Structural insight into enantioselective inversion of an alcohol dehydrogenase reveals a “Polar Gate” in stereo-recognition of diaryl ketones

Conference Dates

September 15-19, 2019


Diaryl ketones are generally regarded as “difficult-to-reduce” ketones due to the large steric hindrance of two bulky aromatic side-chains. Alcohol dehydrogenase from Kluyveromyces polyspora (KpADH) has been identified as a robust biocatalyst due to its high tolerance to diaryl ketone substrate (4-chlorophenyl)-(pyridine-2-yl)-ketone (CPMK), whereas 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 substrate binding pocket were identified. After iterative combinatorial mutagenesis, variants Mu-R2 and Mu-S5 with enhanced (99.2% ee, R) and inverted (97.4% ee, S) stereoselectivity were obtained. Their crystal structures in complex with NADPH were resolved to elucidate the evolution of enantioselective inversion. Based on computational results from MD simulation, an approximate plane formed by α-carbon of four residues (N136, V161, C237 and G214) was identified at the entrance of substrate binding pocket of Mu-S5, which act as a “polar gate” for substrates. Due to the discrepancy in charge characteristics between chlorophenyl and pyridine substituents, pro-S orientation of CPMK is defined when it passing through the “polar gate” in Mu-S5, whereas the similar plane in WT is blocked by several aromatic residues. Our result represents advancement in engineering stereo-complementary ADH toward diaryl ketones, and provides structural insight into the mechanism of stereoselective inversion in KpADH.

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