5ZEC

Crystal structure of Kluyveromyces polyspora ADH (KpADH) mutant (Q136N/F161V/S196G/E214G/S237C)

Summary for 5ZEC

• Entry DOI: 10.2210/pdb5zec/pdb
• Descriptor: Uncharacterized protein ADH, NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE, 1,2-ETHANEDIOL, ... (8 entities in total)
• Functional Keywords: alcohol dehydrogenase(kpadh), nadph, isomerase
• Biological source: Vanderwaltozyma polyspora DSM 70294 (Kluyveromyces polysporus)
• Total number of polymer chains: 2
• Total formula weight: 79765.70

Authors

Wang, Y.,ZHou, J.Y.,Hou, X.D.,Xu, G.C.,Rao, Y.J.,Wu, L.,Zhou, J.H.,Ni, Y. (deposition date: 2018-02-27, release date: 2019-01-02, Last modification date: 2023-11-22)

Primary citation

Zhou, J.Y.,Wang, Y.,Xu, G.C.,Wu, L.,Han, R.Z.,Schwaneberg, U.,Rao, Y.J.,Zhao, Y.L.,Zhou, J.H.,Ni, Y.
Structural Insight into Enantioselective Inversion of an Alcohol Dehydrogenase Reveals a "Polar Gate" in Stereorecognition of Diaryl Ketones.
J. Am. Chem. Soc., 140:12645-12654, 2018

PubMed 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-CPMK and Mu-S5-CPMK 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.

PubMed: 30247889
DOI: 10.1021/jacs.8b08640

Experimental method

X-RAY DIFFRACTION (1.779 Å)