ph 3-phenyl-2-cyclohexen-1-one ND ND O

(5R)-carvone O 2.23 ± 0.10 1.5

(5S)-carvone O 0.83 ± 0.07 0.6

Ketoisophorone 0.02 ± 0.01 ND

2-methylmaleimide 26.85 ± 3.02 194.4

2-methyl-N-phenyl-maleimide 16.90 ± 0.61 213.6

Thymine O NH 0.048 ± 0.004 ND N H O Reactions (0.3 mL) were performed in 50mM KH2PO4/K2HPO4 pH 7.0 containing 100 M NADPH, 1 mM oxidising substrate and 0.1-2 M TOYE for 1 minute at 25 °C. Final ethanol concentration was 5%. a Specificity constants (apparent kcat/Km) were determined from the slope of the rate vs substrate concentration plot at substrate concentrations (5-20 M) well below the estimated Km. ND = No significant activity detected under steady state conditions.

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TOYE showed low rates with methyl-1-cyclohexene-1-carboxylate (0.33 s-1), trans- cinnamaldehyde (0.39 s-1), 2-methylpentenal (0.22 s-1) and citral (0.04 s-1), when compared to PETNR (59) (4.1 s-1, 4.7 s-1, 6.23 s-1 and 0.24 s-1, respectively). No activity was observed for 1-cyclohexene-1-carboxylic acid and a very low rate was obtained with methyl 1-cyclohexene-1-carboxylate. Thymine was tested as a potential substrate as it bears some structural resemblance to 2-cyclohexanone and was the activity towards thymine was very low (0.05 s-1).

Table 5.2. Steady-state kinetics of TOYE with oxidative substrates. 1 -1 -1 Substrate Rate (s ) kcat/KM (s mM )

Methyl 1-cyclohexene-1- carboxylate OHC 0.33 ± 0.02 0.3

OHC trans-cinnamaldehyde 0.39 ±0.01 ND

2-methylpentenal 0.22 ± 0.01 0.14 OHC Citral 0.04 ± 0.01 0.05 OHC

1-cyclohexene-1-carboxylic acid HOOC ND ND

Methyl 1-cyclohexene-1- MeOOC carboxylate ND ND

Reactions (0.3 mL) were performed in 50mM KH2PO4/K2HPO4 pH 7.0 containing 100 µM NADPH, 1 mM oxidising substrate and 0.1-2 µM TOYE for 1 minute at 25 °C. Final ethanol concentration was 5%. a :Specificity constants (apparent kcat/Km) were determined from the slope of the rate vs substrate concentration plot at substrate concentrations well below the estimated Km (5-20 µM). ND = No significant activity detected under steady state conditions.

5.2 Biotransformation studies with TOYE

The biocatalytic potential of TOYE-His6 towards α,β-unsaturated activated alkenes was investigated by determining the yields and identity/enantiopurity of the product(s) formed. Reactions were performed anaerobically at 30 °C using NADH as the source of reducing equivalents due to the high cost of NADPH. The product yields and enantioselectivity of the C=C reduction was studied using GC or HPLC (Table 5.3). 134

5.2.1 2-Methyl-cyclopentenone Cyclic ketones are important synthons or solvents for polymerisations and pharmaceutical drug synthesis (204, 205). Cyclopentanone derivatives are used as intermediates in organic synthesis and fragrances (205). For example, the amination of cyclopentenone results in cyclopentylamine, which is used in the synthesis of N-benzyl-cycloalkylamine fungicides (206).

Alkene 2-methyl-cyclopentenone was reduced to (S)-2-methyl-cyclopentanone (Figure 5.1) with 77% conversion, 73% yield and high enantiopurity of the (S)-product (in 70% enantiomeric excess; ee). These results were similar to the reaction with PETNR, though the yield and conversion were higher (56% and 58% with PETNR, respectively) (59). No significant reaction rate was seen under steady state conditions with PETNR with either cyclopentenone or 2-methyl-cyclohexenone (< 0.01 s-1), while for TOYE a rate of 0.17 s-1 was determined with 2-methyl-cyclopentenone (Section 5.1.1). This shows why we should not rely completely on short steady state reactions to determine the substrate profile towards a variety of substrates while exploring industrial potential of an enzyme, as steady state methods do not always show reactivity towards weaker substrates.

Proton donor CH3 CH3 H3C O O O

2-methyl-cyclopenten-2-one (S)-2-methyl cyclopentanone Hydride from reduced FMN

Figure 5.1 Proposed reaction mechanism of the reduction of 2-methyl-cyclopentenone.

5.2.2 Carvone Substrates which are commonly reduced by OYEs under biotransformation conditions are (R)- and (S)-carvone (59, 203). These substrates are part of a large group of chemicals, terpenoids, which have been used in the flavour and fragrance industries (207), for example (-)-menthol and safranal.

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Table 5.3. Product determination of the reduction of α,β-unsaturated alkene substrates by TOYE.

Conv.[b,c] Yield[c] Ee[d] Substrate Product[a] (%) (%) (%)

O (S)- O 77 73 70

O (5R)- (2R,5R)- O 94 61 95 de

99% ee

O O (5S)- (2R,5S)- 89 77 85 de 99% ee 97 82 55 (S)- OHC OHC 74 23 91 (S)- OHC OHC 82 12 (E)- (S)- 61 O2N Ph

O O 99 74 26 (R)-

O O N >99 90 >99 (R)- Ph

(R)- >99 99 99 O O N H Ph Ph rac 82 80 0 O O O O N N H H Ph Ph

O O rac O O >99 99 0 N N CH CH3 3 [a] Standard reactions (1.0 ml) were performed in 50 mM phosphate buffer pH 7.0 containing alkene (5 mM; added as a 2 % DMF solution), NADH (6 mM) and TOYE (10 µM) at 30 °C for 24 hours at 130 rpm. [b] Conv. = % conversion. [c] by GC using DB-Wax column. [d] by GC or HPLC.

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Although the steady state rates of TOYE with carvones were poor, the reduction of (5R)- carvone and (5S)-carvone was significant under biotransformation conditions (61% and 77% yields, respectively). For both (5R)- and (5S)-carvone the new chiral centres were (2R), resulting in (2R,5R)-dihydrocarvone and (2R,5S)-dihydrocarvone, respectively, with high diastereoisomeric excess (de) (95% de and 85% de, respectively). This suggests that the active site of TOYE allows for bulky substituents at the 5-position. As the stereochemistry at the 2-C centre was identical with both substrates, this suggests the substrates bound in similar conformations in the active site.

The classical OYEs PETNR (59) and OYE1 (203) show a similar activity and similar de values for (2R, 5R)-carvone (95% and 97%, respectively) and (2R,5S)-carvone (88% and 93%, respectively). In contrast, the opposite stereospecificity is seen by the two enone reductases, Reductase-I and Reductase-II, from Astasia Ionga where (5R)- and (5S)- carvone are reduced to (2R,5R)- and (2R,5S)-dihydrocarvone (208).

5.2.3 Maleimide related substrates Maleimides are important chemicals for organic synthesis, as they can be used as precursors for imide polymers and form stable bonds when reacting with hydroxyl, amine and thiol groups. Also, N-substituted maleimides have been shown to have antimicrobial activity (209).

As with other OYEs, the biotransformation of a variety of maleimide substrates by TOYE showed high levels of conversion and yield (Table 5.3). This correlates with the high steady state rates of TOYE with both substrates (Table 5.1). However, there were significant differences in the enantiopurity of the product, as the reduction of N-phenyl-2- methylmaleimide yielded a highly pure (R)-product, while N-methyl-2-phenyl maleimide was reduced to a racemic product. This latter observation could be explained by the presence of non-enzymatic water-mediated racemisation of 2-phenylsuccinimide and 2- phenyl-N-methylsuccinimide, but not with 2-methylsuccinimide and 2-methyl-N- phenylsuccinimide.

The yield and ee were high for the reactions with both 2-methylmaleimide (99% yield and >99% ee) and 2-methyl-N-phenylmaleimide (90% yield and >99% ee). This suggests that TOYE is relatively insensitive to the nature of the N-substituent with 2-methyl maleimide 137

substrates. However, in the case of 2-phenyl maleimides, the nature of the N-substituent (H or methyl) slightly affected the yields, with a higher yield obtained with the N-methyl-2- phenylmaleimide (Table 5.3). These results resemble findings with other OYEs which generally form the (R)-enantiomer with 2-methyl maleimide substrates with high yields and product ee (23, 59, 61, 195, 196, 202), with the exception of MR and EBP1 (210).

5.2.4 2-Methylpentenal Aldehydes are an important chemical group as they are used in reactions such as oxidation to carboxylic acids and the addition of nucleophilic groups. They can be used as dispersants and detergents and in the fragrance industry (207). Among other natural aldehydes, 2-methyl-pentenal has been proven to work as a broad-spectrum postharvest insecticide (211).

TOYE showed remarkable similarity to PETNR in the reduction of 2-methylpentenal by producing the same (S)-enantiomer of 2-methylpentanal with similar yields and ee (82%/85% yield, 55%/66% ee, respectively) (59). The reaction for both enzymes reached completion with 97% and 99% for TOYE and PETNR, respectively. This is despite TOYE -1 having a 28 times lower kcat than PETNR under steady state conditions (0.22 and 6.23 s , respectively). This can be explained by the difference in reaction time between biotransformations and steady state reactions (24 hours and 2 minutes, respectively), which allows the slower reaction to proceed to completion. Similar results from biotransformations have been reported for OYE1-3, OPR1 and YqjM (59, 61, 67) while OPR3 stands out by producing the (R)-enantiomer at a low ee (19%) (61).

5.2.5 Citral Like carvones, citral is a terpenoid and is important in the flavour and fragrance industry (207). The (E)- and (Z)-isomers are known as geranial and neral, respectively. Citral has also been shown to possess fungicidal properties (212).

The reduction of citral by TOYE produced (S)-citronellal to high enantiopurity (91% ee) but low yield (23%) after 74% conversion. This low yield might be explained by non- enzymatic substrate and/or product decomposition in aqueous media. Product analysis by 138

GC-MS did not detect any additional volatile by-products in the organic phase, however this does not exclude the presence of additional non-volatile by-products. PETNR showed a higher conversion and yield of (S)-citronellal (99% and 56%, respectively), (59) with similar product enantiopurities (91% and 87%, respectively). As PETNR converts citral faster than TOYE (kcat of 0.24 against 0.04), it might allow for less time for a nonenzymatic substrate decomposition, resulting in a higher yield.

5.2.6 α,β-Unsaturated nitroalkene Reduction of nitroalkenes leads to nitroalkanes with up to two new chiral centres. Nitroalkanes are important chemicals as they can be modified into amines, aldehydes, carboxylic acids oximes and hydroxylamines (213).

The reduction of the nitroalkene c to (S)-2-phenyl nitropropane by TOYE showed a high conversion (82%) although moderate enantiopurity (61%) of the product. However, the yield was only 12%, and by-products were found both in the enzyme reaction and control reaction (reaction without enzyme). In the enzyme catalysed reaction, 2-phenylpropanal, 2- phenylpropanal oxime and other unidentified products were observed. Acetophenone was also detected in the enzyme and control reactions.

Nitroalkene substrates have been shown to go through an enzyme-catalysed formation of oximes by PETNR (47). Other products can form from the degradation of the oximes. This oxime formation, seen by the wild-type PETNR, can be increased with mutations at H181 and H184 (PETNR numbering), demonstrating that mutations of substrate binding residues can affect product formation.

As the (S)-enantiomer is the main product, TOYE is consistent with the activity of PETNR, OPR3 and YqjM (59, 61, 195). In contrast, OYE1-3 and OPR1 primarily produce the (R)- enantiomeric product (53, 61, 195). The low enantiopurity of the reaction could be improved by changing the conditions of the reaction. Optimisation studies have been performed on PETNR by varying pH, reaction time and enzyme concentration to improve the ee (59). Shortening the reaction time showed a great effect on reactions where water- mediated racemisation was likely to take place. Other conditions such as the presence of oxygen and buffer type have been shown to affect the enantioselectivity of PETNR. In 139

anaerobic conditions and phosphate buffer the (S)-enantiomer is produced (59) while in aerobic conditions and Tris buffer produced the (R)-enantiomer (214). Oxygen competes with the alkene for stripping the hydride from reduced FMN. By-product formation likely occurs by the substrate binding in an alternate conformation allowing the nitro group to be reduced instead of the double bond. Mutations within the active site presumably change the substrate binding site shape sufficiently to increase the likelihood of the substrate binding in the alternate conformation.

5.2.7 Ketoisophorone Ketoisophorone, a diketone, is reduced by a number of OYEs (23, 59, 61) to (R)-levodione which is an important building block for the synthesis of terpenes and carotenoids (215). While TOYE reduces ketoisophorone at a relatively slow rate, during biotransformations the conversion of the substrate was 99%, with a moderately high yield (74%) of levodione as product and low ee of (R)-levodione (26%). The conversion and yield are similar to PETNR reactions, but with a lower ee for the (R)-product (59).

Modelling studies with PETNR have shown two possible binding conformations of ketoisophorone in the active site, both of which would lead to the formation of the (R)- enantiomeric product. This suggests that the low ee is due to water-mediated product racemisation (59). The low enantiopurity was improved by changing the conditions of the biotransformation, such as pH, enzyme concentration and shorter reaction time (59). The use of a coenzyme recycling system has also been shown to improve the ee for both PETNR (from 21% to 57%) (59) and OPR3 (33-55% to 99%) (196).

5.3 Optimisation of biotransformations The optimisation of biotransformation conditions have previously been shown to dramatically improve the enantiopurity of the products for PETNR (59). Therefore, the effects of time, substrate concentration, solvent concentration and the nature of the solvent on the biotransformation of TOYE were investigated to see if product enantiopurities could be improved.

5.3.1 Biocatalysis at elevated temperature The effect of increased temperature on the product yield and enantiopurity of ketoisophorone reduction by TOYE was investigated. This was conducted because of the

140

thermostable nature of TOYE and higher temperature may be an advantage to increase the solubility of substrates (Table 5.4). Reactions were performed with ketoisophorone at both 50 °C and 30 °C. At 50 °C the samples had in most cases lower yields, suggesting significant product evaporation or degradation due to higher temperature or low reaction rates. The reduction of 2-methyl-N-phenyl-maleimide showed very little change in ee (94 – 99%) at different temperatures, but the yields were lower at 50oC, suggesting that the product degrades and/or evaporates at higher temperatures.

