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Publication Abstract from PubMed

Phosphotriesterase (PTE) from soil bacteria is known for its ability to catalyze the detoxification of organophosphate pesticides and chemical warfare agents. Most of the organophosphate chemical warfare agents are a mixture of two stereoisomers at the phosphorus center and the Sp-enantiomers are significantly more toxic than the Rp-enantiomers. In previous investigations PTE variants were created through the manipulation of the substrate binding pockets and these mutants were shown to have greater catalytic activities for the detoxification of the more toxic Sp-enantiomers of nerve agent analogs for GB, GD, GF, VX, and VR than the less toxic Rp- enantiomers. In this investigation alternate strategies were employed to discover additional PTE variants with significant improvements in catalytic activities relative to the wild type enzyme. Screening and selection techniques were utilized to isolate PTE variants from randomized libraries and site specific modifications. The catalytic activities of these newly identified PTE variants towards the Sp- enantiomers of chromophoric analogs of GB, GD, GF, VX, and VR have been improved up to 15,000 fold relative to the wild-type enzyme. The X-ray crystal structures of the best PTE variants were determined. Characterization of these mutants with the authentic G-type nerve agents has confirmed the expected improvements in catalytic activity against the most toxic enantiomers of GB, GD, and GF. The values of kcat/Km for the H257Y/L303T (YT) mutant for the hydrolysis of GB, GD, and GF were determined to be 2 x 106 M-1 s-1, 5 x 105 M-1 s-1, and 8 x 105 M-1 s-1, respectively. The YT mutant is the most proficient enzyme reported thus far for the detoxification of G-type nerve agents. These results support a combinatorial strategy of rational design and directed evolution as a powerful tool to discover more efficient enzymes for the detoxification of organophosphate nerve agents.

Enzymes for the Homeland Defense: Optimizing Phosphotriesterase for the Hydrolysis of Organophosphate Nerve Agents.,Tsai PC, Fox N, Bigley AN, Harvey SP, Barondeau DP, Raushel FM Biochemistry. 2012 Jul 18. PMID:22809162^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.