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

• DOI: 10.1021/jacs.8b08640
• Publication Date: 2018-09-24T20:44:18Z
• Authors: Jieyu Zhou; Yue Wang; Guochao Xu; Lian Wu; Ruizhi Han; Ulrich Schwaneberg; Yijian Rao; Yi-Lei Zhao; Jiahai Zhou; Ye Ni
• Keywords: Binding Sites; Alcohol Dehydrogenase; Stereoisomerism; Ketones; Crystallography, X-Ray; 01 natural sciences; 0104 chemical sciences; Molecular Docking Simulation; Kinetics; Kluyveromyces; Mutagenesis; Benzene Derivatives; NADP

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