Similarly, reactions with citral, (S)-carvone and (R)-carvone suffered losses in yield when increasing temperature from 30 °C to 50 °C, by 18%, 52% and 42%, respectively, while the ee or de only reduced slightly. This again indicates evaporation and/or decomposition of the substrates/products. The reaction with 2-methylpentenal showed a decrease in both ee (from 55% to 10%) and yield (from 82% to 38%), suggesting that racemisation and evaporation and/or decomposition of the substrate/product was present.

In the case of ketoisophorone reduction, the yield was relatively unchanged at different temperatures (74% and 75% at 30 °C and 50 °C, respectively). This suggests no significant decomposition or evaporation took place. However, the product became racemic at 50 °C indicating that the non-enzymatic product racemisation is increased at higher temperatures. This means that reactions generating products that undergo water-mediated racemisation should not be performed at higher temperatures.

No significant changes were seen in the reduction of 3-methyl-cyclopenten-1-one and 3- methyl-cyclohexen-1-one at different temperatures. Reactions of TOYE with these substrates do not exhibit significant rate at 30 °C, and increasing the temperature did not increase the product yield. The reaction of TOYE with 2-methyl-penten-1-one showed a loss of conversion, yield and ee. This suggests that increases in both substrate/product evaporation/decomposition and product racemisation, may be occurring at higher temperatures. The reduction of the nitroalkene 2-phenyl-1-nitropropene was the least affected by a variation in the reaction temperature. The ee decreased slightly, from 61% to 49%, while the yield and conversion were not affected.

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Table 5.4 Product determination of the reduction of -unsaturated alkene substrates by TOYE at 50 °C. Substrate Conf. Product Conditions Conv.b(%) Yieldb eec (%) (%) NADH (30) >99 90 >99 (R)- O N O O N O NADH (50) >99 50 94 Ph Ph NAPDH (50) >99 49 95

(R)- NADH (30) 99 74 26 O O O O NADH (50) 87 75 0

NAPDH (50) 85 73 0 (5R)- (2R,5R)- NADH (30) 94 61 95de O O NADH (50) 91 19 88de 99% ee 99% ee NAPDH (50) 92 18 88de (5S)- (2R, 5S)- NADH (30) 89 77 85de O O NADH (50) 84 25 62de

99% ee 99% ee NAPDH (50) 85 21 62de (S)- NADH (30) 97 82 55

NADH (50) 94 38 10

NAPDH (50) 97 30 5 (S)- NADH (30) 74 23 91

NADH (50) 87 5 85 NAPDH (50) 82 6 86 Not determined NADH (30) 9 <1 nd NADH (50) 15 <1 nd NAPDH (50) 9 <1 nd

Not determined NADH (30) 7 <1 nd NADH (50) 6 <1 nd NAPDH (50) 1 <1 nd

(S)- NADH (30) 77 73 70

NADH (50) 50 44 11 NAPDH (50) 52 42 12 (E)- (S)- NADH (30) 82 12 61 O2N Ph NADH (50) 89 9 49 NAPDH (50) 92 17 50

[a] Standard reactions (1.0 mL) were performed in 50 mM phosphate buffer, pH 7.0, containing alkene (5 mM; added as a DMF solution, 2% final concentration), NAD(P)H (6 mM) and TOYE (10 mM) at 50 °C for 24 hours at 130 rpm. [b] Conv. = % conversion. [c] by GC using DB-Wax column. [d] by GC or HPLC. nd: not detected. 142

These results suggest that biocatalysis of C=C reduction should not be performed at higher temperatures when substrates and/or products are sensitive towards temperatures, even when a thermophilic enzyme is used as a catalyst. As previously described, thermophilic enzymes show increased stability towards solvents and denaturants. These attributes can be applied at ambient temperatures when higher solvent concentration is required for substrate and/or product solubility and stability.

5.3.2 Time An investigation into the effect of reaction time on ketoisophorone reduction by TOYE showed that the enantiopurity of the product (R)-levodione decreases with increased reaction time (Table 5.5). This time-dependent decrease in product ee has previously been shown to be caused by a non-enzymatic water-mediated racemisation of the product (59). When the reaction time was decreased to 2 hours, the final ee improved from 26% (24 h reaction) to 92% (2 h; Table 5.5) with 71% conversion and 68% yield. These results are consistent with the studies with PETNR that showed higher (R)-levodione enantiopurity with shorter reaction times (59). However, the reaction took 6 h to reach completion, which is not surprising given the slow steady state reaction compared to the mesophilic PETNR homologues (59). This suggests that short reaction times are advisable for reactions which are sensitive to water-mediated product racemisation.

5.3.3 Solvent and substrate concentration Studies have shown that thermophilic enzymes tend to be more stable against organic solvents at room temperature than their mesophilic homologues (216-218). This property of thermostable enzymes can be of great advantage in organic synthesis over mesophilic homologues as organic solvents are often required to increase substrate and product solubility and stability.

To determine if TOYE is more solvent stable than an equivalent mesophilic OYE, the stability and activity of TOYE was compared to a mesophilic homologue PETNR in the presence of ethanol using steady-state reactions with 2-cyclohexenone (Figure 5.2). It was found that increasing the concentration of ethanol in the assay led to a decrease in the

143

reduction rate for both TOYE and PETNR, with a 50% loss of activity at 45% and 20% ethanol, respectively. TOYE retained 20% activity at 80% ethanol while PETNR was visibly precipitating above 40% ethanol. This suggests that the thermostable TOYE is more stable against activity loss in the presence of at least one water-miscible organic solvent than a mesophilic counterpart.

Another way to investigate the solvent stability of an enzyme is to determine the remaining activity under standard (low organic solvent) conditions after a preincubation in organic solvent. In this case, comparative steady state reactions with 2-cyclohexenone were performed with TOYE and PETNR following a preincubation for 5 minutes at different ethanol concentrations. Dramatic differences in solvent stability were seen between the enzymes as TOYE retained full activity after incubation in 70% ethanol, while PETNR showed no activity after incubation in 50% ethanol.

This decreased activity in organic solvents by TOYE and PETNR could be attributed to the organic solvent, which can cause enzyme denaturation, as increasing organic solvent concentrations reduce the tendency of hydrophobic residues to pack into the interior of the protein, increasing the likelihood of the protein unfolding. At the lower ethanol concentrations, this unfolding may be only localised and reversible, as the enzymes retained significant activity after the organic solvent concentration was decreased. However, at higher organic solvent concentrations, these effects are likely irreversible as enzyme activity was rapidly lost.

Figure 5.2 showed that an increase in organic solvent concentration decreases the steady- state reaction rate of TOYE. This decrease in rate of the thermophilic TOYE was less than with PETNR, a mesophilic homologue. Similar experiments were performed under biotransformation conditions with increasing concentrations of a water-immiscible solvent dimethylformamide (DMF; 2 – 30%) for 2 hours (Table 5.5). In both cases, the conversion and yields decreased with increased solvent concentration while the enantiopurity of the product was unaffected. This suggests that the rate of the reaction is affected by the solvent concentration and needs to be taken into account when optimising biotransformations. An investigation into the effect of increased solvent and substrate concentration (2% DMF and 5 mM substrate against 4% and 10 mM substrate) on the overall yield of product showed

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that substrate concentrations generated a higher quantity of product (4.3 mM vs 3.4 mM product, respectively).

Figure 5.2. Solvent stability of TOYE and PETNR. Standard reactions (0.3 ml) were performed in 50 mM phosphate buffer pH 7.0 containing NADPH (100 mM), 2-cyclohexenone (1 mM; stock solution in 100 % ethanol) and TOYE/PETNR (0.1 – 2 µM). The percentage of remaining activity of the enzymes after a 5 min incubation at 25 °C in the ethanol:buffer mixes above are shown for TOYE (closed squares) and PETNR (closed circles) with solid lines. The activity of TOYE (open squares) and PETNR (open circles) with 2- cyclohexenone in the presence of increasing concentrations of ethanol are shown as dotted lines.

5.3.4 Solvent type The reduction of ketoisophorone in the presence of a variety of organic solvents (10%) over 2 hours showed distinct solvent-dependent effects on the conversions and yields. The lowest conversion and yield were seen with DMF, acetone and propanol (36 – 54% yield) while reactions with tetrahydrofuran, methanol, ethanol and acetonitrile gave similar yields to the standard reaction with 2% DMF (65 – 69%). This shows that not only is the concentration of cosolvent important, but also that the type of solvent has to be taken into consideration when used in biotransformations.

145

Table 5.5. Product determination of the reduction of ketoisophorone by TOYE in the presence of water-miscible solvent systems.

Solvent Sub. Time Conv. Yield Ee[d] (%)[b] (mM) (h) (%)[c] (%)[c] (%) Reaction Time DMF 2 5 0.5 13 12 >99 DMF 2 5 1.0 37 34 95 DMF 2 5 2 71 68 92 DMF 2 5 3 89 83 88 DMF 2 5 6 96 88 76 DMF 2 5 9 97 85 64 Substrate concentration DMF 4 10 2 54 43 93 Solvent concentration DMF 5 5 2 66 64 92 DMF 10 5 2 48 42 92 DMF 15 5 2 40 32 92 DMF 20 5 2 38 23 93 DMF 30 5 2 32 12 92 Solvent comparison DMF 10 5 2 44 37 93 THF 10 5 2 76 69 93 MeOH 10 5 2 74 66 92 EtOH 10 5 2 70 65 95 PrOH 10 5 2 63 54 90 Acet. 10 5 2 41 36 93 ACN 10 5 2 81 66 93 [a] Standard reactions (1.0 mL) were performed in 50 mM phosphate buffer pH 7.0, containing water miscible organic solvents (2-50 %), ketoisophorone (5-10 mM), NADH (6 mM) and TOYE (10 M) at 30 oC for 0.5-9 hours at 130 rpm. Substrate was added to all reactions as a 2 % final DMF solution. Sub. = substrate concentration; Conv. = % conversion. [b] DMF = dimethyl formamide; THF = tetrahydrofuran; MeOH = methanol; EtOH = ethanol; PrOH = propanol; Acet. = acetone; ACN = acetonitrile. [c] by GC using a DB-Wax column. [d] by GC using a Chirasil-DEX CB column.

Water-miscible and polar solvents, the alcohols and acetonitrile, had high activity, with the exception of acetone. Of the two water-immiscible solvents tested, DMF showed low activity while THF exhibited high activity, similar to the alcohols. As OYEs are known to bind small compounds (15, 24, 38), such as sulphate and glycerol, the structure of the cosolvent is a possible factor where the carbonyl group could be bound in a similar fashion to substrates (Figure 5.3). This could explain the lower activity in DMF and acetone if they were acting as a competitive inhibitor to the reaction and binding tighter to the active site than the other solvents but further studies regarding this are required.

146

O A) O B)

CH3 N CH3 H3C H3C C) D) E) O HO N C CH3 CH3

Figure 5.3 Structures of different solvents. A) Acetone. B) Dimethylformamide. C) Acetonitrile. D) Ethanol. E) Tetrahydrofuran.

5.4 Discussion TOYE shows unique specificity towards oxidative substrates within the OYE family. This difference can be attributed to the identity and position of the residues within the active site and substrate-binding regions. In addition, there may be other effects due to decreased protein flexibility at ambient temperatures compared to mesophilic enzymes, plus other effects of distinct monomer-monomer interactions. The increased solvent stability of TOYE (Section 5.3) would be advantageous for industrial applications where organic solvents are required to increase substrate and product stability and solubility.

In most cases TOYE has, on average, a lower rate and specificity (kcat/KM) than its mesophilic relatives (59), except in the case of the rapidly reduced maleimide derived substrates. This is not surprising given the reactions were performed at a temperature >40oC below its theoretical optimum. TOYE shows a preference towards methyl substitution at the C-2 rather than the C-3 position of 2-cyclopentenone and 2- cyclohexenone substrates (Table 5.1). The reduction of 2-phenyl maleimide and 2-phenyl- N-methyl maleimide resulted in the prduction of racemic forms of phenylsuccinimide and N-methyl-α-phenylsuccinimide, respectively. Alkane N-methyl-α-phenylsuccinimide, also known as phensuximide, is a member of a large group of succinimide anticonvulsant drugs (219, 220). This suggests a possible use for TOYE and other OYEs in the production of some pharmaceutical drugs.

Unlike PETNR and other OYEs (59), TOYE exhibits poor activity towards ketoisophorone, which contains a dimethyl substitution at C6. The reaction time is critical in determining the enantiopurity of (R)-levodione as the loss of ee is time-dependent

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(Table 5.5). This is likely due to the increased time the product is exposed to water, leading to an increase in the amount of non-enzymatic racemisation (59).

Reactions in the presence of ethanol showed that increasing the concentration of solvent decreased the steady-state rate with both TOYE and PETNR (Section 5.3). However, TOYE displayed a higher tolerance towards ethanol and was still active at concentrations that precipitated PETNR (40%). In addition, TOYE retained about 20% activity in the presence of 70% ethanol. However, the effects of 70% ethanol on TOYE were reversible as, after the preincubation, the activity was regained under the standard reaction conditions (5% ethanol). Therefore, TOYE shows the typical increase in solvent stability at ambient temperatures commonly seen with other thermostable enzymes. This solvent concentration-dependent decrease in rate of TOYE was also evident during the biotransformation reactions (Table 5.5). On the other hand, increasing solvent concentrations had no effect on the enantiopurity of the product. Even though TOYE did not show the highest activity in DMF, this demonstrates that biotransformations with high concentration of water-miscible organic solvents can yield a highly enantiopure product. Further reaction optimisations, such as increased reaction time or enzyme concentration would be required to make up for the reduced reaction rate due to the organic solvents.

This organic solvent stability of TOYE can prove useful in industrial biotransformations where increased substrate solubility is needed, and mesophilic biocatalysts would be structurally compromised. They could also prove useful in biphasic reactions as the increased solvent stability would increase the product yield, and the sequestering of the substrate/product into the organic phase would dramatically decrease the likelihood of unfavourable water-mediated product decomposition, such as hydrolysis, and non- enzymatic racemisation (for example of 2-phenylsuccinimide and 2-phenyl-N- methylsuccinimide; Section 5.2.3).

