Active site arginine controls the stereochemistry of hydride transfer in cyclohexanone monooxygenase

Cyclohexanone monooxygenase (CHMO) uses NADPH and O 2 to insert oxygen into an array of (a)cyclic ketones to form esters or lactones. Herein, the role of two conserved active site residues (R327 and D57) in controlling the binding mode of NADP(H) was investigated. Wild type CHMO elicits a kinetic isotope effect (KIE) of 4.7 ± 0.1 and 1.1 ± 0.1 with 4( R )-[4- 2 H]NADPH and 4( S )-[4- 2 H]NADPH, respectively, consistent with transfer of the pro R hydrogen to FAD. Strikingly, the R327K variant appears to lack stereospecificity for hydride transfer as a KIE of 1.5 ± 0.1 and 2.5 ± 0.1 was observed for the pro R and pro S deuterated forms of NADPH. 1 H NMR of the NADP + products confirmed that the R327K variant abstracts either the pro R or pro S hydrogen from NADPH. While the D57A variant retained stereospecificity for the pro R hydrogen, this substitution resulted in slow decomposition of the C4a-peroxyflavin intermediate in the presence of cyclohexanone. Based on published structures of a related flavin monooxygenase, we suggest that elimination of the hydrogen bond between D57 and R327 in the D57A variant causes R327 to adopt a substrate-induced conformation that slows substrate access to the active site, thereby prolonging the lifetime of the C4a-peroxyflavin intermediate.