Altering the substrate specificity of methyl parathion hydrolase with directed evolution

Archives of Biochemistry and Biophysics
Tee-Kheang NgDavid L Ollis

Abstract

Many organophosphates (OPs) are used as pesticides in agriculture. They pose a severe health hazard due to their inhibitory effect on acetylcholinesterase. Therefore, detoxification of water and soil contaminated by OPs is important. Metalloenzymes such as methyl parathion hydrolase (MPH) from Pseudomonas sp. WBC-3 hold great promise as bioremediators as they are able to hydrolyze a wide range of OPs. MPH is highly efficient towards methyl parathion (1 × 10(6) s(-1) M(-1)), but its activity towards other OPs is more modest. Thus, site saturation mutagenesis (SSM) and DNA shuffling were performed to find mutants with improved activities on ethyl paraxon (6.1 × 10(3) s(-1) M(-1)). SSM was performed on nine residues lining the active site. Several mutants with modest activity enhancement towards ethyl paraoxon were isolated and used as templates for DNA shuffling. Ultimately, 14 multiple-site mutants with enhanced activity were isolated. One mutant, R2F3, exhibited a nearly 100-fold increase in the kcat/Km value for ethyl paraoxon (5.9 × 10(5) s(-1) M(-1)). These studies highlight the 'plasticity' of the MPH active site that facilitates the fine-tuning of its active site towards specific substrates with only minor changes required...Continue Reading

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Citations

Sep 30, 2015·Biotechnology and Bioengineering·Immacolata Del GiudiceGiuseppe Manco
May 18, 2016·Biochemistry·Anna PabisShina C L Kamerlin
Oct 6, 2016·Enzyme and Microbial Technology·Xu-Yun LiuJian-He Xu
Oct 5, 2016·Philosophical Transactions. Series A, Mathematical, Physical, and Engineering Sciences·Miha PurgShina Caroline Lynn Kamerlin
Jan 10, 2017·Chemistry : a European Journal·Yayun GuYijun Chen
Jun 3, 2017·Dalton Transactions : an International Journal of Inorganic Chemistry·Marcelo Monteiro PedrosoGerhard Schenk
Jan 12, 2017·Scientific Reports·Chun-Feng D HouDavid L Ollis
Nov 19, 2019·Journal of Agricultural and Food Chemistry·Taozhong ShiRimao Hua

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