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Chapter 6 – Temperature-dependence of 1° KIE of TOYE

Table of Contents 6.1 Transient state traces and interpretation ...... 150

6.2 Coenzyme specificity of TOYE ...... 154

6.2.1 Coenzyme kinetics 154

6.2.2 Binding of nonreactive coenzyme analogues and CT-complex formation 155

6.3 Temperature dependence of the reductive half-reaction ...... 156

6.3.1 Temperature dependences of hydride transfer with NAD(P)H 156

6.3.2 Temperature dependence of 1° KIEs 158

6.3.3 Temperature dependence of 2° KIEs 161

6.3.4 Temperature dependence of 1° KIE in steady-state 164

6.5 Discussion ...... 168

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Temperature dependence studies using isotopically labelled coenzymes and substrates have provided new insights into hydrogen tunnelling and the role of motion during catalysis (86, 90, 93, 103, 107, 221). Stopped-flow techniques have been used to investigate complex enzyme kinetics and measure single reaction phases. Within the OYE family, the mesophilic PETNR and MR have been extensively studied (40, 130, 222-226). In this chapter, the reductive half-reaction of TOYE will be examined and comparison made with the mesophilic OYE homologues and other thermophilic enzymes. The temperature dependences of 1° KIEs and α-2° KIEs were used to examine the presence of tunnelling and active site geometry during catalysis. Due to the complexity of the data processing the 1° KIE temperature dependence was compared to a simpler steady-state approach. This is the first member of the thermophilic-like subclass of OYE studied in this fashion.

6.1 Transient state traces and interpretation The hydride-transfer in the reductive half-reaction of TOYE was studied by stopped-flow, as described in Section 2.16.2. The reducing coenzymes used were NADH, (R)-[4-2H]- NADH, (S)-[4-2H]-NADH, NADPH, (R)-[4-2H]-NADPH and (S)-[4-2H]-NADPH. As described in Section 3.4, the reduction of FMN can proceed with both NADH and NADPH. As with other OYEs, the reduction of the enzyme bound FMN by NAD(P)H showed a reduction in the absorbance peak at 456 nm (Figure 6.1). The formation of a charge-transfer complex was also observed with NAD(P)H at around 555 nm (Figure 6.1 and Section 6.2.2), similar to PETNR (26, 40, 224) and MR (25, 130, 223), with NADPH and NADH, respectively, suggesting that the mechanism is similar.

Saturating concentrations of coenzymes (>10x KS) were used to guarantee pseudo-first order rates during temperature studies. The results in this thesis are the first reported analysis of pre-steady state kinetics for TOYE and compared to the well characterised homologues, PETNR and MR. Sample reaction transients for NADH and NADPH are shown in Figures 6.2 and 6.3, respectively.

150

0.6

0.4

Absorbance 0.2

0.0 300 350 400 450 500 550 600 Wavelength (nm)

B) C)

0.40 0.030 0.35 0.025 0.30

0.25 0.020

0.20 0.015

0.15 Absorbance Absorbance 0.010 0.10 0.005 0.05 0.00 0.000 1E-3 0.01 0.1 1 1E-3 0.01 0.1 1 Time (sec) Time (sec)

Figure 6.1. Reduction of TOYE by NADPH. A) Selected spectra captured using a multiple wavelength stopped-flow spectrophotometer (280-600 nm) showing the bleaching of flavin absorbance resulting from reduction by NADPH. Red, green and turquoise lines show the absorbance at the start of the reaction, during maximal point of the 555 nm absorbance (9 ms) and at the end of the reaction observation (1 second). B) The decrease in 456 nm absorbance during reduction C) CT complex formation and decay during the observed time. 400 data points were collected over 1 second. Conditions: 100 µM NADPH and 40 µM TOYE in 50 mM potassium phosphate buffer pH 7.0 at 25 °C.

151

A) B)

1.0 1.0

0.8 0.8

0.6 0.6

0.4 0.4

0.2 0.2

RelativeAbsorbance RelativeAbsorbance 0.0 0.0 1E-3 0.01 0.1 1 10 100 1E-3 0.01 0.1 1 10 100

0.01 Time (s) 0.01 Time (s)

0.00 0.00 Residuals -0.01 Residuals -0.01 1E-3 0.01 0.1 1 10 100 1E-3 0.01 0.1 1 10 100 Time (s) Time (s)

1.0

0.8

0.6

0.4

0.2 RelativeAbsorbance 0.0 1E-3 0.01 0.1 1 10 100

0.01 Time (s)

0.00

Residuals -0.01 1E-3 0.01 0.1 1 10 100 Time (s)

Figure 6.2. Representative traces for FMN reduction of TOYE by NADH at different temperatures. Stopped- flow traces are shown by a black line and a double exponential fit is represented by a red line. The residuals of that fit are shown below as a red line. A) 5 °C B) 25 °C C) 50 °C. Conditions used were 20 µM TOYE-

His6, 5 mM NADH in 50 mM potassium phosphate buffer pH 7.0.

152

A) B)

1.0 1.0

0.8 0.8

0.6 0.6

0.4 0.4

0.2 0.2

RelativeAbsorbance RelativeAbsorbance 0.0 0.0 1E-3 0.01 0.1 1 10 100 1E-3 0.01 0.1 1 10 100

0.04 Time (s) 0.04 Time (s)

0.02 0.02

0.00 0.00

-0.02 -0.02 Residuals Residuals -0.04 -0.04 1E-3 0.01 0.1 1 10 100 1E-3 0.01 0.1 1 10 100 Time (s) Time (s)

1.0

0.8

0.6

0.4

0.2 RelativeAbsorbance 0.0 1E-3 0.01 0.1 1 10 100

0.04 Time (s) 0.02

0.00 -0.02

Residuals -0.04 1E-3 0.01 0.1 1 10 100 Time (s)

Figure 6.3. Representative traces for FMN reduction of TOYE by NADPH at different temperatures. Stopped-flow traces are shown by a black line and a triple exponential fit is represented by a red line. The residuals of that fit are shown below as a red line. A) 5 °C B) 25 °C C) 50 °C. Conditions used were 20 µM

TOYE-His6, 5 mM NADH in 50 mM potassium phosphate buffer pH 7.0.

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The stopped-flow transients were fit to Equation 2.10 using a double and triple exponential function for NADH and NADPH, respectively. Multiple exponentials were observed for TOYE and were more prominent at higher temperatures. In MR, multiple reactive conformations (MRC) in active site mutants have been used to explain multiple exponentials (130, 223) where the wild type enzyme proceeds through a single kinetic phase (225). Another possible reason for multiple exponents is an allosteric effect between monomeric units of the active dimers as they take part in the active site formation of their neighbouring monomer. Change in amplitudes was observed with change in temperature, suggesting that the MRCs or allosteric effects could have different temperature dependences. Therefore, the analysis within this thesis will be limited to the major exponential phase (>75% of the total amplitude), as described by Pudney et al. (130), to provide the best analysis of the H-transfer chemistry and participating dynamics.

6.2 Coenzyme specificity of TOYE

6.2.1 Coenzyme kinetics As demonstrated in Section 3.4, TOYE is reduced at a faster rate and binds more tightly to -1 -1 NADPH than NADH (klim of 31.4 s and 3.8 s and Ks of 49.9 and 102 µM, respectively). -1 The preference to NADPH is similar to PETNR (klim of 34 s and Ks of 73 µM) (40, 130) -1 while MR solely reduces NADH (with klim of 56 s and Ks of 101 µM) (40, 225) (Table

6.1). In Section 4.3.5 it was shown that TOYE binds NADH4 in a different way compared to the mesophilic homologues.

Table 6.1. Substrate specificity of OYEs. TOYE PETNR YqjM TOYE PETNR MR

NADPH NADPH NADPH NADH NADH NADH a -1 b b c kobs (s ) 30.6 ± 1.0 33.5 ± 0.2 - 3.68 ± 0.04 2.0 ± 0.02 56.4 ± 0.77 -1 d e f d klim (s ) 31.37 ± 1.2 34 ± 0.4 22.2 3.77 ± 0.08 2.0 ± 0.4 56 ± 0.6 d e f d Ks (µM) 49.9 ± 11.8 73 ± 0.4 333 102 ± 12.1 1000 ± 100 101 ± 7

klim/Kd 630 ± 150 470 ± 0.1 66.7 40 ± 4 2 ± 0.4 554 ± 0.1 (mM-1s-1) a at 25 °C b (130) c (225) d (40) e (55) f (224)

154

Structural differences within the OYE family have previously been used to explain the specificity of PETNR (224). TOYE exhibits a preference towards NADPH over NADH, with similar kcat and klim as PETNR (Table 6.1). The coenzyme preference of classical OYEs has been attributed to the presence of two arginine residues that could interact with the 2’-phosphate group of the ribose connected to the adenine moiety (14) in enzymes showing preference towards NADPH, such as PETNR. Other residues have also been implicated in coenzyme binding. Enzymes preferring NADH, for example MR, have aspartate and glutamate instead that can interact with the 2’-hydroxygroup of NADH. The

NADH4-bound structure of TOYE (Section 4.3.5 and Figure 4.13), has flexible ribose units which could not be modelled in all 4 subunits. On the other hand, the adenosine moiety could be modelled as it takes part in the coenzyme binding. This suggests that, at least in the case of NADH4, the ribose does not take part in the NADH4 binding.

At the present time, the lack of other thermophilic-like OYE structures with coenzymes bound makes it impossible to determine if the way TOYE binds NADH4 is typical for the subclass or not (Section 4.3.5). A crystal structure with bound NADPH4 would also help to explain fully the coenzyme specificity of TOYE and if there is a difference in binding between these two coenzymes. The preference of YqjM is not known as only results from stopped-flow studies with NADPH have been published (55) while NADH and NADPH has been used during biotransformations (61). Steady-state rate constants, with Cr(VI) as oxidative substrate, have been reported for CrS. NADPH was the preferred coenzyme, with similar KM, but higher specific activity than with NADH, although NADPH did exhibit some substrate inhibition at concentrations above 100 µM.

6.2.2 Binding of nonreactive coenzyme analogues and CT-complex formation The formation and decay of a CT complex during the hydride transfer of the C4 pro-R- hydrogen of the coenzyme to N5 of the FMN was monitored at 555 nm. The decay had a similar rate as the FMN reduction, which suggests that they are kinetically linked. The CT complex can be formed and stably maintained by titrating TOYE with non-reactive

NAD(P)H4. The titration resulted in hyperbolic concentration-dependencies (Figure 6.4) -1 -1 and gives a maximal CT absorbance (ε555 max) of 0.050 and 0.058 mM cm for NADH4 and NADPH4, respectively. The dissociation constant for NADH4 and NADPH4 are 54 ± 4 and 31.1 ± 6 µM, respectively. These values are similar to those previously reported for 155

PETNR and MR, and data presented in section 6.1 and 6.2 suggests that the equilibrium binary complex and FMN reduction in TOYE is comparable to other OYEs.

1.0

0.8 )

-1 0.6

cm -1 0.4

0.2

555(mM  0.0

0 1 2 3 4 5 [NAD(P)H ] (mM) 4

Figure 6.4. Binding titration of TOYE with NAD(P)H4. Red and black data points and fits are for NADPH4 and NADH4, respectively. Conditions: 50 µM TOYE, 50 mM potassium phosphate pH 7.0 at 25 °C.

6.3 Temperature dependence of the reductive half-reaction Measurements were carried out under anaerobic conditions and rates were observed at 5 °C intervals over the temperature range of 5 – 50 °C. As shown in Section 3.5, TOYE is fully stable over this temperature range. Higher temperatures were not measured due to limitations of the instruments. The rates and KIEs referred to here are for the hydride- transfer step in the reductive half-reaction of TOYE using NAD(P)H.

6.3.1 Temperature dependences of hydride transfer with NAD(P)H A link between motions and the formation and cleavage of bonds during hydrogen transfer has been shown in recent years (91, 93, 108, 227). The temperature dependencies of the reduction of TOYE by protiated coenzymes are shown in Figure 6.5 for NADH and NADPH and parameters are shown in Table 6.2. The temperature dependence of TOYE with NADH is log-linear, with ΔH‡ = 43.9 ± 0.46 kJ mol-1, similar to the mesophilic homologues compared to 35.8 and 34.2 kJ mol-1 for MR (225) and PETNR (224), respectively. On the other hand, the temperature dependencies of TOYE with NADPH did 156

not fit well to a linear fit (Figure 6.5 B) and a breakpoint was introduced to the fit (Section 2.18), which was at 302 K. The temperature dependence exhibited a large change in ΔH‡ before and after the breakpoint, from 18.6 ± 3.1 kJ mol-1 below 25 °C to 32.8 ± 1.0 kJ mol- 1 above 25 °C. As the only structural difference between the coenzymes is the 2’-phosphate group of the ribose on NADPH the breakpoint could be attributed to different binding modes that are affected by temperature. This has been seen with other enzymes, such as dihydrofolate reductase (DHFR) from E. coli. Crystallography of the active site surface loop of DHFR showed different conformations throughout the reaction pathway depending on the ligand (126).

A) B)

-3.0 -1.6 -1.8 -3.5 K1 NADH-2.0 -4.0 lnA -2.213.2 ±0.19 dH 43.9 ±0.46 -2.4 -4.5

-2.6 ln(k/T)

-5.0 ln(k1/T) -2.8 -3.0 -5.5 -3.2 -6.0 -3.4 0.0031 0.0032 0.0033 0.0034 0.0035 0.0036 0.0031 0.0032 0.0033 0.0034 0.0035 0.0036 1/T (K-1) -1 1/T (K )

Figure 6.5. Temperature dependence of FMN reduction of TOYE-His6 by NADH and NADPH. A) Changes in rate of the major amplitude over the measured temperature range during reduction by NADPH. B) Temperature dependence of the rate of the major amplitude during reduction by NADPH. Linear fit (Eq. 2.12) is shown as a blue line and fit using a breakpoint (Eq. 2.13) is shown as red. Conditions used were 20

µM TOYE-His6, 5 mM NAD(P)H in 50 mM potassium phosphate buffer pH 7.0 over the range of 5 – 50 °C.

157

6.3.2 Temperature dependence of 1° KIEs The temperature dependencies with (R)-[4-2H]-NADH and (R)-[4-2H]-NADPH were measured over the temperature range of 5 – 50 °C at 5 °C intervals (Figure 6.6 and Table 6.2). With (R)-[4-2H]-NADH the KIE of between 4.5 and 6.5 (6.2 at 25 °C) is essentially temperature independent (ΔΔH‡ = -3.0 ± 1.2 kJ mol-1). The temperature dependence for the deuterated substrate, like the protiated substrate, does not exhibit a break point over the temperature range. The ΔΔH‡ of a linear fit is -3.0 ± 1.2 kJ mol-1 and at 25 °C the 1° KIE is 6.2. Given the relatively high error this could be interpreted as a temperature independent KIE. The trend and values are similar to the mesophilic homologues PETNR (52) with 1° KIE of 8.1 at 25 °C and ΔΔH‡ = -1.1 ± 2.1. Temperature independent 1° KIE and low ΔH‡ is expected for a reaction proceeding through pure tunnelling as the barrier crossing is temperature independent while a semiclassical model ascribes different activation energies for the isotopologues and temperature dependent KIEs. Temperature-independent KIEs have been explained as tunnelling with no or little affect from enzyme dynamics (93, 107).

