Customizing the Enantioselectivity of a Cyclohexanone Monooxygenase by a Strategy Combining “Size-Probes” with in silico Study

Graphical Customizing the enantioselectivity : WT CHMO Acineto leads to enantioselectivity up to 99 % ee ( S ) in the Baeyer-Villiger oxidation of 4-substituted cyclohexanones. By probing the binding pocket of rationally designed variants with different-sized 4-substituted cyclohexanones, single, double and triple mutants were identified as the best mutants providing highly reversed enantioselectivity for these probing molecules. Enzymatic Baeyer-Villiger oxidation provides a promising green route utilizing molecular oxygen as the oxidant to produce chiral lactones. Wild-type (WT) CHMO Acineto leads to enantioselectivity up to 99 % ee ( S ) in the synthesis of substituted ϵ-caprolactones. To reverse the inherent enantiopreference of CHMO Acineto toward an array of cyclohexanones with various chain length, we herein reshaped the binding pocket with a minimal number of mutations by a rational design strategy combining “size-probes” with in silico study, which drastically reduces the screening effort. By probing the binding pocket of variants with different-sized 4-substituted cyclohexanones substrates, single, double and triple mutants were identified as the best mutants providing highly reversed enantioselectivity for these probing molecules, respectively. The successful demonstration of the strategy combining “size-probes” with in silico study in the protein engineering of CHMO Acineto may provide a valuable guidance for facile engineering other BVMOs with customized enantiopreference for the same classified substrates with their substituents on the chiral or prochiral central atom.