The long-term objective for the research described in this proposal is the design and construction of a robust catalytic system that can be exploited for the decontamination, destruction, and detection of organophosphate nerve agents. The nerve agents sarin (GB), soman (GD), and VX are among the most toxic and deadly nerve agents ever reported. Since these compounds are easy to synthesize and distribute, they represent a serious threat to the health and well being of civilized societies. In order to prepare a system that can detoxify organophosphate nerve agents, we will manipulate and enhance the enzymatic power of wild type proteins to serve as catalysts for the recognition and hydrolytic turnover of these highly toxic materials. It has been demonstrated that the catalytic machinery embedded within the active sites of the bacterial phosphotriesterase and alpha-prolidase are capable of hydrolyzing certain organophosphate nerve agents such as paraoxon at the diffusion controlled limit. The active site structures of these enzymes will be re- engineered through rational and combinatorial mutagenesis techniques to create libraries of mutant enzymes with altered catalytic properties. These enzyme libraries will be efficiently evaluated with high throughput screening protocols using fluorescence and visible spectroscopy with chiral analogs of GB, GD, and VX. The catalytic activities with the restricted nerve agents will be optimized through a direct collaboration with the DeFrank group at the Aberdeen Proving Ground. The structural analyses of the wild type and mutant proteins will be conducted by the Holden group at Wisconsin.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM068550-04S1
Application #
7548097
Study Section
Biochemistry Study Section (BIO)
Program Officer
Ikeda, Richard A
Project Start
2003-07-01
Project End
2008-06-30
Budget Start
2006-07-01
Budget End
2008-06-30
Support Year
4
Fiscal Year
2008
Total Cost
$116,400
Indirect Cost
Name
Texas A&M University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
078592789
City
College Station
State
TX
Country
United States
Zip Code
77845
Bigley, Andrew N; Xu, Chengfu; Henderson, Terry J et al. (2013) Enzymatic neutralization of the chemical warfare agent VX: evolution of phosphotriesterase for phosphorothiolate hydrolysis. J Am Chem Soc 135:10426-32
Meier, Monika M; Rajendran, Chitra; Malisi, Christoph et al. (2013) Molecular engineering of organophosphate hydrolysis activity from a weak promiscuous lactonase template. J Am Chem Soc 135:11670-7
Bigley, Andrew N; Raushel, Frank M (2013) Catalytic mechanisms for phosphotriesterases. Biochim Biophys Acta 1834:443-53
Tsai, Ping-Chuan; Fox, Nicholas; Bigley, Andrew N et al. (2012) Enzymes for the homeland defense: optimizing phosphotriesterase for the hydrolysis of organophosphate nerve agents. Biochemistry 51:6463-75
Tsai, Ping-Chuan; Fan, Yubo; Kim, Jungwook et al. (2010) Structural determinants for the stereoselective hydrolysis of chiral substrates by phosphotriesterase. Biochemistry 49:7988-97
Tsai, Ping-Chuan; Bigley, Andrew; Li, Yingchun et al. (2010) Stereoselective hydrolysis of organophosphate nerve agents by the bacterial phosphotriesterase. Biochemistry 49:7978-87
Kim, Jungwook; Tsai, Ping-Chuan; Chen, Shi-Lu et al. (2008) Structure of diethyl phosphate bound to the binuclear metal center of phosphotriesterase. Biochemistry 47:9497-504
Li, Yingchun; Raushel, Frank M (2007) Differentiation of chiral phosphorus enantiomers by P and H NMR spectroscopy using amino acid derivatives as chemical solvating agents. Tetrahedron Asymmetry 18:1391-1397
Ghanem, Eman; Li, Yingchun; Xu, Chengfu et al. (2007) Characterization of a phosphodiesterase capable of hydrolyzing EA 2192, the most toxic degradation product of the nerve agent VX. Biochemistry 46:9032-40
Samples, Cynthia R; Raushel, Frank M; DeRose, Victoria J (2007) Activation of the binuclear metal center through formation of phosphotriesterase-inhibitor complexes. Biochemistry 46:3435-42

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