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PEDS Advance Access originally published online on January 19, 2006
Protein Engineering Design and Selection 2006 19(3):99-105; doi:10.1093/protein/gzj007
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Functional analysis of organophosphorus hydrolase variants with high degradation activity towards organophosphate pesticides

Catherine Mee-Hie Cho1,2, Ashok Mulchandani1 and Wilfred Chen1,3

1Department of Chemical and Environmental Engineering and 2Department of Biochemistry, University of California, Riverside, CA 92521, USA

3 To whom correspondence should be addressed. E-mail: wilfred{at}engr.ucr.edu

Organophosphorus hydrolase (OPH, also known as phosphotriesterase) is a bacterial enzyme that is capable of degrading a wide range of neurotoxic organophosphate nerve agents. Directed evolution has been used to generate one variant (22A11) with up to 25-fold improved hydrolysis of methyl parathion. Surprisingly, this variant also degraded all other substrates (paraoxon, parathion and coumaphos) tested 2- to 10-fold faster. Since only one mutation (H257Y) is directly located in the active site, site-directed mutagenesis and saturation mutagenesis were used to identify the role of the other distal substitutions (A14T, A80V, K185R, H257Y, I274N) on substrate specificity and activity. Sequential site-directed mutagenesis indicated that K185R and I274N are the most important substitutions, leading to an improvement not only in the hydrolysis of methyl parathion but also the overall hydrolysis rate of all other substrates tested. Using structural modeling, these two mutations were shown to favor the formation of hydrogen bonds with nearby residues, resulting in structural changes that could alter the overall substrate hydrolysis.

Keywords: degradation/directed evolution/methyl parathion/organophosphate pesticides/organophosphorus hydrolase/site-directed mutagenesis

Received July 8, 2005; revised November 23, 2005; accepted December 1, 2005.

Edited by Dan Tawfik


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