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Transient expression of organophosphorus hydrolase to enhance the degrading activity of tomato fruit on coumaphos.

ABSTRACT: We constructed an expression cassette of the organophosphorus pesticide degrading (opd) gene under the control of the E8 promoter. Then opd was transformed into tomato fruit using an agroinfiltration transient expression system. beta-Glucuronidase (GUS) staining, reverse transcription-polymerase chain reaction (RT-PCR), wavelength scanning, and fluorescent reaction were performed to examine the expression of the opd gene and the hydrolysis activity on coumaphos of organophosphorus hydrolase (OPH) in tomato fruit. The results show that the agroinfiltrated tomato fruit-expressed OPH had the maximum hydrolysis activity of about 11.59 U/mg total soluble protein. These results will allow us to focus on breeding transgenic plants that could not only enhance the degrading capability of fruit and but also hold no negative effects on pest control when spraying organophosphorus pesticides onto the seedlings in fields.

SUBMITTER: Zhao JH

PROVIDER: S-EPMC2644755 | biostudies-literature | 2009 Feb

REPOSITORIES: biostudies-literature

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Transient expression of organophosphorus hydrolase to enhance the degrading activity of tomato fruit on coumaphos.

Zhao Jie-hong JH Zhao De-gang DG

Journal of Zhejiang University. Science. B 20090201 2

We constructed an expression cassette of the organophosphorus pesticide degrading (opd) gene under the control of the E8 promoter. Then opd was transformed into tomato fruit using an agroinfiltration transient expression system. beta-Glucuronidase (GUS) staining, reverse transcription-polymerase chain reaction (RT-PCR), wavelength scanning, and fluorescent reaction were performed to examine the expression of the opd gene and the hydrolysis activity on coumaphos of organophosphorus hydrolase (OPH ...[more]

PMID: 19235273

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Project description:The bacterial enzyme organophosphorus hydrolase (OPH) exhibits both catalytic and substrate promiscuity. It hydrolyzes bonds in a variety of phosphotriester (P-O), phosphonothioate (P-S), phosphofluoridate (P-F), and phosphonocyanate (F-CN) compounds. However, its catalytic efficiency varies markedly for different substrates, limiting the broad-range application of OPH as catalyst in the bioremediation of pesticides and chemical war agents. In the present study, pK(a) calculations and multiple explicit-solvent molecular dynamics (MD) simulations were performed to characterize and contrast the structural dynamics of OPH bound to two substrates hydrolyzed with very distinct catalytic efficiencies: the nerve agent soman (O-pinacolylmethylphosphonofluoridate) and the pesticide paraoxon (diethyl p-nitrophenyl phosphate). pK(a) calculations for the substrate-bound and unbound enzyme showed a significant pK(a) shift from standard values (ΔpK(a) = ±3 units) for residues His254 and Arg275. MD simulations of protonated His254 revealed a dynamic hydrogen bond network connecting the catalytic residue Asp301 via His254 to Asp232, Asp233, Arg275, and Asp235, and is consistent with a previously postulated proton relay mechanism to ferry protons away from the active site with substrates that do not require activation of the leaving group. Hydrogen bonds between Asp301 and His254 were persistent in the OPH-paraoxon complex but not in the OPH-soman one, suggesting a potential role for such interaction in the more efficient hydrolysis of paraoxon over soman by OPH. These results are in line with previous mutational studies of residue His254, which led to an increase of the catalytic efficiency of OPH over soman yet decreased its efficiency for paraoxon. In addition, comparative analysis of the molecular trajectories for OPH bound to soman and paraoxon suggests that binding of the latter facilitates the conformational transition of OPH from the open to the closed substate promoting a tighter binding of paraoxon.

Dataset Information

Transient expression of organophosphorus hydrolase to enhance the degrading activity of tomato fruit on coumaphos.

Publications

Transient expression of organophosphorus hydrolase to enhance the degrading activity of tomato fruit on coumaphos.

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