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

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.