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Altering the substrate specificity of methyl parathion hydrolase with directed evolution

Ng, Tee-Kheang; Gahan, Lawrence; Schenk, Gerhard; Ollis, David

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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 × 106 s-1 M-1), but its activity...[Show more]

dc.contributor.authorNg, Tee-Kheang
dc.contributor.authorGahan, Lawrence
dc.contributor.authorSchenk, Gerhard
dc.contributor.authorOllis, David
dc.date.accessioned2015-12-10T23:13:13Z
dc.identifier.issn0003-9861
dc.identifier.urihttp://hdl.handle.net/1885/64325
dc.description.abstractAbstract 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 × 106 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 × 103 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 × 105 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. MPH is thus an ideal candidate for the development of an enzyme-based bioremediation system.
dc.publisherAcademic Press
dc.sourceArchives of Biochemistry and Biophysics
dc.titleAltering the substrate specificity of methyl parathion hydrolase with directed evolution
dc.typeJournal article
local.description.notesImported from ARIES
local.identifier.citationvolume573
dc.date.issued2015
local.identifier.absfor039999 - Chemical Sciences not elsewhere classified
local.identifier.ariespublicationu4005981xPUB917
local.type.statusPublished Version
local.contributor.affiliationNg, Tee-Kheang, College of Physical and Mathematical Sciences, ANU
local.contributor.affiliationGahan, Lawrence, University of Queensland
local.contributor.affiliationSchenk, Gerhard, University of Queensland
local.contributor.affiliationOllis, David, College of Physical and Mathematical Sciences, ANU
local.description.embargo2037-12-31
local.bibliographicCitation.startpage59
local.bibliographicCitation.lastpage68
local.identifier.doi10.1016/j.abb.2015.03.012
local.identifier.absseo970106 - Expanding Knowledge in the Biological Sciences
dc.date.updated2015-12-10T09:37:35Z
local.identifier.scopusID2-s2.0-84925850321
CollectionsANU Research Publications

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