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Title: Investigating nicotinamide cofactor promiscuity in a new class of flavoprotein monooxygenases
Author: Jensen-Loughrey, Chantel N.
ISNI:       0000 0004 5348 6198
Awarding Body: University of York
Current Institution: University of York
Date of Award: 2014
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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.
Supervisor: Grogan, Gideon Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available