NDLI: A 5000-Fold Increase in the Specificity of a Bacterial Phosphotriesterase for Malathion through Combinatorial Active Site Mutagenesis

Content Provider	PubMed Central
Author	Naqvi, Tatheer Warden, Andrew C. French, Nigel Sugrue, Elena Carr, Paul D. Jackson, Colin J. Scott, Colin
Editor	Dobson, Renwick
Copyright Year	2014
Abstract	Phosphotriesterases (PTEs) have been isolated from a range of bacterial species, including Agrobcaterium radiobacter (PTE Ar ), and are efficient enzymes with broad substrate ranges. The turnover rate of PTEAr for the common organophosphorous insecticide malathion is lower than expected based on its physical properties; principally the pka of its leaving group. In this study, we rationalise the turnover rate of PTE Ar for malathion using computational docking of the substrate into a high resolution crystal structure of the enzyme, suggesting that malathion is too large for the PTE Ar binding pocket. Protein engineering through combinatorial active site saturation testing (CASTing) was then used to increase the rate of malathion turnover. Variants from a CASTing library in which Ser308 and Tyr309 were mutated yielded variants with increased activity towards malathion. The most active PTE Ar variant carried Ser308Leu and Tyr309Ala substitutions, which resulted in a ca. 5000-fold increase in k cat/K M for malathion. X-ray crystal structures for the PTE Ar Ser308Leu\Tyr309Ala variant demonstrate that the access to the binding pocket was enhanced by the replacement of the bulky Tyr309 residue with the smaller alanine residue.
Related Links	http://dx.doi.org/10.1371/journal.pone.0094177
Starting Page	94177
File Format	PDF
ISSN	19326203
e-ISSN	19326203
Journal	PLoS ONE
Issue Number	4
Volume Number	9
Language	English
Publisher	Public Library of Science
Publisher Date	2014-04-01
Access Restriction	Open
Rights Holder	Public Library of Science
Subject Keyword	Biochemistry, Genetics and Molecular Biology(all) Agricultural and Biological Sciences(all) Medicine(all) Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Multidisciplinary

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