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Investigating Nicotinamide Cofactor Promiscuity in a New Class of Flavoprotein Monooxygenases

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Investigating Nicotinamide Cofactor Promiscuity in a New Class of Flavoprotein Monooxygenases Investigating Nicotinamide Cofactor Promiscuity in a New Class of Flavoprotein Monooxygenases Chantel Nixon Jensen-Loughrey Doctor of Philosophy The University of York Department of Chemistry August 2014 Abstract This thesis concerns the investigation into the structure and function of the 38.6 kDa FAD-containing flavoprotein Stenotrophomonas maltophilia flavin-containing monooxygenase, SMFMO, which was encoded from a gene from the marine bacterium Stenotrophomonas maltophilia. The enzyme was found to catalyse the asymmetric oxidation of prochiral sulfides and the regioselective Baeyer-Villiger oxidation of bicyclo[3.2.0]hept-2-en-6-one. SMFMO was unusual amongst FPMOs as it demonstrated an ability to employ either NADH or NADPH as nicotinamide cofactor in order to reduce the flavin for catalysis. In an effort to determine the residues responsible for the cofactor promiscuity of SMFMO the structure of SMFMO was determined and revealed that the cofactor promiscuity of SMFMO may be due to the substitution of an arginine residue, responsible for the recognition of the 2’-phosphate on the NADPH ribose in related NADPH dependent FMOs, with a glutamine residue in SMFMO. In an attempt to explore the cofactor determinants in SMFMO, the two residues Gln193 and His194 in the cofactor binding site of SMFMO were mutated in order to mimic the cofactor binding site of the NADPH-dependent FMO, mFMO, from Methylophaga aminisulfidivorans sp. SK1, in which structurally homologous residues Arg234 and Thr235 bind the 2’-phosphate on NADPH. mFMO possesses an asparagine residue which is thought to be involved in the stabilisation of the flavin hydroperoxide intermediate, in SMFMO this residue is replaced by Phe52. Mutation of the Phe52 residue revealed that this residue is a determinant in enantioselectivity. The natural variants of SMFMO, PFMO from Pseudomonas stutzeri and CFMO from Cellvibrio sp., also had the ability to use both nicotinamide cofactors equally to reduce the flavin. The structure of PFMO revealed that the residues Gln194 and Glu195, structurally homologous to Gln193 and His194 in SMFMO, were orientated away from the 2’- phosphate site and thus the Glu195 would not repel the negatively charged phosphate as originally thought. i ii Table of Contents Abstract .............................................................................................................................. i Table of Contents ............................................................................................................. iii List of Figures .................................................................................................................. ix List of Tables................................................................................................................... xv Acknowledgements ....................................................................................................... xvii Declaration ..................................................................................................................... xix Chapter 1: Introduction ..................................................................................................... 1 1.1 Enzymes within chemistry ...................................................................................... 1 1.1.1 The history of enzymes ..................................................................................... 1 1.1.2 Biocatalysts within organic chemistry .............................................................. 1 1.2 Biocatalysis using monooxygenases ....................................................................... 4 1.3 Flavoprotein monooxygenases ................................................................................ 5 1.3.1 Introduction ....................................................................................................... 5 1.3.2 The mechanism of a FPMO .............................................................................. 6 1.3.3 Cofactors and their roles ................................................................................... 9 1.3.4 Classification of FPMOs ................................................................................. 12 1.4 Class B flavoprotein monooxygenases .................................................................. 14 1.4.1 Baeyer-Villiger monooxygenases ................................................................... 14 1.4.2 Flavin monooxygenases .................................................................................. 22 1.4.3 N-hydroxylating monooxygenases ................................................................. 26 1.4.4 The drawback of class B FPMOs ................................................................... 27 1.5 Applications of class B FPMOs ............................................................................ 28 Chapter 2: Preliminary Investigation of SMFMO .......................................................... 31 2.1 Identification and isolation of SMFMO gene ........................................................ 31 iii 2.2 Cloning, expression and purification..................................................................... 32 2.3 Enzyme assays ....................................................................................................... 33 2.4 Crystallisation and structure determination ........................................................... 34 2.5 PhD project Aims .................................................................................................. 35 Chapter 3: Methods ......................................................................................................... 37 3.1 Gene Cloning ......................................................................................................... 37 3.1.1 Polymerase chain reaction (PCR) ................................................................... 37 3.1.2 Ligation independent cloning (LIC) ............................................................... 40 3.1.3 Agarose gel electrophoresis ............................................................................ 42 3.1.4 LIC T4 polymerase reactions .......................................................................... 44 3.1.5 LIC annealing reaction ................................................................................... 44 3.1.6 Transformation and miniprep ......................................................................... 44 3.1.7 Restriction Digest ........................................................................................... 45 3.2 Protein Production ................................................................................................. 45 3.2.1 Transformation with SMFMO plasmid .......................................................... 45 3.2.2 Preparation of starter culture .......................................................................... 46 3.2.3 Small scale expression tests ............................................................................ 46 3.2.4 Sodium dodecyl sulphate-polyacrylamide electrophoresis (SDS-PAGE) ...... 46 3.2.5 Large scale gene expression ........................................................................... 48 3.3 Protein purification ................................................................................................ 49 3.3.1 Cell lysis by sonication ................................................................................... 50 2+ 3.3.2 Nickel (Ni ) affinity chromatography ........................................................... 50 3.3.3 Size exclusion chromatography (SEC) ........................................................... 52 3.3.4 His6-tag cleavage ............................................................................................ 53 3.4 Enzyme assays ....................................................................................................... 53 3.4.1 Spectrophotometric assays.............................................................................. 54 iv 3.4.2 NADH/NADPH oxidation assays ................................................................... 56 3.4.3 Bicyclo[3.2.0]hept-2-en-6-one (substrate 1) oxidation assays ....................... 56 3.4.4 Biotransformations.......................................................................................... 58 3.4.5 Gas chromatography analysis ......................................................................... 60 3.4.6 Gas chromatography-mass spectrometry (GC-MS) ....................................... 62 3.4.7 Cyclohexanone monooxygenase (CHMO) –Assignment of configuration. ... 63 3.5 Mutagenesis ........................................................................................................... 64 3.5.1 Site-directed mutagenesis (SDM) ................................................................... 64 3.5.2 Site saturation mutagenesis (SSM) ................................................................. 68 3.6 Chemical synthesis of sulfoxides .......................................................................... 70 3.7 Protein crystallisation ............................................................................................ 71 Chapter 4: Characterisation of SMFMO ......................................................................... 73 4.1 Introduction ........................................................................................................... 73 4.2 Aims .....................................................................................................................
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