Unraveling the substrate-binding pocket: Endowing carbonyl reductase BaSDRX with intriguing inconsistent stereoselectivity towards similar structured β-Ketoesters.

Tailoring of carbonyl reductase with desired stereoselectivity for the asymmetric reduction of given β-ketoesters continues to pose a significant challenge. The reconstruction of substrate-binding pocket endowed carbonyl reductase BaSDRX with inconsistent stereoselectivity towards similar structured β-ketoesters. The variant F86C/E142A showed reversed stereoselectivity towards β-ketoesters with a larger substituent on the carbonyl side, giving antiPrelog alcohols with ee values varied from 52.8 % to 95.8 %. However, it displayed only a decreased stereoselectivity for β-ketoesters that contain a smaller methyl group at the same position. Analysis of enzyme-substrate complexes showed that a newly formed groove provided more spaces and chances for β-ketoester with a larger substituent on the carbonyl side to approach the catalytic triad of F86C/E142A in antiPrelog-preferred binding modes. For the β-ketoesters with a smaller methyl group on the carbonyl side, the introduction of mutations exerted less effect on the proximity of the substrate with antiPrelog-preferred binding modes to the catalytic residues. Meanwhile, both β-ketoesters with smaller group and larger group on carbonyl side underwent minor alterations in Prelog-preferred conformation. All of these made the variant F86C/E142A exhibited inconsistent stereoselectivity towards β-ketoesters possessing analogous structure. These results offered detailed mechanisms that govern the stereoselectivity in the enzyme-mediated asymmetric reduction of β-ketoesters.