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PEDS Advance Access published online on January 19, 2006
Protein Engineering Design and Selection, 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
Received July 8, 2005
Revised November 23, 2005
Accepted December 1, 2005

Article

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

Catherine Mee-Hie Cho 1, Ashok Mulchandani 2, and Wilfred Chen 2 *

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

* To whom correspondence should be addressed.
Wilfred Chen, E-mail: wilfred{at}engr.ucr.edu


  Abstract

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.
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