Quantum Mechanical/Molecular Mechanical Study on the Enantioselectivity of the Enzymatic Baeyer–Villiger Reaction of 4‑Hydroxycyclohexanone

We report a combined quantum mechanical/molecular mechanical (QM/MM) study of the effect of mutations of the Phe434 residue in the active site of cyclohexanone monooxygenase (CHMO) on its enantioselectivity toward 4-hydroxycyclohexanone. In terms of our previously established model of the enzymatic Baeyer–Villiger reaction, enantioselectivity is governed by the preference toward the equatorial ((<i>S</i>)-selectivity) or axial ((<i>R</i>)-selectivity) conformation of the substituent at the C4 carbon atom of the cyclohexanone ring in the Criegee intermediate and the subsequent rate-limiting transition state for migration (TS2). We assess the enantiopreference by locating all relevant TS2 structures at the QM/MM level. In the wild-type enzyme we find that the axial conformation is energetically slightly more stable, thus leading to a small excess of (<i>R</i>)-product. In the Phe434Ser mutant, there is a hydrogen bond between the serine side chain and the equatorial substrate hydroxyl group that is retained during the whole reaction, and hence there is pronounced reverse (<i>S</i>)-enantioselectivity. Another mutant, Phe434Ile, is shown to preserve and enhance the (<i>R</i>)-selectivity. All these findings are in accordance with experiment. The QM/MM calculations allow us to explain the effect of point mutations on CHMO enantioselectivity for the first time at the molecular level by an analysis of the specific interactions between substrate and active-site environment in the TS2 structures that satisfy the basic stereoelectronic requirement of anti-periplanarity for the migrating σ-bond