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 (kcat /Km ) 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. © 2021 Wiley-VCH GmbH.

Citation

Aipeng Li, Ting Wang, Qing Tian, Xiaohong Yang, Dongming Yin, Yong Qin, Lianbing Zhang. Single-Point Mutant Inverts the Stereoselectivity of a Carbonyl Reductase toward β-Ketoesters with Enhanced Activity. Chemistry (Weinheim an der Bergstrasse, Germany). 2021 Apr 07;27(20):6283-6294

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PMID: 33475219

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