With (R)-[4-2H]-NADPH the KIE is temperature dependent over the whole temperature range and the temperature dependence of the rate exhibits a breakpoint at around 25 °C, similarly to the temperature dependence with the protonated coenzyme. The ΔΔH‡ below and above 25 °C are 27.7 ± 2.2 kJ mol-1 and 11.7 ± 4.5 kJ mol-1, respectively. At 25 °C and below the temperature dependence of rates for NADPH and (S)-[4-2H]-NADPH and the ΔΔH‡ increase but this breakpoint suggests a temperature dependent change takes place in the enzyme dynamics (Figure 6.6 B).

The parameters above the breakpoint of TOYE are similar to PETNR (52) and MR (130) with their preferred coenzyme, NADPH and NADH respectively (ΔΔH‡ values of 6.5 ± 2.8 kJ mol-1 and 7.4 ± 1.5 kJ mol-1 for PETN and MR, respectively), though neither has a breakpoint in their temperature dependence. Both experimental work and computational simulations on MR suggest that the 1° KIE and ΔΔH‡ reflect the qualitative magnitude of a compressive mode of a reaction coordinate, affecting the donor-acceptor distance (130, 226, 228). Larger ΔΔH‡ are thought to suggest larger H-transfer distance and relatively ‘softer’ compressive mode. This would mean that above 25 °C, where the ΔΔH‡ is smaller, the H-transfer distance is smaller and the donor-acceptor distance is affected by a higher force constant (‘stiffer’ compression).

158

A) B)

-3 -1.5 -2.0 -4 -2.5 -3.0 -5 -3.5 -6 -4.0

ln(k/T) -4.5 -7 ln(k/T) -5.0 -8 -5.5 -6.0 -9 0.0031 0.0032 0.0033 0.0034 0.0035 0.0036 0.0031 0.0032 0.0033 0.0034 0.0035 0.0036 -1 -1 1/T (K ) 1/T (K ) C)

KIE 8

0.0031 0.0032 0.0033 0.0034 0.0035 0.0036 -1 1/T (K )

Figure 6.6 Temperature dependence on the FMN reduction of TOYE by NADH and (R)-[4-2H]-NADH and the KIE. A) Eyring plot of the reductive half-reaction of TOYE with NADH and (R)-[4-2H]-NADPH, black squares and red circles, respectively. Linear fit shown as blue line. B) Eyring plot of the reductive half- reaction of TOYE with NADPH and (R)-[4-2H]-NADPH, shown as black squares and red circles, respectively. Linear fit and fit with a break point are shown as black dashed lines and blue lines, respectively. C) Temperature dependence of the 1° KIE of (R)-[4-2H]-NADH and (R)-[4-2H]-NADPH, shown as red fill circles and black filled squares, respectively. Conditions used were 20 µM TOYE-His6, 5 mM NADH in 50 mM potassium phosphate buffer pH 7.0 over the range of 5 – 50 °C.

159

Table 6.2 Parameters from temperature dependence of FMN reduction of TOYE by NAD(P)H and (R)-[4-2H]-NAD(P)H. TOYE TOYE TOYE TOYE NADH NADPH NADPH NADPH (linear) (linear) (< 25°C) (> 25 °C) ‡ -1 ΔH H (kJ mol ) 43.9 ± 0.5 28.4 ± 1.3 18.6 ± 3.1 32.8 ± 1.0

ln A’H 13.2 ± 0.2 9.1 ± 0.5 11.0 ± 0.4 5.0 ± 1.2 ‡ -1 ΔH D (kJ mol ) 40.8 ± 0.7 46.5 ± 2.6 60.5 ± 1.2 30.3 ± 1.4 ‡ -1 ΔΔH D-H (kJ mol ) -3.0 ± 1.2 11.7 ± 2.0 41.9 ± 3.3 2.5 ± 1.7

ln A’D 10.2 ± 0.3 14.4 ± 2.6 20.2 ± 0.5 8.2 ± 0.6 KIE (25 °C) 1.16 ± 0.01 6.7 ± 0.5

A’H : A’D 21.9 0.005 0.0001 0.04 Equations 2.12 and 2.13 were used for linear and breakpoint fit, respectively.

Biphasic temperature dependencies such as presented here have been reported for thermophilic enzymes (129, 221, 229, 230), with a breakpoint at temperatures near mesophilic temperatures where thermophilic enzymes are more rigid (227, 230-232). Thermophilic enzymes, such as DHFR (129) from Thermotoga maritime (tmDHFR) and alcohol dehydrogenase (221) from Bacillus stearothermophilus (htADH), have a similar breakpoint in their temperature dependence to TOYE.

The 1° KIEs for hydrogen transfer by tmDHFR during the oxidative half-reaction, hydrogen transfer from the cofactor to 7,8-dihydrofolate, exhibits a break point at around 25 °C (129). Above 25 °C the KIE is temperature independent while below 25 °C the KIE ‡ ‡ was temperature dependent with values similar to TOYE, ΔH H = 47.4, ΔH D = 66.7 and ΔΔH‡ = 19.3. Studies on htADH have demonstrated that significant tunnelling takes place at high temperatures (221), as high as the physiological temperature of 65 °C, which is similar to its mesophilic homologue at lower temperatures (87). The temperature dependence breakpoint of htADH is around 30 °C for both protiated and deuterated substrates. Below the breakpoint, the KIE increases with lower temperature as well with a similar pattern as seen with TOYE (Figure 6.6). This suggests an increase in tunnelling or

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H-transfer distance at lower temperatures and a role of temperature dependent enzyme dynamics in the tunnelling reaction. For example, amide protium/deuterium studies on htADH (227) and 3-isopropylmalate dehydrogenase from Th. Thermophilus (231) and their mesophilic homologues show a comparable level of global flexibility at the physiological temperatures. Also, both thermophiles were more rigid than the mesophilic homologues at 25 °C (227, 231).

The Arrhenius pre-exponential factor ratio (A’H/A’D) has also been used to indicate the presence of tunnelling during hydrogen transfer (129, 233-235) and is the semi-classical range 0.7 – 1.2, values outside of are considered to suggest tunnelling during catalysis (236). For TOYE, this ratio is well outside the semi-classical range with both NADH

(A’H/A’D of 21.9) and NADPH (Table 6.2). The reaction with NADPH has A’H/A’D ratio of 1*10-4 at temperatures below 25 °C and 0.04 above. The difference between coenzymes is similar to PETNR where the A’H/A’D ratios are 5.2 and 0.51 for NADH and NADPH, respectively (52). In htADH which exhibits a breakpoint around 30 °C the A’H/A’D ratio changes from 1*10-5 below 30°C to 2.2 above the breakpoint (233), both values outside the semi-classical range. In the case of tmDHFR the enzyme exhibits a ratio of ~1 at higher temperatures but a value of 0.002 below the breakpoint of 25 °C (129).

A change in KIE has been interpreted as enzyme dynamics playing an important role in hydrogen tunnelling, affecting the donor-acceptor distance. If the tunnelling was considered to proceed through a barrier affected by stiffer compressive motions then the ΔΔH‡ values would be around zero and KIE large. These enzymes also exhibit a temperature dependence, which indicates ‘stiffer’ compressive motions and shorter donor- acceptor distance at higher temperatures. Unlike TOYE, the enzymes showed temperature independent KIE and near zero ΔΔH‡ values after the breakpoint. The ΔΔH‡ of TOYE at higher temperatures (ΔΔH‡ = 11.7 ± 4.5 kJ mol-1 over 25 – 50 °C) is similar to the values for the mesophilic homologues, PETNR (52) and MR (130) at mesophilic temperatures (ΔΔH‡ = 6.5 ± 2.8 kJ mol-1 and 7.4 ± 1.5 kJ mol-1, respectively, over 5 – 35 °C).

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6.3.3 Temperature dependence of 2° KIEs Secondary KIEs have been used to analyse the nature and geometry of transition states (111, 237, 238) and tunnelling ready configuration (TRC) (130). The TRC is one of possibly many conformations of the enzyme and substrate(s) where the reaction can proceed through tunnelling (225). The KIE is considered to be sensitive to the change in bending modes during sp3 to sp2 hybridisation during the rate determining step (95). A transition state with a similar structure as the reactant and thus no major change in the hybridisation would show a 2° KIE around 1. 2° KIEs are normally between unity and EIE, for TS this is considered as reactant- and product-like TS states, respectively (239). When the 2° KIE is close to or equal to the EIE the transition state is considered to be similar to the product and the change from sp3 to sp2 has occurred.

Using TST a low 2° KIE would suggest a late transition state (240, 241) while secondary KIEs above the 2° EIE of 1.13 (inflated KIEs) are explained as cases where the secondary and primary hydrogen, which is transferred, have coupled motion affecting the tunnelling of the primary hydrogen (87, 111, 237, 238, 242-244). The magnitude of the 2° KIE has been considered to reflect the tunnelling ready configuration of the coenzyme-enzyme complex. The 2° KIE has a classical limit, similar to 1° KIE (Section 1.5.3.2). Outside the classical limit the TST cannot fully explain the rate differences. For example, the yeast alcohol dehydrogenase has a 2° KIE value (1.05 ± 0.01) outside of unity (245). Other examples of 2° KIEs outside of the semiclassical range have been linked to coupled motion (225, 237, 246, 247).

The breakpoint for (S)-[4-2H]-NADPH at 300.4 ± 2.0 K is similar to the breakpoints seen for NADPH (302 K) and (S)-[4-2H]-NADPH (300 K; Figure 6.7 and Table 6.4). The normal semiclasscal limits for 2° KIE are considered 1 – 1.13 and TOYE the 2° KIE for NADPH is within that limit between 5 - 25 °C (2° KIEs between 1.11 – 1.00) while the 2° KIE for NADH is above the EIE between 5 – 35 °C (2° KIEs between 1.24 – 1.16), indicating a tunnelling in the reaction with NADH and an earlier TS compared to a reaction NADPH. As the temperature increases the 2° KIE becomes inverted, above 25 °C and 35 °C for NADPH and NADH, respectively. This inverted isotope effect at higher temperatures may be explained by a change in the rate limiting step or change in the multiple exponents, as seen in Figure 6.2 and 6.3, causing difficulties during data processing.

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A) B)

-3.0 -1.5 -3.5 -2.0 -4.0

-4.5 -2.5

-5.0

-3.0

ln(k/T) ln(k/T) -5.5 -3.5 -6.0

-6.5 -4.0 0.0031 0.0032 0.0033 0.0034 0.0035 0.0036 0.0031 0.0032 0.0033 0.0034 0.0035 0.0036 -1 -1 1/T (K ) 1/T (K ) C)

1.5 1.4 1.3 1.2 1.1 1.0

KIE 0.9 0.8 0.7 0.6 0.5 0.0031 0.0032 0.0033 0.0034 0.0035 0.0036 -1 1/T (K )

Figure 6.7 Temperature dependence on the FMN reduction of TOYE by NADH and (S)-[4-2H]-NADH and the 2° KIE. A) Eyring plot of the reductive half-reaction of TOYE with NADH and (S)-[4-2H]-NADH, black squares and red circles, respectively. Linear fit shown as blue line. B) Eyring plot of the reductive half- reaction of TOYE with NADPH and (S)-[4-2H]-NADPH, shown as black squares and red circles, respectively. Linear fit for the (S)-[4-2H]-NADPH and fit with a break point are shown as black dashed lines and blue lines, respectively. Equations 2.12 and 2.13 were used for linear and breakpoint fit, respectively. C) Temperature dependence of the 1° KIE of (S)-[4-2H]-NADH and (S)-[4-2H]-NADPH, shown as red fill circles and black filled squares, respectively. Conditions used were 20 µM TOYE-His6, 5 mM NADH in 50 mM potassium phosphate buffer pH 7.0 over the range of 5 – 50 °C.

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Table 6.3. Parameters for 2° KIE temperature dependence studies of TOYE. (S)-[4-2H]- (S)-[4-2H]- (S)-[4-2H]- (S)-[4-2H]- NADPH NADPH NADH NADPH linear < 25 °C > 25 °C ΔH‡ (kJ mol-1) 48.6 ± 1.1 35.3 ± 2.1 45.9 ± 2.0 22.9 ± 2.4 ΔΔH‡ (kJ mol-1) 4.7 ± 1.6 6.9 ± 3.4 13.1 ± 3.0 4.3 ± 5.5 ln A’ 15.0 ± 0.4 11.8 ± 0.8 7.0 ± 0.9 16.2 ± 0.8 KIE (25 °C) 1.16 ± 0.01 1.05 ± 0.03

6.3.4 Temperature dependence of 1° KIE in steady-state In Section 5.1.2, it was shown that 2-methyl maleimide was reduced at higher rate (26.85 -1 -1 s ) compared to other OYEs (for example the kcat for PETNR is 7.65 s (59)). As the reduction by TOYE was similar to the rate of TOYE reduction by NADPH the oxidative half-reaction was examined in stopped-flow studies to determine which half-reaction was rate-limiting. The concentration dependence for 2-methyl maleimide was not completed as the reaction finished at higher concentrations (>1500 µM) mostly before the 2 ms deadtime of the instrument (Figure 6.8), leaving little amplitude from which to extract a reliable rate. An observed rate of reduction at 1000 µM concentration was 703 ± 58 s-1, which is more than 20x higher than the klim for the faster reductive coenzyme, NADPH. The preliminary -1 parameters of the concentration dependence at 25 °C were klim of 1508 ± 155 s and KS of 1247 ± 22 µM, demonstrating that reduction of 2-methyl maleimide is faster than oxidation of both coenzymes, suggesting that during steady-state the reductive half-reaction is rate limiting.

It has been reported that due to a lack of oxidative substrates which are not rate limiting during steady-state turnover that the methodology is not appropriate to study KIEs of the reductive half-reaction (103). With the rates of 2-methyl maleimide in stopped flow suggesting that it would not be rate limiting during steady-state turnover the 1° KIE of NADPH was investigated under those conditions to be compared to the stopped-flow technique. Concentration dependencies were performed at 25 and 50 °C and compared to rates from stopped-flow temperature dependence studies. The concentration parameters for steady-state conditions at 25 °C (Figure 6.9 and Table 6.5) were within error from the ones obtained from stopped-flow from Section 3.2.

