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

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.