Single‐Point Mutant Inverts the Stereoselectivity of a Carbonyl Reductase toward β‐Ketoesters with Enhanced Activity

Abstract Enzyme stereoselectivity control is still a major challenge. To gain insight into the molecular basis of enzyme stereo‐recognition and expand the source of antiPrelog carbonyl reductase toward β‐ketoesters, rational enzyme design aiming at stereoselectivity inversion was performed. The designed variant Q139G switched the enzyme stereoselectivity toward β‐ketoesters from Prelog to antiPrelog, providing corresponding alcohols in high enantiomeric purity (89.1–99.1 % ee ). More importantly, the well‐known trade‐off between stereoselectivity and activity was not found. Q139G exhibited higher catalytic activity than the wildtype enzyme, the enhancement of the catalytic efficiency ( k cat / K m ) varied from 1.1‐ to 27.1‐fold. Interestingly, the mutant Q139G did not lead to reversed stereoselectivity toward aromatic ketones. Analysis of enzyme–substrate complexes showed that the structural flexibility of β‐ketoesters and a newly formed cave together facilitated the formation of the antiPrelog‐preferred conformation. In contrast, the relatively large and rigid structure of the aromatic ketones prevents them from forming the antiPrelog‐preferred conformation.