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A) B)

1000 0.25

800 0.20

600 )

0.15 -1

(s 400

obs k Absorbance 0.10 200

0.05 0 1E-3 0.01 0.1 1 10 0 500 1000 1500 2000 Time (s) [2-methyl maleimide] (µM)

1000 60

800 40 )

-1 30 (s

600 obs 20

k )

-1 10

(s 400 0

0 1000 2000 3000 4000 5000 obs

k [Substrate or coenzyme] (µM) 200

0 0 1000 2000 3000 4000 5000 [Substrate or coenzyme] (µM)

Figure 6.8. Stopped-flow studies of 2-methyl maleimide. A) Transient state traces of 0, 50 and 2500 µM 2- methyl maleimide. B) Incomplete concentration dependence of 2-methyl maleimide. C) Comparison of rates in stopped-flow for 2-methyl maleimide, NADPH and NADH, shown in black, red and green, respectively.

Conditions used were 20 µM TOYE-His6 in 50 mM potassium phosphate buffer pH 7.0 at 25 °C. For 2- methyl maleimide TOYE was reduced by equimolar amount of dithionite.

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Table 6.4. Comparison of concentration dependences performed under steady-state and stopped-flow conditions. 25 °C 50 °C -1 kcat (s ) ss 36.3 ± 1.6 70.7 ± 4.3 -1 klim (s ) sf 36.1 ± 0.5 -

KM (µM) ss 40.0 ± 6.7 125 ± 27

KS (µM) sf 33.1 ± 3.9 - ss: steady-state reaction, sf: stopped-flow reaction

) -1

(s obs

k 20

0 0 200 400 600 800 1000 [NADPH] (µM)

Figure 6.9. Concentration dependences at steady-state conditions for NADPH. Shown are concentration dependences for 25 and 50 °C in black squares and red circles, respectively. Horizontal lines indicate rates from stopped-flow temperature dependence studies at 25 °C (black line) and 50 °C (red line).

The temperature dependence of NADPH and (S)-[4-2H]-NADPH are similar to the stopped-flow data at mesophilic temperatures. Due to difficulties keeping high and low temperatures stable in the steady-state spectrometer the measurements are limited to mid- range temperatures. The temperature dependence seen for the protonated coenzyme is linear, but it appears to have a break point at 29.5 °C for the deuterated coenzyme (Table 6.5 and Figure 6.10). This was also seen in the stopped-flow experiments (Sections 6.3.1 and 6.3.2). The observation of a breakpoint is debatable over the temperature range due to the error at lower temperatures (for example the data shown for 15 °C). The lack of a break-point for NADPH may be due to the fact that the steady-state technique covers the whole reaction while in stopped-flow rate constants for MRCs are separated. This method is not comparable to the stopped-flow for TOYE but might be more reliable for enzymes that proceed through the reductive half-reaction in a single phase (single exponential fit), such as wild type PETNR (38). 166

A) B)

-1.4 -1.6 -3.0 -1.8 -3.5 -2.0 -2.2 -4.0 -2.4 -4.5 -2.6

ln(k/T) -5.0

-2.8 ln(k1/T)

-3.0 -5.5 -3.2 -6.0 -3.4 0.0031 0.0032 0.0033 0.0034 0.0035 0.0036 0.0031 0.0032 0.0033 0.0034 0.0035 0.0036 -1 1/T (K ) 1/T C)

KIE 8

0.0031 0.0032 0.0033 0.0034 0.0035 0.0036 -1 1/T (K )

Figure 6.10. Temperature dependences at steady-state conditions. A) Comparison of temperature dependences of NADPH. Stopped-flow and steady-state data shown in red and black. Linear fit for steady- state data is shown with red line and the break-point fit for stopped flow is shown with a blue line. B) Comparison of temperature dependences of (R)-[4-2H]-NADPH. Stopped-flow and steady-state data shown in red and black. Linear fit for steady-state data is shown with red line and the break-point fit for stopped flow is shown with a blue line. C) Equations 2.12 and 2.13 were used for linear and breakpoint fit, respectively. Comparison of 1° KIEs from steady-state and stopped-flow. Steady-state and stopped-flow data are shown as black squares and red circles, respectively.

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6.4 Discussion As thermophilic enzymes have been shown to be more rigid at mesophilic temperatures than at their physiological temperatures (151, 152, 227, 231), it can be inferred that TOYE is likely to have greater flexibility at the higher temperatures and be more comparable to the mesophilic homologues at physiological temperatures. It was not possible to conduct the experiments in this thesis at the physiological temperature of the natural host of TOYE (69 °C (153)) due to instrument limitations.

The analysis of coenzyme analogue binding and specificity is interesting due to the fact that NADPH is reduced almost 10 times faster than NADH. This resembles the cofactor specificity described for PETNR (52) while the cofactor analogue binding appears to be different (Section 4.3.5). The 1° KIE of TOYE by NADH exhibits a very small temperature dependence or is temperature independent, while PETNR has a temperature independent 1° KIE with NADH (52). The 1° KIE for NADPH is temperature dependent, and this was also seen with PETNR and MR towards the preferred coenzymes, NADPH and NADH, respectively (Section 6.3.1).

As the magnitude of the KIE is determined in part by the tunnelling distance and that the 1° KIE of (S)-[4-2H]-NADH does not change significantly over the temperature range tested, it can be inferred that there is no major thermally activated motion that takes part in the chemical step. The 1° KIE with (S)-[4-2H]-NADPH changes from 15.0 ± 0.8 at 5 °C down to 4.6 ± 0.1 at 50 °C, suggesting that some temperature dependent promoting motions take part in the reaction for NADPH but not NADH. The 1° KIEs for NADH and NADPH at higher temperatures are similar. This is in contrast to PETNR, where the 1° KIE is similar for both NADH and NADPH, suggesting a similar tunnelling distance. If the trend seen around 50 °C extends to the physiological temperature of TOYE for NADPH it would suggest that the OYE family applies a compressive mode with qualitatively similar force constants for this chemical step. This hypothesis could be further supported by a comparative study on a psychrophilic homologue at lower temperatures.

At lower temperatures, the enzyme dynamics which facilitate tunnelling decrease. This increases the ΔH‡, and decreases the rate compared to what would be expected from a linear trend based on high temperature rates. TOYE exhibits a temperature dependent change which affects hydrogen tunnelling. The higher ΔH‡ values and KIE around the semi-classical range at the higher temperatures for NADPH and over the whole 168

temperature range for NADH suggest a vibrationally enhanced mechanism. These results are in agreement with the theory that thermophilic enzymes apply similar mechanisms to mesophilic homologues for catalysis at their (higher) physiological temperatures (231).

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7. Conclusions

In this thesis, a novel thermophilic OYE was introduced and the characteristics detailed. The enzyme originates from the anaerobic bacterium T. pseudethanolicus E39 and was given the name ‘Thermophilic Old Yellow Enzyme’ (TOYE). Prior to the characterisation of TOYE, the OYE-family was generally considered as being one group, with only one family member differing significantly in structure. The thermophilic-like subclass was presented with YqjM, TOYE and the subsequent thermophilic OYEs. This is a group of enzymes within the OYE-family with unique structural features.

In recent years, the use of enzymes as biocatalysts for industry has increased as they often offer higher efficiency and stereo- and enantioselectivity to conventional chemical catalysts. Thermophilic enzymes have offered a great solution where there is a need to keep a substrate or product soluble with organic solvents, extreme pH values or increased temperature (248-250). Though OYEs are heavily investigated for potential use as biocatalysts, there have been no similar studies of thermophilic OYEs as they were not available at the time. Same applies to temperature dependences of the hydride transfer, which have been studied for thermophilic enzymes in other enzyme families, but no data for thermophilic OYE was previously available to compare the heavily studied mesophilic homologues. As the quantum tunnelling is sensitive towards movement within the enzyme, a relatively rigid OYE homologue would be a valuable tool for tunnelling studies. The three aspects of this thesis (structural findings, biocatalysis and the effect of structure on the hydride transfer) will be discussed separately below.

7.1 Connection between structure and thermal stability of TOYE In Chapters 3 and 4, the cloning, purification and characterisation of the recombinant TOYE was described for the first time. Although the overexpression of TOYE was successful, only 25% of the purified enzyme was flavinated after the purification. This meant that addition of excess FMN was required during the purification process to achieve ~80% flavination. This is similar to the closely related homologues YqjM (30, 55) and CrS 170

(187), where the wild-type enzymes require an addition of FMN during the purification process to achieve high flavination. Low affinity towards the FMN may not be a structural feature of the thermophilic-like OYEs. This issue has not been reported in previous work by Schittmayer et al. with the thermophilic-like OYE, GkOYE (155). This can be attributed to the high level of over expression of the recombinant protein which could exceed the FMN production of the expression host. This is plausible considering that the IPTG induced expression system does not increase the synthesis of FMN by the host. The over expression can also hinder other cell functions, such as the synthesis of cofactors. It is therefore important to include the addition of FMN in the purification of OYEs when the over expression is high.

The structure of TOYE was examined in detail in an attempt to understand the nature of the oligomeric states and the structural adaptation employed by the enzyme to increase stability at high temperatures. These findings could prove useful in future studies where the mesophilic homologues would be made more heat resistant for biocatalytic use. Native SDS-PAGE gel electrophoresis suggested that the lowest oligomeric state of TOYE is a tetramer, similarly to the mesophilic homologue YqjM. The classical OYEs are either monomers or dimers (16, 21, 36, 37, 39, 157) and previously only YqjM had shown a tetrameric state (24, 30). Because of high sequence similarity, YqjM was used as a base model for the TOYE structure with the assumption that close homologous relationship translates into high structural similarities.

The crystal structure of TOYE showed a structure formed by a dimer of dimers, and is the first OYE to be shown to form larger complexes than tetramers in solution. The crystal structure of the tetramer (Figure 7.1) is similar to the mesophilic homologue YqjM and shows a large active site cavity when compared to classical OYEs due to the shorter loops surrounding it. The other thermophilic OYEs also have an intriguing oligomeric state. The homologues enzyme, CrS has been described as an active dimer (154) in solution, similar to the classical OYEs, though results are not published. While CrS is said to crystalise as an octomer (187) the published crystal structure (pdb accession code: 3HF3) shows 4 monomers, where 3 monomers are arranged in a similar pattern to the tetramer form. The other described thermophilic homologue, GkOYE has been shown to be a tetramer at

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physiological temperatures (155). These findings are in line with the notion that thermophilic enzymes employ higher oligomeric state as one way of increasing stability (146, 251).

A) B)

Figure 7.1. Structure of TOYE. A) Overall tetrameric structure of TOYE. B) TOYE monomer. Residues are shown as rainbow cartoons from N- (blue) to C-terminus (orange) in each monomer. Each FMN molecule is shown as atom coloured sticks with cyan carbons.

The larger active site cavity of TOYE is also a result from another adaptation to high temperatures: the shorter surface loops. The higher oligomeric states of TOYE, such as octamer and dodecamer, seen in solution and on gels by various techniques have previously not been seen within the OYE-family. The nature of these higher oligomeric states merits a further investigation both with TOYE and the other thermophilic-like OYEs to investigate the importance of oligomerisation for stability and if allosteric interactions between monomers affect the catalysis.

The tetrameric structure and shorter surface loops of TOYE would be expected to yield a resistance towards high temperatures. An important step in the characterisation of enzymes is investigation of the stability to ensure their shelf-life does not limit the experiments performed, when long experiment time or high temperatures are required. The secondary structure of TOYE was shown to be stable up to 70 °C at which point the enzyme proceeded to precipitate. These findings are similar to the thermostable GkOYE, where unfolding commences at 75 °C and is followed by aggregation (155), whereas YqjM has limited thermal stability and denatures at 50 °C. Problems arise if detailed unfolding studies were to be performed on TOYE. The hydrophobic areas clump together during the 172

unfolding process as TOYE aggregates, making the use of spectroscopic techniques difficult to show the unfolding process quantifiably by calculating the Tm.

Despite being extensively studied for industrial use, there is distinct lack of published research regarding the stability of the commonly used OYEs, such as the mesophilic OYE1, PETNR and YqjM. Detailed studies regarding the stability of these enzymes would be expected as they are subjected to long incubation times (24 hours to 7 days) at or above 25 °C (47, 48, 53, 252, 253). Partial denaturation of the wild-type PETNR has been mentioned by Pudney et al. (225) at 40 °C after a short incubation time but temperatures of 35 – 40 °C are often used during sensitive stopped-flow studies relating to quantum tunnelling (40, 52, 225), where instruments require time to reach the desired temperatures and enzyme samples need time to equilibrate at that temperature, often several minutes. These factors require consideration when working with mesophilic enzymes, especially potentially unstable mutated enzymes, to distinguish enzyme activity from substrate denaturation or activity of minor contaminating enzymes. This increased thermal stability presents a new OYE-family member which has potential in biocatalysis.

7.2 Biocatalytical potential of TOYE Enzymes within the OYE family have been extensively studied regarding potentials for biocatalytical use (47, 48, 53, 59, 75, 78, 195, 214, 253-255). As organic chemicals often require organic solvents to stay soluble it is advantageous for the enzyme applied to be stable in the presence of solvents. The mesophilic PETN reductase has been studied in detail regarding biocatalysis (47, 48, 53, 59) and the solvent stability of PETN reductase was compared to TOYE in Chapter 5. Similar to stability at elevated temperatures, there is a lack of published results regarding the stability of OYEs in organic solvents. Increased stability towards organic solvents compared to a mesophilic homologue was shown, which would be advantageous for industrial application where solvents are required for substrate and/or product stability and solubility. As predicted, TOYE was more resistant towards ethanol than PETN reductase. This property is likely to apply to other organic solvents as well and further studies are required to fully demonstrate the capacity TOYE has as biocatalyst over the mesophilic homologues.

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The biocatalytical potentials of TOYE were explored in Chapter 5 by steady-state and biotransformations. As expected of an OYE-family member, TOYE performed well as a biocatalyst. TOYE exhibited unique substrate specificity and this is expected to be caused by the residues lining the active site. An example was the formation of a succinimide anticonvulsant drug, phensuximide (N-methyl-α-phenylsuccinimide), from 2-phenyl-N- methyl maleimide which was achieved by TOYE with >99% conversion. This reaction makes TOYE an attractive target for pharmaceutical applications.

