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

Aipeng Li, Ting Wang, Qing Tian, Xiaohong Yang, Dongming Yin, Yong Qin, Lianbing Zhang

Research output: Contribution to journalArticlepeer-review

16 Scopus citations

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 (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.

Original languageEnglish
Pages (from-to)6283-6294
Number of pages12
JournalChemistry - A European Journal
Volume27
Issue number20
DOIs
StatePublished - 7 Apr 2021

Keywords

  • carbonyl reductase
  • chirality
  • protein engineering
  • stereoselectivity inversion
  • β-ketoesters

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