PDBsum entry 5x8h

Oxidoreductase

PDB id: 5x8h

Contents

Protein chains

248 a.a.

Waters ×131

References listed in PDB file

Key reference

• Title: Crystal structure and iterative saturation mutagenesis of chkred20 for expanded catalytic scope.
• Authors: F.J.Zhao, Y.Jin, Z.Liu, C.Guo, T.B.Li, Z.Y.Li, G.Wang, Z.L.Wu.
• Ref.: Appl Microbiol Biotechnol, 2017, 101, 8395-8404. [DOI no: 10.1007/s00253-017-8556-2]
• PubMed id: 29067484

Abstract

ChKRED20 is an efficient and robust anti-Prelog ketoreductase that can catalyze the reduction of ketones to chiral alcohols as pharmaceutical intermediates with great industrial potential. To overcome its limitation on the bioreduction of ortho-substituted acetophenone derivatives, the X-ray crystal structure of the apo-enzyme of ChKRED20 was determined at a resolution of 1.85 Å and applied to the molecular modeling and reshaping of the catalytic cavity via three rounds of iterative saturation mutagenesis together with alanine scanning and recombination. The mutant Mut3B was achieved with expanded catalytic scope that covered all the nine substrates tested as compared with two substrates for the wild type. It exhibited 13-20-fold elevated k_cat/K_mvalues relative to the wild type or to the first gain-of-activity mutant, while retaining excellent stereoselectivity toward seven of the substrates (98-> 99% ee). Another mutant 29G10 displayed complementary selectivity for eight of the ortho-substituted acetophenone derivatives, with six of them delivering excellent stereoselectivity (90-99% ee). Its k_cat/K_mvalue toward 1-(2-fluorophenyl)ethanone was 5.6-fold of the wild type. The application of Mut3B in elevated substrate concentrations of 50-100 g/l was demonstrated in 50-ml reactions, achieving 75-> 99% conversion and > 99% ee.