Though TOYE showed on average a lower activity than PETNR and other mesophilic homologues, it was highly active towards maleimide substrates. The reaction with these substrates in a steady state was at a similar rate as the reduction of NADPH, suggesting that the reductive half-reaction might be the rate limiting factor. This was verified using -1 stopped-flow, and the reduction of 2-methyl maleimide by TOYE (klim of 1508 s ) was at -1 25 °C faster than the reduction of GTN by PETNR (klim of 518 s ) and codeinone by MR -1 (kobs of 128 s at 2 mM substrate concentration), respectively (14, 26). This is an unprecedented rate by OYE and notably at 40 °C below the physiological temperature for TOYE. On the other hand, YqjM has been reported to have a faster reoxidation rate in the presence of N-Ethylmaleimide than could be measured in stopped-flow. The reaction had finished during the dead time of the instrument used (55). Normally, it is accepted that thermophilic enzymes operate at a lower rate at mesophilic temperatures due to their rigid structure. This extreme reaction rate by TOYE with maleimide substrates may not hint at what the physiological substrate is but highlights its potential as a high through-put green catalyst in the pharmaceutical industry for drug synthesis, phensuximide for example.

The application of environmentally-friendly chemistry is growing in the pharmaceutical and chemical industry, allowing for less waste, energy consumption and toxic side- products (256-262). For example the use of alcohol dehydrogenase from Rhodococcus erythropolis for the reduction of 3,5-bistriﬂuoromethyl acetophenone (263) to form an intermediate for the synthesis of the antiemetic drug, Aprepitant (264). TOYE could be used in a ‘green synthesis’ of succinimide drugs (such as the anticonvulsant phensuximide, mesuximide and ethosuximide), where simple organic chemicals could be built up to the final product in steps of enzyme or mild chemical reactions.

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The interesting substrate specificity makes it important to explore the role of key amino acids inside the active site as has been done for the classical OYEs (35, 47, 51, 203, 265, 266) for comparison. They could also provide a useful mutant library as directed evolution studies similar to PETNR (48) and YqjM (49) have been successfully used to improve catalysis of poor substrates and to change or improve enantiospecificity of a reaction.

Directed evolution could also be used to improve the already good stability of TOYE in order to increase the shelf-life should it be used comercially as a catalyst, for example by improving the monomer interactions by point mutations. The aggregation of monomers is accepted as an evolutionary strategy to stabilise an enzyme (267-270). For example the tetrameric β-glucoronidase which was greatly stabilised when a number of mutations on the monomer interfaces worked together in a directed evolution study (271).

The stability of TOYE makes it suitable for new applications, such as installation on monoliths (272-274) or surfaces (275-277) or microfluid reactors (278-281). As previously stated, if the catalyst is required to be functional for an extended time, the enzyme stability is important. Thermophilic enzymes are well suited for these applications as they are more stable than mesophilic enzymes. TOYE would have an added linker to allow it to be absorbed to a surface. The increased stability would not only mean higher tolerance towards solvents but also a longer life-time and thus TOYE would last longer than mesophilic homologues. The increased stability of TOYE would make it a cheaper alternative to the mesophilic homologues as well, as life-time of enzymes is a problem in biocatalysis and the regeneration of the enzyme would be required less frequently (282). Potential use would be as a biosensor. Similarly to FAD-dependent glucose dehydrogenase from Aspergillus species in amperometric glucose biosensors (283).

7.3 Effects of structural adaptation on hydrogen tunnelling It has been more accepted in recent years that protein dynamics do not only take part in substrate binding and product release but also in the catalysis of the enzyme reaction (284- 286). The structural results in Chapter 4 were applied during analysis in Chapter 6 which is summarized below. It was expected that minor structural changes between TOYE and mesophilic homologues would yield notable differences, as quantum tunnelling is a 175

sensitive phenomenon. Over the last few years, the importance of protein dynamics in catalysis has been studied in detail and is becoming more apparent. The mobility of proteins allows them to explore high energy conformational states of a multi-dimensional free energy landscape and this protein motion takes influence substrate binding, catalysis and release of product (284, 285, 287). Though it is difficult to directly measure protein dynamics and directly link them to catalysis, advances are being made (4, 288-291). An example is recent work where a large scale domain motion has been monitored upon ligand binding in the human cytochrome P450 reductase, using a time-resolved fluorescence resonance energy transfer linked with stopped-flow spectroscopy (288). This suggested that the electron transfer is coupled to the conformational changes.

Hydrogen tunnelling has been used to connect enzyme catalysis and protein motion (290, 292, 293). When inspecting the hydride transfer by TOYE and potential tunnelling, MR and PETNR were used as benchmark enzymes for comparison as they have been studied in great detail (40, 130, 222-226). The reductive half-reaction of TOYE was examined through temperature dependence studies using isotopically labelled coenzymes with both stopped-flow and steady-state techniques. High primary and secondary kinetic isotope effects suggest that the reductive half-reaction proceeds through tunnelling during catalysis for NADPH and NADH, respectively. While the primary KIE of NADH was temperature independent, there is a large change with NADPH where there is a breakpoint in the temperature dependence around 25 °C. This suggests that thermally activated motion takes part in the reduction by NADPH but not NADH though the coenzymes are structurally similar. At higher temperatures the ΔH‡ and KIEs for TOYE are similar to the values for mesophilic homologues at 25 °C which supports the hypothesis that thermophiles employ similar mechanisms for catalysis as mesophilic homologues at their physiological temperatures (144).

While the active site residues are similar to other OYEs with conserved residues which take part in catalysis, the binding of the NADH4 inhibitor is different from its homologues. The active site structure of TOYE is similar to PETNR and MR and thus does not explain the different behaviour with NADPH (14, 15, 23). The breakpoint visible in the temperature dependence with NADPH is unique within the OYE-family. The findings with

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NADH are similar to PETNR and MR. This suggests that the breakpoint is not caused by a structural difference around the active site, as the nicotine amide moiety of the coenzymes is identical. The two coenzymes differ by one phosphate group on the ribose ring. The kinetic findings indicate that thermally induced motions play different roles in binding and catalysis with NADH and NADPH. On the other hand, to fully support this proposal there is a need of a crystal structure of TOYE with NADPH4 bound in the future.

These findings are similar to the suggested temperature dependant conformational changes of htADH. Temperature-dependent hydrogen-deuterium (H/D) exchange experiments on htADH have shown that regions taking part in cofactor and substrate binding become more mobile at increased temperatures, compared to other parts of the enzyme (294). The change in mobility is also in the area where a breakpoint is visible in the temperature dependence (221). This suggests a link between the enzyme mobility and hydrogen tunnelling. The effect of temperature below the break point is similar for htADH and TOYE, where the KIE increases as the enzymes become more rigid (235, 294).

A problematic feature of TOYE is the multi-exponential traces during stopped-flow experiments as they make analysis of data difficult. These are not seen with the wild type MR and PETNR. This complexity may cause the unexpected temperature dependence of the 2°KIEs, where the KIE becomes inverted with increased temperature. Though other 2° KIEs outside of the semiclassical range have been linked to coupled motion of the two hydrogen bonds (225, 237, 246, 247) further studies are required to elucidate the meaning or relevance of inverted KIEs which TOYE exhibits.

This thesis provided a more precise understanding of the OYE-family, more specifically the newly defined thermophilic-like subclass and a new OYE-family member was introduced and characterised. It was shown that protein mobility plays a key role in modulating hydrogen transfer. The work sets TOYE up for further studies in biocatalysis and deeper analysis of the role of motion during the reductive half-reaction.

177

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Appendix A

A.1 Gene optimisation

The original sequence of TOYE and the optimised sequence are listed below, labelled with 'Org:' and 'Opt:' respectively. Where both are identical, the original sequence contains dots instead of nucleotides ('.'). The names of the restriction enzymes start at the first nucleotide after the restriction cut in the forward strand.

5 10 15 20 25 30 35 40 45 ORF: M S I L H M P L K I K D I T I Org: ...... T ... T.A ..T ... ..T T.A ..G ..A ..G ..T ..T ..A ..A Opt: CAT ATG AGC ATT CTG CAC ATG CCG CTG AAA ATC AAA GAC ATC ACC ATC NdeI

50 55 60 65 70 75 80 85 90 95 ORF: K N R I M M S P M C M Y S A S T Org: ... ..T A.A ...... T ..T ... ..T ... ..C ..A ..T ... ..A Opt: AAA AAC CGC ATC ATG ATG TCC CCG ATG TGC ATG TAT TCT GCC TCT ACC

100 105 110 115 120 125 130 135 140 ORF: D G M P N D W H I V H Y A T R A Org: ... ..G ...... T ...... A ... ..T ..C ..C ..A A.G ..T Opt: GAT GGT ATG CCA AAC GAC TGG CAT ATC GTT CAC TAT GCG ACT CGT GCG

145 150 155 160 165 170 175 180 185 190 ORF: I G G V G L I M Q E A T A V E S Org: ..T ... ..A ... ..A ..T ...... A ..A ..C ..A ...... G AGC Opt: ATC GGT GGT GTA GGT CTG ATT ATG CAG GAG GCT ACT GCT GTT GAA TCT

195 200 205 210 215 220 225 230 235 240 ORF: R G R I T D H D L G I W N D E Q Org: A.A ..A A.A ..A ..T ... ..T ..C ..T ..C ..A ... ..T ..T ... ..A Opt: CGT GGT CGT ATT ACG GAT CAC GAT CTG GGT ATC TGG AAC GAC GAA CAG

245 250 255 260 265 270 275 280 285 ORF: V K E L K K I V D I C K A N G A Org: ..T ... ..A T.A ...... T ..A ... ..T ..T ... ..A ..T ..C ..T Opt: GTC AAA GAG CTG AAA AAA ATC GTT GAC ATC TGC AAA GCC AAC GGT GCA

290 295 300 305 310 315 320 325 330 335 ORF: V M G I Q L A H A G R K C N I S Org: ..G ... ..A ..A ... ..T ..T ...... A A.A ...... T ..A ... Opt: GTT ATG GGT ATT CAG CTG GCG CAT GCA GGT CGC AAA TGT AAC ATC TCC

340 345 350 355 360 365 370 375 380 ORF: Y E D V V G P S P I K A G D R Y Org: ... ..G ...... A ..A ..T ..C ..T ..C ...... A ... ..C ... Opt: TAC GAA GAT GTC GTT GGT CCG TCT CCG ATT AAA GCA GGT GAC CGT TAC

196

385 390 395 400 405 410 415 420 425 430 ORF: K L P R E L S V E E I K S I V K Org: ... ..T ..A A.A ... T.A ..A ...... A ... TCT ..A ..A ... Opt: AAA CTG CCG CGT GAA CTG TCC GTT GAG GAA ATC AAA AGC ATC GTG AAA

435 440 445 450 455 460 465 470 475 480 ORF: A F G E A A K R A N L A G Y D V Org: ..T ... ..G ..A ... ..T ... A.G ...... T.A ..A ... ..T ..T ..A Opt: GCG TTT GGT GAG GCT GCG AAA CGT GCT AAC CTG GCT GGT TAC GAC GTT

485 490 495 500 505 510 515 520 525 ORF: V E I H A A H G Y L I H E F L S Org: ..T ... ..A ... ..A ..T ..C ..C ..T T.A ... ..C ... ..T ..T ... Opt: GTA GAA ATC CAT GCG GCA CAT GGT TAC CTG ATC CAT GAA TTC CTG TCT

530 535 540 545 550 555 560 565 570 575 ORF: P L S N K R K D E Y G N S I E N Org: ..T ..T ..A ..T ... ..A ... ..T ..A ...... T AG...... T Opt: CCG CTG TCC AAC AAA CGT AAA GAC GAG TAC GGC AAC TCC ATT GAA AAC

580 585 590 595 600 605 610 615 620 ORF: R A R F L I E V I D E V R K N W Org: A.A ..A A.A ..T T.A ..T ...... A ...... T A.A ... ..T ... Opt: CGT GCT CGT TTC CTG ATC GAA GTG ATC GAT GAA GTG CGC AAA AAC TGG

625 630 635 640 645 650 655 660 665 670 ORF: P E N K P I F V R V S A D D Y M Org: ..T ... ..T ... ..T ..T ... ..G ..G ..A ... ..A ...... C ... Opt: CCG GAA AAC AAA CCA ATC TTC GTT CGT GTG TCT GCG GAT GAT TAT ATG

675 680 685 690 695 700 705 710 715 720 ORF: E G G I N I D M M V E Y I N M I Org: ..A ... ..A ..A ... ..A ..T ...... A ... ..T ...... A Opt: GAG GGC GGC ATT AAC ATC GAC ATG ATG GTG GAA TAC ATC AAC ATG ATC

725 730 735 740 745 750 755 760 765 ORF: K D K V D L I D V S S G G L L N Org: ...... T ... T.A ..T ... ..A ... AG. ..A ..A ..T T.A ..T Opt: AAA GAC AAA GTA GAT CTG ATC GAT GTC AGC TCT GGC GGT CTG CTG AAC

770 775 780 785 790 795 800 805 810 815 ORF: V D I N L Y P G Y Q V K Y A E T Org: ..T ... ..A ..T ..A ..T ..T ..A ..T ..A ..T ...... T ... ..A Opt: GTA GAT ATC AAC CTG TAC CCG GGT TAC CAG GTG AAA TAC GCC GAA ACG

820 825 830 835 840 845 850 855 860 ORF: I K K R C N I K T S A V G L I T Org: ..T ... ..G ... ..T ..T ..A ... ..T ... ..G ... ..A T.A ..A ..G Opt: ATC AAA AAA CGC TGC AAC ATC AAA ACC TCT GCA GTA GGT CTG ATC ACT

865 870 875 880 885 890 895 900 905 910 ORF: T Q E L A E E I L S N E R A D L Org: ..A ..A ..G ..T ...... T ..T ..A ..T ... A.G ..A ... T.A Opt: ACC CAG GAA CTG GCA GAA GAA ATC CTC TCT AAC GAA CGT GCT GAC CTG

915 920 925 930 935 940 945 950 955 960 ORF: V A L G R E L L R N P Y W V L H Org: ... ..A ..T ..A A.A ... ..T T.A A.A ..T ..C ...... T Opt: GTT GCG CTG GGT CGT GAA CTG CTG CGT AAC CCG TAT TGG GTT CTG CAC

197

965 970 975 980 985 990 995 1000 1005 ORF: T Y T S K E D W P K Q Y E R A F Org: ... ..C ..T TCA ..G ...... A ... ..A ..T ... A.A ..T ..T Opt: ACC TAT ACC AGC AAA GAA GAC TGG CCG AAA CAG TAC GAA CGT GCC TTC

1010 1015 1020 1025 1030 1035 1040 ORF: K K L E H H H H H H ! Org: ...... Opt: AAA AAA CTC GAG CAC CAC CAC CAC CAC CAC TGA XhoI

A.2 Codon Usage This is a comparison of the codon frequency of the original sequence vs. the optimised sequence. As a reference, the codon usage of the target organism (E. coli) is included.

