Active site variants provide insight into the nature of conformational changes that accompany the cyclohexanone monooxygenase catalytic cycle.

Baeyer-Villiger monooxygenases are flavoenzymes that use NADPH and O2 to convert ketones to esters or lactones. A diagnostic feature of BVMO catalysis is the dual role of the pyridine nucleotide: NADPH functions as a reductant of the FAD cofactor and the resulting NADP+ acts to stabilize the ensuing C4a-peroxyflavin intermediate. Using cyclohexanone monooxygenase from Acinetobacter sp. NCIMB 9871 as a model system, we investigated the catalytic role of T187 and W490, which hydrogen bond to the phosphate and ribose of the nicotinamide mononucleotide half of NADP(H), respectively. Eliminating either hydrogen bond through creation of a T187A or a W490F variant leads to a 15-fold reduction in turnover of cyclohexanone. Substitution of either residue does not affect the rate of FAD reduction or the coupling efficiency. Rather, T187A and W490F disrupt distinct steps of the oxidative half-reaction. Kinetic and spectroscopic analysis of T187A reveals that this residue is critical for locking NADP+ in a configuration that dramatically accelerates O2 activation by the reduced flavin. W490 also promotes O2 activation (albeit less so than T187) and accelerates the reaction between the C4a-peroxyflavin and cyclohexanone. The results provide insight into the conformation of CHMO and the coenzyme for optimal catalysis.