Stereodivergent evolution of KpADH for the asymmetric reduction of diaryl ketones with para-substituents

Kp ADH is a promising biocatalyst for the synthesis of chiral bulky-bulky alcohols. Substrate specificity toward diaryl ketone substrates with different substituents at phenyl group was investigated. The results reveal that the para -position of diaryl ketones plays important role in manipulating the stereoselectivity of Kp ADH. Two vital residues, E214 and T215, were identified through single-point mutagenesis, and saturation mutagenesis was performed to evaluate their contributions to the stereoselectivity. Furthermore, stereodivergent evolution of Kp ADH was achieved through combinatorial mutagenesis. Among them, E214I/T215S/S237A (ISA) and F161V/S196G/E214G (VGG) exhibited e.e. values of >99% ( S ) and 99% ( R ) toward [4-(trifluoromethyl)phenyl]-2-pyridinylmethanone ( 5a ), and e.e. values of >99% ( S ) and 98% ( R ) toward (4-methylphenyl)-2-pyridinylmethanone ( 6a ), respectively. Kinetic characterization and interaction analysis further prove that the eliminated side-chain collision and enhanced electrostatic interactions are mainly responsible for the increased catalytic efficiency and stereoselectivity. This study provides beneficial mutants with complementary stereoselectivity toward diaryl ketones with para -substituents and also provides guidance for the engineering of homologous alcohol dehydrogenases toward bulky-bulky ketones.