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

Abstract

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.

Crystal Structure of NADPH-Dependent Methylglyoxal Reductase Gre2 from Candida Albicans

“…The observation of salt-bridges supports a higher affinity as well as preferential binding of NADPH over NADH in these enzymes, which is contrary to the reported data for HsUGE an EcUGE [31,32]. Additionally, the catalytic triad Ser129-Tyr167-Lys171 in CaGre2 is well conserved in yeast (ScGre2, SsCR, KpADH) and plant homologs (MtCAD2, SbCCR1); this catalytic triad has also been reported to play crucial roles in the catalytic pathway [2,27,28,33,34].…”

“…To uncover to the key active site residues of CaGre2, a comparison of sequences was performed using the protein sequences of CaGre2, ScGre2, SsCR, and Kluyveromyces polyspora alcohol dehydrogenase (KpADH; PDB code, 5Z2X) ( Figure 3F). The Ser129, Tyr167, and Lys171 residues of CaGre2 are well conserved among the four enzymes and are reported as being the core catalytic residues in those structures [2,27,28]. This suggests that these three residues most likely act as a crucial catalytic triad in CaGre2.…”

Evolutionary coupling saturation mutagenesis: Coevolution‐guided identification of distant sites influencing Bacillus naganoensis pullulanase activity

“…b). Thus, it is conclusive that the distal mutations can significantly promote the reactive population of prereaction state and global effect might be very important in enzyme engineering .…”

Confining enzyme clusters in supramolecular nanoreactors enhances cofactor-dependent cascade for chiral alcohol synthesis

“…Besides, the high stereoselectivity of SsCR towards 2a, 2b, 2d and 2m were maintained by P22-SP-BmGDH-SsCR (96% 99% ee). In previous studies, the precise control over the stereoselectivity of ketoreductases is a major challenge, and time-consumed protein evolution is usually needed to acquire the desired stereopreferences 50 . In contrast, we showed that encapsulation of a ketoreductase in P22 nanoparticles can enhance its stereoselectivity without directed evolution, which, to our knowledge, has not been reported before.…”