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Enzymes for the homeland defense: optimizing phosphotriesterase for the hydrolysis of organophosphate nerve agents.

ABSTRACT: 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 S(P)-enantiomers are significantly more toxic than the R(P)-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 S(P)-enantiomers of nerve agent analogues for GB, GD, GF, VX, and VR than the less toxic R(P)-enantiomers. In this investigation, alternate strategies were employed to discover additional PTE variants with significant improvements in catalytic activities relative to that of 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 toward the S(P)-enantiomers of chromophoric analogues of GB, GD, GF, VX, and VR have been improved up to 15000-fold relative to that of 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 k(cat)/K(m) for the H257Y/L303T (YT) mutant for the hydrolysis of GB, GD, and GF were determined to be 2 × 10(6), 5 × 10(5), and 8 × 10(5) 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 for the discovery of more efficient enzymes for the detoxification of organophosphate nerve agents.

SUBMITTER: Tsai PC

PROVIDER: S-EPMC3447986 | biostudies-literature | 2012 Aug

REPOSITORIES: biostudies-literature

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Enzymes for the homeland defense: optimizing phosphotriesterase for the hydrolysis of organophosphate nerve agents.

Tsai Ping-Chuan PC Fox Nicholas N Bigley Andrew N AN Harvey Steven P SP Barondeau David P DP Raushel Frank M FM

Biochemistry 20120731 32

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 S(P)-enantiomers are significantly more toxic than the R(P)-enantiomers. In previous investigations, PTE variants were created through the manipulation of the substrate binding pockets and these mutants were shown to hav ...[more]

PMID: 22809162

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Project description:The organophosphorus chemical warfare agents were initially synthesized in the 1930's and are some of the most toxic compounds ever discovered. The standard means of decontamination are either harsh chemical hydrolysis or high temperature incineration. Given the continued use of chemical warfare agents there are ongoing efforts to develop gentle environmentally friendly means of decontamination and medical counter measures to chemical warfare agent intoxication. Enzymatic decontamination offers the benefits of extreme specificity and mild conditions, allowing their use for both environmental and medical applications. The most promising enzyme for decontamination of the organophosphorus chemical warfare agents is the enzyme phosphotriesterase from Pseudomonas diminuta. However, the catalytic activity of the wild-type enzyme with the chemical warfare agents falls far below that seen with its best substrates, and its stereochemical preference is for the less toxic enantiomer of the chiral phosphorus center found in most chemical warfare agents. Rational design efforts have succeeded in the dramatic improvement of the stereochemical preference of PTE for the more toxic enantiomers. Directed evolution experiments, including site-saturation mutagenesis, targeted error-prone PCR, computational design, and quantitative library analysis, have systematically improved the catalytic activity against the chemical warfare nerve agents. These efforts have resulted in greater than 4-orders of magnitude improvement in catalytic activity and have led to the identification of variants that are highly effective at detoxifying both G-type and V-type nerve agents. The best of these variants have the ability to prevent intoxication when delivered as a post-exposure treatment for VX and as a pre-exposure treatment for G-agent intoxication with observed protective factors up to 60-fold. Combining the best variant, H257Y/L303T, with a PCB polymer coating has enabled the development of a long lasting circulating prophylactic treatment that is highly effective against sarin.

Dataset Information

Enzymes for the homeland defense: optimizing phosphotriesterase for the hydrolysis of organophosphate nerve agents.

Publications

Enzymes for the homeland defense: optimizing phosphotriesterase for the hydrolysis of organophosphate nerve agents.

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