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Investigating the catalytic mechanism if Class B flavin-dependent monooxygenases

University of British Columbia

Baeyer-Villiger monooxygenases (BVMOs) and N-hydroxylating monooxygenases (NMO) belong to the Class-B flavin-dependent monooxygenases family of enzymes. Members of this family use NADPH, O₂ and an FAD cofactor to form a reactive C4a-(hydro)peroxyflavin intermediate for selective substrate oxidation. BVMOs are potential biocatalysts as they can catalyze Baeyer-Villiger oxidations, N-hydroxylations and sulfoxidations with exquisite chemo-, regio- and enantioselectivity. NMOs are involved in the biosynthesis of siderophores, having the potential to serve as drug targets for treatment of microbial diseases. Cyclohexanone monooxygenase (CHMO) from Acinetobacter sp. NCIMB 9871 serves as the prototypic member of BVMOs. In Chapters 2 and 3, it was used as a model system to investigate the role of active site residues in mediating discrete steps of the BVMO catalytic cycle. T187 and W490, which hydrogen bond to the phosphate and ribose of the nicotinamide mononucleotide half of NADP(H), respectively, were mutated to alanine and phenylalanine. Through a combination of steady-state kinetic studies, stopped-flow spectrophotometry, and product analysis, T187 was shown to be critical for locking NADP⁺ in a conformation that promotes oxygen activation by the reduced flavin. Alternatively, W490 facilitates the reaction of the C4a-peroxyflavin intermediate with cyclohexanone through stabilization of the Criegee intermediate. Kinetic isotope effects, molecular modelling and ¹H-NMR analysis revealed that the invariant R327 active site residue single-handedly controls the stereospecificity of NADPH hydride transfer, while D57 mediates substrate entry into the active site. In Chapter 4, the catalytic mechanism of acetone monooxygenase, a BVMO with the potential to serve as an acetone biosensor, was investigated. Based on our kinetic analysis, ACMO has a broad substrate specificity and a weak binding affinity for NADPH, resulting in a high rate of uncoupled NADPH oxidation leading to excess H₂O₂ production. In Chapter 5, the catalytic mechanism of cadaverine monooxygenase (DfoA), an NMO from the fire blight pathogen, Erwinia amylovora was investigated. DfoA has a narrow substrate specificity and a very high dissociation constant for NADPH. Hence, DfoA was not able to stabilize the C4a-hydroperoxyflavin intermediate. It is hoped that the strict substrate specificity elicited by DfoA can be exploited in designing new compounds to control E. amylovora virulence.

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2019-07-31

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/71164

Biochemistry and Molecular Biology

University of British Columbia

UBCO

http://creativecommons.org/licenses/by-nc-nd/4.0/