Dataset Information

Multiple Reaction Products from the Hydrolysis of Chiral and Prochiral Organophosphate Substrates by the Phosphotriesterase from Sphingobium sp. TCM1.

ABSTRACT: The phosphotriesterase from Sphingobium sp. TCM1 ( Sb-PTE) is notable for its ability to hydrolyze organophosphates that are not substrates for other enzymes. In an attempt to determine the catalytic properties of Sb-PTE for hydrolysis of chiral phosphotriesters, we discovered that multiple phosphodiester products are formed from a single substrate. For example, Sb-PTE catalyzes the hydrolysis of the RP-enantiomer of methyl cyclohexyl p-nitrophenyl phosphate with exclusive formation of methyl cyclohexyl phosphate. However, the enzyme catalyzes hydrolysis of the SP-enantiomer of this substrate to an equal mixture of methyl cyclohexyl phosphate and cyclohexyl p-nitrophenyl phosphate products. The ability of this enzyme to catalyze the hydrolysis of a methyl ester at the same rate as the hydrolysis of a p-nitrophenyl ester contained within the same substrate is remarkable. The overall scope of the stereoselective properties of this enzyme is addressed with a library of chiral and prochiral substrates.

SUBMITTER: Bigley AN

PROVIDER: S-EPMC5938755 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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Publications

Multiple Reaction Products from the Hydrolysis of Chiral and Prochiral Organophosphate Substrates by the Phosphotriesterase from Sphingobium sp. TCM1.

Bigley Andrew N AN Narindoshvili Tamari T Xiang Dao Feng DF Raushel Frank M FM

Biochemistry 20180313 12

The phosphotriesterase from Sphingobium sp. TCM1 ( Sb-PTE) is notable for its ability to hydrolyze organophosphates that are not substrates for other enzymes. In an attempt to determine the catalytic properties of Sb-PTE for hydrolysis of chiral phosphotriesters, we discovered that multiple phosphodiester products are formed from a single substrate. For example, Sb-PTE catalyzes the hydrolysis of the R<sub>P</sub>-enantiomer of methyl cyclohexyl p-nitrophenyl phosphate with exclusive formation o ...[more]

PMID: 29513982

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Project description: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.

Dataset Information

Multiple Reaction Products from the Hydrolysis of Chiral and Prochiral Organophosphate Substrates by the Phosphotriesterase from Sphingobium sp. TCM1.

Publications

Multiple Reaction Products from the Hydrolysis of Chiral and Prochiral Organophosphate Substrates by the Phosphotriesterase from Sphingobium sp. TCM1.

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