Structural Insight into Enantioselective Inversion of an Alcohol Dehydrogenase Reveals a “Polar Gate” in Stereorecognition of Diaryl Ketones

化学 对映选择合成 反演(地质) 醇脱氢酶 极地的 组合化学 立体化学 有机化学 催化作用 天文 生物 构造盆地 物理 古生物学
作者
Jieyu Zhou,Yue Wang,Guochao Xu,Lian Wu,Ruizhi Han,Ulrich Schwaneberg,Yijian Rao,Yi‐Lei Zhao,Jiahai Zhou,Ye Ni
出处
期刊:Journal of the American Chemical Society [American Chemical Society]
卷期号:140 (39): 12645-12654 被引量:101
标识
DOI:10.1021/jacs.8b08640
摘要

Diaryl ketones are important building blocks for synthesizing pharmaceuticals and are generally regarded as "difficult-to-reduce" ketones due to the large steric hindrance of their two bulky aromatic side chains. Alcohol dehydrogenase from Kluyveromyces polyspora (KpADH) has been identified as a robust biocatalyst due to its high conversion of diaryl ketone substrate (4-chlorophenyl)(pyridine-2-yl)ketone (CPMK) with a moderate R-selectivity of 82% ee. To modulate the stereoselectivity of KpADH, a "polarity scanning" strategy was proposed, in which six key residues inside and at the entrance of the substrate binding pocket were identified. After iterative combinatorial mutagenesis, variants Mu-R2 and Mu-S5 with enhanced (99.2% ee, R) and inverted (97.8% ee, S) stereoselectivity were obtained. The crystal structures of KpADH and two mutants in complex with NADPH were resolved to elucidate the evolution of enantioselective inversion. Based on MD simulation, Mu-R2–CPMKProR and Mu-S5–CPMKProS were more favorable in the formation of prereaction states. Interestingly, a quadrilateral plane formed by α-carbons of four residues (N136, V161, C237, and G214) was identified at the entrance of the substrate binding pocket of Mu-S5; this plane acts as a "polar gate" for substrates. Due to the discrepancy in charge characteristics between chlorophenyl and pyridine substituents, the pro-S orientation of CPMK is defined when it passes through the "polar gate" in Mu-S5, whereas the similar plane in wild-type is blocked by several aromatic residues. Our result paves the way for engineering stereocomplementary ADH toward bulky diaryl ketones and provides structural insight into the mechanism of stereoselective inversion.
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