Key residues for controlling enantioselectivity of Halohydrin dehalogenase from Arthrobacter sp. strain AD2, revealed by structure-guided directed evolution.

Applied and Environmental Microbiology
Lixia TangMaja Majeric Elenkov

Abstract

Halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) is a valuable tool in the preparation of R enantiomers of epoxides and β-substituted alcohols. In contrast, the halohydrin dehalogenase from Arthrobacter sp. AD2 (HheA) shows a low S enantioselectivity toward most aromatic substrates. Here, three amino acids (V136, L141, and N178) located in the two neighboring active-site loops of HheA were proposed to be the key residues for controlling enantioselectivity. They were subjected to saturation mutagenesis aimed at evolving an S-selective enzyme. This led to the selection of two outstanding mutants (the V136Y/L141G and N178A mutants). The double mutant displayed an inverted enantioselectivity (from S enantioselectivity [E(S)] = 1.7 to R enantioselectivity [E(R)] = 13) toward 2-chloro-1-phenylethanol without compromising enzyme activity. Strikingly, the N178A mutant showed a large enantioselectivity improvement (E(S) > 200) and a 5- to 6-fold-enhanced specific activity toward (S)-2-chloro-1-phenylethanol. Further analysis revealed that those mutations produced some interference for the binding of nonfavored enantiomers which could account for the observed enantioselectivities. Our work demonstrated that those three a...Continue Reading

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Mar 21, 2006·Biochemical Society Transactions·D B JanssenJ H Lutje Spelberg
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Citations

Nov 1, 2012·Applied Microbiology and Biotechnology·Zhong-Yu YouYu-Guo Zheng
Jan 13, 2015·Current Opinion in Chemical Biology·Carl A DenardHuimin Zhao
Feb 11, 2015·Enzyme and Microbial Technology·Marcus SchallmeyDick B Janssen
Aug 10, 2016·Applied Microbiology and Biotechnology·Anett Schallmey, Marcus Schallmey
Sep 23, 2014·Applied and Environmental Microbiology·Marcus SchallmeyAnett Schallmey
Apr 16, 2013·Chembiochem : a European Journal of Chemical Biology·Marcus SchallmeyDick B Janssen
Feb 18, 2020·Biotechnology and Applied Biochemistry·Ijaz GulLixia Tang
May 6, 2020·Applied Microbiology and Biotechnology·Ijaz GulLixia Tang
Apr 15, 2021·Applied Microbiology and Biotechnology·Feng XueQing Xu

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