Switching catalysis from hydrolysis to perhydrolysis in Pseudomonas fluorescens esterase.

Biochemistry
De Lu Tyler YinRomas J Kazlauskas

Abstract

Many serine hydrolases catalyze perhydrolysis, the reversible formation of peracids from carboxylic acids and hydrogen peroxide. Recently, we showed that a single amino acid substitution in the alcohol binding pocket, L29P, in Pseudomonas fluorescens (SIK WI) aryl esterase (PFE) increased the specificity constant of PFE for peracetic acid formation >100-fold [Bernhardt et al. (2005) Angew. Chem., Int. Ed. 44, 2742]. In this paper, we extend this work to address the three following questions. First, what is the molecular basis of the increase in perhydrolysis activity? We previously proposed that the L29P substitution creates a hydrogen bond between the enzyme and hydrogen peroxide in the transition state. Here we report two X-ray structures of L29P PFE that support this proposal. Both structures show a main chain carbonyl oxygen closer to the active site serine as expected. One structure further shows acetate in the active site in an orientation consistent with reaction by an acyl-enzyme mechanism. We also detected an acyl-enzyme intermediate in the hydrolysis of epsilon-caprolactone by mass spectrometry. Second, can we further increase perhydrolysis activity? We discovered that the reverse reaction, hydrolysis of peracetic aci...Continue Reading

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Citations

Jul 10, 2013·Journal of Biotechnology·Dragana DespotovicUlrich Schwaneberg
Feb 7, 2012·Nature Chemical Biology·Sagar D KhareDavid Baker
Nov 4, 2015·Frontiers in Bioengineering and Biotechnology·Jennifer A Littlechild
Jan 18, 2013·Chemistry : a European Journal·DeLu Tyler YinRomas J Kazlauskas
Feb 26, 2011·Chembiochem : a European Journal of Chemical Biology·Yun JiangRomas J Kazlauskas
Jul 2, 2010·Chembiochem : a European Journal of Chemical Biology·Robert KouristUwe T Bornscheuer
May 24, 2012·Chemistry : a European Journal·DeLu Tyler Yin, Romas J Kazlauskas
Apr 6, 2016·Journal of Biotechnology·Zeinab AssafMélanie Hall
Jan 13, 2015·New Biotechnology·Ljubica VojcicUlrich Schwaneberg
May 27, 2015·The FEBS Journal·Christopher SayerJennifer A Littlechild
Feb 25, 2011·Bioresource Technology·DeLu Tyler YinRomas J Kazlauskas
Mar 19, 2015·Biochemical and Biophysical Research Communications·Xuping WangYonghua Wang
Mar 7, 2012·Journal of Biomolecular Screening·Dragana DespotovicUlrich Schwaneberg
Mar 25, 2018·Angewandte Chemie·JiaJia DongWuyuan Zhang
Apr 13, 2017·Journal of Industrial Microbiology & Biotechnology·J A Littlechild
Feb 24, 2012·Organic & Biomolecular Chemistry·Leticia L TorresAurelio Hidalgo
Oct 26, 2018·Journal of Industrial Microbiology & Biotechnology·Shaozhou Zhu, Guojun Zheng
Oct 28, 2017·Journal of Chemical Theory and Computation·Esra BozkurtUrsula Rothlisberger

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