Hinge loop reconstruction facilitates hydride transfer in Baeyer-Villiger monooxygenases

Abstract

To speed up biocatalytic redox reactions that use nicotinamide cofactors, previous researchers reshaped the cofactor-binding pocket, but this study focuses on the modification of a hinge loop outside the active site. This loop connects the FAD- and NADP-binding domains of AcPSMO (Acinetobacter calcoaceticus prazole sulfide monooxygenase). One penta-site mutant increased the catalytic efficiency with NADPH 87.5-fold more than the wild type, and another penta-site mutant increased the catalytic efficiency with NADH 12.9-fold more than the L143P mutant (the wild type was undetectable). These AcPSMO mutants also accepted two previously unreactive nicotinamide cofactor biomimetics as hydride donors. X-ray crystal structures and molecular dynamics simulations reveal that these substitutions move the bound NAD(P)H and FAD closer to one another. Introducing similar mutations in several other Baeyer-Villiger monooxygenases (BVMOs) also enhanced their catalytic activity. Thus, hinge-loop engineering is an effective alternative strategy to improve the reactivity of nicotinamide cofactors in BVMOs.