GGG 9.10% 0.00% 0.00% A.2.1 A - Alanine: Codon Original Target Optimised A.2.7 H - Histidine: GCU 48.00% 28.00% 28.00% Codon Original Target Optimised GCC 8.00% 16.00% 16.00% CAU 46.70% 30.00% 33.30% GCA 40.00% 24.00% 24.00% CAC 53.30% 70.00% 66.70% GCG 4.00% 32.00% 32.00% A.2.8 I - Isoleucine: A.2.2 C - Cysteine: Codon Original Target Optimised Codon Original Target Optimised AUU 36.40% 33.00% 21.20% UGU 100.00% 39.00% 25.00% AUC 12.10% 67.00% 78.80% UGC 0.00% 61.00% 75.00% AUA 51.50% 0.00% 0.00%

A.2.3 D - Aspartic acid: A.2.9 K - Lysine: Codon Original Target Optimised Codon Original Target Optimised GAU 66.70% 46.00% 47.60% AAA 85.20% 100.00% 100.00% GAC 33.30% 54.00% 52.40% AAG 14.80% 0.00% 0.00% A.2.4 E - Glutamic acid: A.2.10 L - Leucine: Codon Original Target Optimised Codon Original Target Optimised GAA 81.50% 75.00% 74.10% UUA 46.20% 0.00% 0.00% GAG 18.50% 25.00% 25.90% UUG 0.00% 0.00% 0.00% CUU 42.30% 0.00% 0.00% A.2.5 F - Phenylalanine: CUC 3.80% 8.00% 7.70% Codon Original Target Optimised CUA 3.80% 0.00% 0.00% UUU 80.00% 28.00% 20.00% CUG 3.80% 92.00% 92.30% UUC 20.00% 72.00% 80.00% A.2.6 G - Glycine: A.2.11 M - Methionine: Codon Original Target Optimised Codon Original Target Optimised GGU 9.10% 57.00% 81.80% AUG 100.00% 100.00% 100.00% GGC 22.70% 43.00% 18.20% GGA 59.10% 0.00% 0.00% 198

A.2.12 N - Asparagine: GUC 4.20% 13.00% 12.50% Codon Original Target Optimised GUA 37.50% 20.00% 20.80% AAU 83.30% 0.00% 0.00% GUG 12.50% 27.00% 25.00% AAC 16.70% 100.00% 100.00%

A.2.13 P - Proline: A.2.19 W - Tryptophan: Codon Original Target Optimised Codon Original Target Optimised CCU 66.70% 0.00% 0.00% UGG 100.00% 100.00% 100

CCC 8.30% 0.00% 0.00% CCA 25.00% 15.00% 16.70% A.2.20 Y - Tyrosine: CCG 0.00% 85.00% 83.30% Codon Original Target Optimised A.2.14 Q - Glutamine: UAU 46.70% 35.00% 33.30% UAC 53.30% 65.00% 66.70% Codon Original Target Optimised CAA 83.30% 0.00% 0.00% CAG 16.70% 100.00% 100.00% A.2.21 ! - Stop: Codon Original Target Optimised A.2.15 R - Arginine: UAA 0.00% 72.00% 0.00% Codon Original Target Optimised UGA 100.00% 28.00% 100.00% CGU 0.00% 67.00% 77.80% UAG 0.00% 0.00% 0.00% CGC 11.10% 33.00% 22.20% CGA 5.60% 0.00% 0.00% CGG 5.60% 0.00% 0.00%

AGA 61.10% 0.00% 0.00% AGG 16.70% 0.00% 0.00%

A.2.16 S - Serine: Codon Original Target Optimised UCU 33.30% 49.00% 50.00% UCC 11.10% 27.00% 27.80% UCA 27.80% 0.00% 0.00% UCG 0.00% 0.00% 0.00% AGU 11.10% 0.00% 0.00% AGC 16.70% 24.00% 22.20%

A.2.17 T - Threonine: Codon Original Target Optimised ACU 27.30% 28.00% 27.30% ACC 9.10% 57.00% 54.50% ACA 54.50% 0.00% 0.00% ACG 9.10% 15.00% 18.20%

A.2.18 V - Valine: Codon Original Target Optimised GUU 45.80% 40.00% 41.70% 199

A.3 Optimised sequence

The final gene sequence of the optimised TOYE gene (including the His6-tag).

CATAT GAGCA TTCTG CACAT GCCGC TGAAA ATCAA AGACA TCACC ATCAA AAACC GCATC ATGAT GTCCC CGATG TGCAT GTATT CTGCC TCTAC CGATG GTATG CCAAA CGACT GGCAT ATCGT TCACT ATGCG ACTCG TGCGA TCGGT GGTGT AGGTC TGATT ATGCA GGAGG CTACT GCTGT TGAAT CTCGT GGTCG TATTA CGGAT CACGA TCTGG GTATC TGGAA CGACG AACAG GTCAA AGAGC TGAAA AAAAT CGTTG ACATC TGCAA AGCCA ACGGT GCAGT TATGG GTATT CAGCT GGCGC ATGCA GGTCG CAAAT GTAAC ATCTC CTACG AAGAT GTCGT TGGTC CGTCT CCGAT TAAAG CAGGT GACCG TTACA AACTG CCGCG TGAAC TGTCC GTTGA GGAAA TCAAA AGCAT CGTGA AAGCG TTTGG TGAGG CTGCG AAACG TGCTA ACCTG GCTGG TTACG ACGTT GTAGA AATCC ATGCG GCACA TGGTT ACCTG ATCCA TGAAT TCCTG TCTCC GCTGT CCAAC AAACG TAAAG ACGAG TACGG CAACT CCATT GAAAA CCGTG CTCGT TTCCT GATCG AAGTG ATCGA TGAAG TGCGC AAAAA CTGGC CGGAA AACAA ACCAA TCTTC GTTCG TGTGT CTGCG GATGA TTATA TGGAG GGCGG CATTA ACATC GACAT GATGG TGGAA TACAT CAACA TGATC AAAGA CAAAG TAGAT CTGAT CGATG TCAGC TCTGG CGGTC TGCTG AACGT AGATA TCAAC CTGTA CCCGG GTTAC CAGGT GAAAT ACGCC GAAAC GATCA AAAAA CGCTG CAACA TCAAA ACCTC TGCAG TAGGT CTGAT CACTA CCCAG GAACT GGCAG AAGAA ATCCT CTCTA ACGAA CGTGC TGACC TGGTT GCGCT GGGTC GTGAA CTGCT GCGTA ACCCG TATTG GGTTC TGCAC ACCTA TACCA GCAAA GAAGA CTGGC CGAAA CAGTA CGAAC GTGCC TTCAA AAAAC TCGAG CACCA CCACC ACCAC CACTG A

200

A.4 Vector map of pET-21b

pET-21a(+) sequence landmarks f1 origin T7 promoter 311-327 Multiple Cloning T7 transcription start 310 Region T7•Tag coding sequence 207-239 bal coding Multiple cloning sites sequence (BamH I - Xho I) 158-203 pET-21b His•Tag coding sequence 140-157 T7 terminator 26-72 lacI coding sequence 714-1793 lacI coding pBR322 origin 3227 ori bla coding sequence 3988-4845 sequence f1 origin 4977-5432

The expression plasmid which TOYE was inserted into, via Nde I and Xho I restriction sites, for growth and expression.

pET21b (+) Multiple Cloning site region (158-203)

Bgl II T7 promoter lac operator AGATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCC … Xba I rbs Nde I Nhe I T7 tag …CCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGGCTAGCATGACTGGTG … Eag I Ava I T7 tag BamH I EcoR I Sac I Sal I Hind III Not I Xho I …GACAGCAAATGGGTCGCGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGA … His Tag …GCACCACCACCACCACCACTGAGGTCGGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGG … His Tag End …CCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAA … Bpu1102 I T7 terminator …GCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACG G… T7 terminator …GTCTTGAGGGGTTTTTTG

201

Appendix B The gene of TOYE was sequenced forwards and backwards using T7 and T7 terminal primers to verify that the gene was only mutated at the desired location of the new stop codon.

B.1 Gene Sequences

>TOYE-His6 CATATGAGCATTCTGCACATGCCGCTGAAAATCAAAGACATCACCATCAAAAACCGCATCATGATGTCCCCGA TGTGCATGTATTCTGCCTCTACCGATGGTATGCCAAACGACTGGCATATCGTTCACTATGCGACTCGTGCGAT CGGTGGTGTAGGTCTGATTATGCAGGAGGCTACTGCTGTTGAATCTCGTGGTCGTATTACGGATCACGATCTG GGTATCTGGAACGACGAACAGGTCAAAGAGCTGAAAAAAATCGTTGACATCTGCAAAGCCAACGGTGCAGTTA TGGGTATTCAGCTGGCGCATGCAGGTCGCAAATGTAACATCTCCTACGAAGATGTCGTTGGTCCGTCTCCGAT TAAAGCAGGTGACCGTTACAAACTGCCGCGTGAACTGTCCGTTGAGGAAATCAAAAGCATCGTGAAAGCGTTT GGTGAGGCTGCGAAACGTGCTAACCTGGCTGGTTACGACGTTGTAGAAATCCATGCGGCACATGGTTACCTGA TCCATGAATTCCTGTCTCCGCTGTCCAACAAACGTAAAGACGAGTACGGCAACTCCATTGAAAACCGTGCTCG TTTCCTGATCGAAGTGATCGATGAAGTGCGCAAAAACTGGCCGGAAAACAAACCAATCTTCGTTCGTGTGTCT GCGGATGATTATATGGAGGGCGGCATTAACATCGACATGATGGTGGAATACATCAACATGATCAAAGACAAAG TAGATCTGATCGATGTCAGCTCTGGCGGTCTGCTGAACGTAGATATCAACCTGTACCCGGGTTACCAGGTGAA ATACGCCGAAACGATCAAAAAACGCTGCAACATCAAAACCTCTGCAGTAGGTCTGATCACTACCCAGGAACTG GCAGAAGAAATCCTCTCTAACGAACGTGCTGACCTGGTTGCGCTGGGTCGTGAACTGCTGCGTAACCCGTATT GGGTTCTGCACACCTATACCAGCAAAGAAGACTGGCCGAAACAGTACGAACGTGCCTTCAAAAAACTCGAGCA CCACCACCACCACCACTGA

> TOYE_T7 -- 29..763 of sequence TAATTTTGTTTAACTTTAAGAAGGAGATATACATATGAGCATTCTGCACATGCCGCTGAAAATCAAAGA CATCACCATCAAAAACCGCATCATGATGTCCCCGATGTGCATGTATTCTGCCTCTACCGATGGTATGCC AAACGACTGGCATATCGTTCACTATGCGACTCGTGCGATCGGTGGTGTAGGTCTGATTATGCAGGAGGC TACTGCTGTTGAATCTCGTGGTCGTATTACGGATCACGATCTGGGTATCTGGAACGACGAACAGGTCAA AGAGCTGAAAAAAATCGTTGACATCTGCAAAGCCAACGGTGCAGTTATGGGTATTCAGCTGGCGCATGC AGGTCGCAAATGTAACATCTCCTACGAAGATGTCGTTGGTCCGTCTCCGATTAAAGCAGGTGACCGTTA CAAACTGCCGCGTGAACTGTCCGTTGAGGAAATCAAAAGCATCGTGAAAGCGTTTGGTGAGGCTGCGAA ACGTGCTAACCTGGCTGGTTACGACGTTGTAGAAATCCATGCGGCACATGGTTACCTGATCCATGAATT CCTGTCTCCGCTGTCCAACAAACGTAAAGACGAGTACGGCAACTCCATTGAAAACCGTGCTCGTTTCCT GATCGAAGTGATCGATGAAGTGCGCAAAAACTGGCCGGAAAACAAACCAATCTTCGTTCGTGTGTCTGC GGATGATTATATGGAGGGCGGCATTAACATCGACATGATGGGTGG

> TOYE_T7term -- 9..1043 of sequence CTTCTTTCGGGCTTTGTTAGCAGCCGGATCTCAGTGGTGGTGGTGGTGGTGCTCTCATTTTTTGAAGGCACGT TCGTACTGTTTCGGCCAGTCTTCTTTGCTGGTATAGGTGTGCAGAACCCAATACGGGTTACGCAGCAGTTCAC GACCCAGCGCAACCAGGTCAGCACGTTCGTTAGAGAGGATTTCTTCTGCCAGTTCCTGGGTAGTGATCAGACC TACTGCAGAGGTTTTGATGTTGCAGCGTTTTTTGATCGTTTCGGCGTATTTCACCTGGTAACCCGGGTACAGG TTGATATCTACGTTCAGCAGACCGCCAGAGCTGACATCGATCAGATCTACTTTGTCTTTGATCATGTTGATGT ATTCCACCATCATGTCGATGTTAATGCCGCCCTCCATATAATCATCCGCAGACACACGAACGAAGATTGGTTT GTTTTCCGGCCAGTTTTTGCGCACTTCATCGATCACTTCGATCAGGAAACGAGCACGGTTTTCAATGGAGTTG CCGTACTCGTCTTTACGTTTGTTGGACAGCGGAGACAGGAATTCATGGATCAGGTAACCATGTGCCGCATGGA TTTCTACAACGTCGTAACCAGCCAGGTTAGCACGTTTCGCAGCCTCACCAAACGCTTTCACGATGCTTTTGAT TTCCTCAACGGACAGTTCACGCGGCAGTTTGTAACGGTCACCTGCTTTAATCGGAGACGGACCAACGACATCT TCGTAGGAGATGTTACATTTGCGACCTGCATGCGCCAGCTGAATACCCATAACTGCACCGTTGGCTTTGCAGA TGTCAACGATTTTTTTCAGCTCTTTGACCTGTTCGTCGTTCCAGATACCCAGATCGTGATCCGTAATACGACC ACGAGATTCAACAGCAGTAGCCTCCTGCATAATCAGACCTACACCACCGATCGCACGAGTCGCATAGTGAACG ATATGCCAGTCGTTTGGCATACCATCGGTAGAGGCAGAATACATGCACATCGGGGACATCATGATGCGGTTTT TGATGGTGATGTT

202

> TOYE_T7term RC

AACATCACCATCAAAAACCGCATCATGATGTCCCCGATGTGCATGTATTCTGCCTCTACCGATGGTATGCCAA ACGACTGGCATATCGTTCACTATGCGACTCGTGCGATCGGTGGTGTAGGTCTGATTATGCAGGAGGCTACTGC TGTTGAATCTCGTGGTCGTATTACGGATCACGATCTGGGTATCTGGAACGACGAACAGGTCAAAGAGCTGAAA AAAATCGTTGACATCTGCAAAGCCAACGGTGCAGTTATGGGTATTCAGCTGGCGCATGCAGGTCGCAAATGTA ACATCTCCTACGAAGATGTCGTTGGTCCGTCTCCGATTAAAGCAGGTGACCGTTACAAACTGCCGCGTGAACT GTCCGTTGAGGAAATCAAAAGCATCGTGAAAGCGTTTGGTGAGGCTGCGAAACGTGCTAACCTGGCTGGTTAC GACGTTGTAGAAATCCATGCGGCACATGGTTACCTGATCCATGAATTCCTGTCTCCGCTGTCCAACAAACGTA AAGACGAGTACGGCAACTCCATTGAAAACCGTGCTCGTTTCCTGATCGAAGTGATCGATGAAGTGCGCAAAAA CTGGCCGGAAAACAAACCAATCTTCGTTCGTGTGTCTGCGGATGATTATATGGAGGGCGGCATTAACATCGAC ATGATGGTGGAATACATCAACATGATCAAAGACAAAGTAGATCTGATCGATGTCAGCTCTGGCGGTCTGCTGA ACGTAGATATCAACCTGTACCCGGGTTACCAGGTGAAATACGCCGAAACGATCAAAAAACGCTGCAACATCAA AACCTCTGCAGTAGGTCTGATCACTACCCAGGAACTGGCAGAAGAAATCCTCTCTAACGAACGTGCTGACCTG GTTGCGCTGGGTCGTGAACTGCTGCGTAACCCGTATTGGGTTCTGCACACCTATACCAGCAAAGAAGACTGGC CGAAACAGTACGAACGTGCCTTCAAAAAATGAGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAA AGCCCGAAAGAAG

B.2 Sequence Alignments Forward Sequencing from T7:

TOYE ------CATATGAGCATTCTGCACATGCCGCTGAA 29 TOYE_T7 TAATTTTGTTTAACTTTAAGAAGGAGATATACATATGAGCATTCTGCACATGCCGCTGAA 60 *****************************

TOYE AATCAAAGACATCACCATCAAAAACCGCATCATGATGTCCCCGATGTGCATGTATTCTGC 89 TOYE_T7 AATCAAAGACATCACCATCAAAAACCGCATCATGATGTCCCCGATGTGCATGTATTCTGC 120 ************************************************************

TOYE CTCTACCGATGGTATGCCAAACGACTGGCATATCGTTCACTATGCGACTCGTGCGATCGG 149 TOYE_T7 CTCTACCGATGGTATGCCAAACGACTGGCATATCGTTCACTATGCGACTCGTGCGATCGG 180 ************************************************************

TOYE TGGTGTAGGTCTGATTATGCAGGAGGCTACTGCTGTTGAATCTCGTGGTCGTATTACGGA 209 TOYE_T7 TGGTGTAGGTCTGATTATGCAGGAGGCTACTGCTGTTGAATCTCGTGGTCGTATTACGGA 240 ************************************************************

TOYE TCACGATCTGGGTATCTGGAACGACGAACAGGTCAAAGAGCTGAAAAAAATCGTTGACAT 269 TOYE_T7 TCACGATCTGGGTATCTGGAACGACGAACAGGTCAAAGAGCTGAAAAAAATCGTTGACAT 300 ************************************************************

TOYE CTGCAAAGCCAACGGTGCAGTTATGGGTATTCAGCTGGCGCATGCAGGTCGCAAATGTAA 329 TOYE_T7 CTGCAAAGCCAACGGTGCAGTTATGGGTATTCAGCTGGCGCATGCAGGTCGCAAATGTAA 360 ************************************************************

TOYE CATCTCCTACGAAGATGTCGTTGGTCCGTCTCCGATTAAAGCAGGTGACCGTTACAAACT 389 TOYE_T7 CATCTCCTACGAAGATGTCGTTGGTCCGTCTCCGATTAAAGCAGGTGACCGTTACAAACT 420 ************************************************************

TOYE GCCGCGTGAACTGTCCGTTGAGGAAATCAAAAGCATCGTGAAAGCGTTTGGTGAGGCTGC 449 TOYE_T7 GCCGCGTGAACTGTCCGTTGAGGAAATCAAAAGCATCGTGAAAGCGTTTGGTGAGGCTGC 480 ************************************************************

TOYE GAAACGTGCTAACCTGGCTGGTTACGACGTTGTAGAAATCCATGCGGCACATGGTTACCT 509 TOYE_T7 GAAACGTGCTAACCTGGCTGGTTACGACGTTGTAGAAATCCATGCGGCACATGGTTACCT 540 ************************************************************

TOYE GATCCATGAATTCCTGTCTCCGCTGTCCAACAAACGTAAAGACGAGTACGGCAACTCCAT 569 TOYE_T7 GATCCATGAATTCCTGTCTCCGCTGTCCAACAAACGTAAAGACGAGTACGGCAACTCCAT 600 ************************************************************

203

TOYE TGAAAACCGTGCTCGTTTCCTGATCGAAGTGATCGATGAAGTGCGCAAAAACTGGCCGGA 629 TOYE_T7 TGAAAACCGTGCTCGTTTCCTGATCGAAGTGATCGATGAAGTGCGCAAAAACTGGCCGGA 660 ************************************************************

TOYE AAACAAACCAATCTTCGTTCGTGTGTCTGCGGATGATTATATGGAGGGCGGCATTAACAT 689 TOYE_T7 AAACAAACCAATCTTCGTTCGTGTGTCTGCGGATGATTATATGGAGGGCGGCATTAACAT 720 ************************************************************

TOYE CGACATGATGGTGGAATACATCAACATGATCAAAGACAAAGTAGATCTGATCGATGTCAG 749 TOYE_T7 CGACATGATGGGTGG------735 *********** *

Backward sequencing from T7 terminal:

TOYE-His6 CATATGAGCATTCTGCACATGCCGCTGAAAATCAAAGACATCACCATCAAAAACCGCATC 60 TOYE_T7term ------AACATCACCATCAAAAACCGCATC 24 ***********************

TOYE-His6 ATGATGTCCCCGATGTGCATGTATTCTGCCTCTACCGATGGTATGCCAAACGACTGGCAT 120 TOYE_T7term ATGATGTCCCCGATGTGCATGTATTCTGCCTCTACCGATGGTATGCCAAACGACTGGCAT 84 ************************************************************

TOYE-His6 ATCGTTCACTATGCGACTCGTGCGATCGGTGGTGTAGGTCTGATTATGCAGGAGGCTACT 180 TOYE_T7term ATCGTTCACTATGCGACTCGTGCGATCGGTGGTGTAGGTCTGATTATGCAGGAGGCTACT 144 ************************************************************

TOYE-His6 GCTGTTGAATCTCGTGGTCGTATTACGGATCACGATCTGGGTATCTGGAACGACGAACAG 240 TOYE_T7term GCTGTTGAATCTCGTGGTCGTATTACGGATCACGATCTGGGTATCTGGAACGACGAACAG 204 ************************************************************

TOYE-His6 GTCAAAGAGCTGAAAAAAATCGTTGACATCTGCAAAGCCAACGGTGCAGTTATGGGTATT 300 TOYE_T7term GTCAAAGAGCTGAAAAAAATCGTTGACATCTGCAAAGCCAACGGTGCAGTTATGGGTATT 264 ************************************************************

TOYE-His6 CAGCTGGCGCATGCAGGTCGCAAATGTAACATCTCCTACGAAGATGTCGTTGGTCCGTCT 360 TOYE_T7term CAGCTGGCGCATGCAGGTCGCAAATGTAACATCTCCTACGAAGATGTCGTTGGTCCGTCT 324 ************************************************************

TOYE-His6 CCGATTAAAGCAGGTGACCGTTACAAACTGCCGCGTGAACTGTCCGTTGAGGAAATCAAA 420 TOYE_T7term CCGATTAAAGCAGGTGACCGTTACAAACTGCCGCGTGAACTGTCCGTTGAGGAAATCAAA 384 ************************************************************

TOYE-His6 AGCATCGTGAAAGCGTTTGGTGAGGCTGCGAAACGTGCTAACCTGGCTGGTTACGACGTT 480 TOYE_T7term AGCATCGTGAAAGCGTTTGGTGAGGCTGCGAAACGTGCTAACCTGGCTGGTTACGACGTT 444 ************************************************************

TOYE-His6 GTAGAAATCCATGCGGCACATGGTTACCTGATCCATGAATTCCTGTCTCCGCTGTCCAAC 540 TOYE_T7term GTAGAAATCCATGCGGCACATGGTTACCTGATCCATGAATTCCTGTCTCCGCTGTCCAAC 504 ************************************************************

TOYE-His6 AAACGTAAAGACGAGTACGGCAACTCCATTGAAAACCGTGCTCGTTTCCTGATCGAAGTG 600 TOYE_T7term AAACGTAAAGACGAGTACGGCAACTCCATTGAAAACCGTGCTCGTTTCCTGATCGAAGTG 564 ************************************************************

TOYE-His6 ATCGATGAAGTGCGCAAAAACTGGCCGGAAAACAAACCAATCTTCGTTCGTGTGTCTGCG 660 TOYE_T7term ATCGATGAAGTGCGCAAAAACTGGCCGGAAAACAAACCAATCTTCGTTCGTGTGTCTGCG 624 ************************************************************

TOYE-His6 GATGATTATATGGAGGGCGGCATTAACATCGACATGATGGTGGAATACATCAACATGATC 720 TOYE_T7term GATGATTATATGGAGGGCGGCATTAACATCGACATGATGGTGGAATACATCAACATGATC 684 ************************************************************

TOYE-His6 AAAGACAAAGTAGATCTGATCGATGTCAGCTCTGGCGGTCTGCTGAACGTAGATATCAAC 780 TOYE_T7term AAAGACAAAGTAGATCTGATCGATGTCAGCTCTGGCGGTCTGCTGAACGTAGATATCAAC 744 ************************************************************

204

TOYE-His6 CTGTACCCGGGTTACCAGGTGAAATACGCCGAAACGATCAAAAAACGCTGCAACATCAAA 840 TOYE_T7term CTGTACCCGGGTTACCAGGTGAAATACGCCGAAACGATCAAAAAACGCTGCAACATCAAA 804 ************************************************************

TOYE-His6 ACCTCTGCAGTAGGTCTGATCACTACCCAGGAACTGGCAGAAGAAATCCTCTCTAACGAA 900 TOYE_T7term ACCTCTGCAGTAGGTCTGATCACTACCCAGGAACTGGCAGAAGAAATCCTCTCTAACGAA 864 ************************************************************

TOYE-His6 CGTGCTGACCTGGTTGCGCTGGGTCGTGAACTGCTGCGTAACCCGTATTGGGTTCTGCAC 960 TOYE_T7term CGTGCTGACCTGGTTGCGCTGGGTCGTGAACTGCTGCGTAACCCGTATTGGGTTCTGCAC 924 ************************************************************

TOYE-His6 ACCTATACCAGCAAAGAAGACTGGCCGAAACAGTACGAACGTGCCTTCAAAAAACTCGAG 1020 TOYE_T7term ACCTATACCAGCAAAGAAGACTGGCCGAAACAGTACGAACGTGCCTTCAAAAAATGAGAG 984 ****************************************************** ***

TOYE-His6 CACCACCACCACCACCACTGA------1041 TOYE_T7term CACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGAAG 1035 *********************

205

Appendix C

C.1 Potentiometry titrations 1.0 1.0

0.8 0.8

0.6 0.6

0.4 0.4

Absorbance Absorbance 0.2 0.2

0.0 0.0 300 400 500 600 300 400 500 600 Wavelength (nm) Wavelength (nm)

1.0 1.0

0.8 0.8

0.6 0.6

0.4 0.4

Absorbance Absorbance 0.2 0.2

0.0 0.0 300 400 500 600 300 400 500 600 Wavelength (nm) Wavelength (nm)

1.0 1.0

0.8 0.8

0.6 0.6

0.4 0.4

Absorbance Absorbance

0.2 0.2

0.0 0.0 300 400 500 600 300 400 500 600 Wavelength (nm) Wavelength (nm)

Figure B.1. Redox potentiometric titration of TOYE. Spectral changes during reductive titration of TOYE with sodium dithionite at A) 6 °C, B) 10 °C, C) 15 °C, D) 20 °C, E) 25 °C and F) 30 °C in 50 mM potassium phosphate buffer, pH 7.0.

206

100 100

80 80

60 60

40 40

20 20 PercentageOxidised PercentageOxidised 0 0

-400 -300 -200 -100 -400 -300 -200 -100 Potential versus hydrogen electrode (mV) Potential versus hydrogen electrode (mV)

100 100

80 80

60 60

40 40

20 20 PercentageOxidised PercentageOxidised 0 0

-400 -300 -200 -100 -400 -300 -200 -100 Potential versus hydrogen electrode (mV) Potential versus hydrogen eletrode (mV)

100 100

80 80

60 60

40 40

20 20 PercentageOxidised PercentageOxidised 0 0

-400 -350 -300 -250 -200 -150 -100 -300 -250 -200 -150 -100 Potential versus hydrogen eletrode (mV) Potential versus hydrogen eletrode (mV)

Figure B.2. Redox potentiometric titration of TOYE. Absorbance changes at 456 nm as a function of observed potential for redox potentiometric titrations of TOYE with sodium dithionite at A) 6 °C, B) 10 °C, C) 15 °C, D) 20 °C, E) 25 °C and F) 30 °C. Starting and final absorbance values were used as 100% and 0%, respectively, for the application of the Nernst equation.

207

Appendix D

D.1 Rejected hydrodynamic bead models

Below are shown the models of the quaternary structure of TOYE that did not fit the hydrodynamic data from Chapter 4.5. The bead models of the quaternary structures for TOYE were generated using the PDB file and the SOMO solution bead modelling program as described in Chapter 2.10.

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