Dataset Information

Aminoalcohol-Induced Activation of Organophosphorus Hydrolase (OPH) towards Diisopropylfluorophosphate (DFP).

ABSTRACT: Aminoalcohols have been addressed as activating buffers for alkaline phosphatase. However, there is no record on the buffer activation regarding organophosphorus hydrolase (OPH). Here we reported the activating effects of aminoalcohols on OPH-catalyzed hydrolysis of diisopropylfluorophosphate (DFP), an analog molecule of G-type warfare agents. The kinetic parametors kcat, Vmax and kcat/Km in the OPH reaction were remarkably increased in the buffers (pH 8.0, 25°C) containing aminoalcohols with C2 between nitrogen (N) and oxygen (O) in their structures, including triethanolamine (TEA), diethanolamine, monoethanolamine, 1-amino-2-propanol, 2-amino-2-methyl-1-propanol, and triisopropanolamine. In contrast, much lower or no rate-enhancing effects were observed in the adding of amines, alcohols, amine/alcohol mixtures, or 3-amino-1-propanol (C3 between N and O). The 300 mM TEA further increased DFP-degrading activities of OPH mutants F132Y and L140Y, the previously reported OPH mutants with desirable activities towards DFP. However, the treatment of ethylenediaminetetraacetate (EDTA) markedly abolished the TEA-induced activation of OPH. The product fluoride effectively inhibited OPH-catalyzed hydrolysis of DFP by a linear mixed inhibition (inhibition constant Ki ~ 3.21 mM), which was partially released by TEA adding at initial or later reaction stage. The obtained results indicate the activation of OPH by aminoalcohol buffers could be attributed to the reduction of fluoride inhibition, which would be beneficial to the hydrolase-based detoxification of organophosphofluoridate.

SUBMITTER: Li D

PROVIDER: S-EPMC5234802 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Aminoalcohol-Induced Activation of Organophosphorus Hydrolase (OPH) towards Diisopropylfluorophosphate (DFP).

Li Dandan D Zhang Yunze Y Song Haitao H Lu Liangqiu L Liu Deli D Yuan Yongze Y

PloS one 20170113 1

Aminoalcohols have been addressed as activating buffers for alkaline phosphatase. However, there is no record on the buffer activation regarding organophosphorus hydrolase (OPH). Here we reported the activating effects of aminoalcohols on OPH-catalyzed hydrolysis of diisopropylfluorophosphate (DFP), an analog molecule of G-type warfare agents. The kinetic parametors kcat, Vmax and kcat/Km in the OPH reaction were remarkably increased in the buffers (pH 8.0, 25°C) containing aminoalcohols with C2 ...[more]

PMID: 28085964

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Project description:Preclinical efforts to improve medical countermeasures against organophosphate (OP) chemical threat agents have largely focused on adult male models. However, age and sex have been shown to influence the neurotoxicity of repeated low-level OP exposure. Therefore, to determine the influence of sex and age on outcomes associated with acute OP intoxication, postnatal day 28 Sprague-Dawley male and female rats were exposed to the OP diisopropylfluorophosphate (DFP; 3.4 mg/kg, s.c.) or an equal volume of vehicle (∼80 µL saline, s.c.) followed by atropine sulfate (0.1 mg/kg, i.m.) and pralidoxime (2-PAM; 25 mg/kg, i.m.). Seizure activity was assessed during the first 4 h post-exposure using behavioral criteria and electroencephalographic (EEG) recordings. At 1 d post-exposure, acetylcholinesterase (AChE) activity was measured in cortical tissue, and at 1, 7, and 28 d post-exposure, brains were collected for neuropathologic analyses. At 1 month post-DFP, animals were analyzed for motor ability, learning and memory, and hippocampal neurogenesis. Acute DFP intoxication triggered more severe seizure behavior in males than females, which was supported by EEG recordings. DFP caused significant neurodegeneration and persistent microglial activation in numerous brain regions of both sexes, but astrogliosis occurred earlier and was more severe in males compared to females. DFP males and females exhibited pronounced memory deficits relative to sex-matched controls. In contrast, acute DFP intoxication altered hippocampal neurogenesis in males, but not females. These findings demonstrate that acute DFP intoxication triggers seizures in juvenile rats of both sexes, but the seizure severity varies by sex. Some, but not all, chronic neurotoxic outcomes also varied by sex. The spatiotemporal patterns of neurological damage suggest that microglial activation may be a more important factor than astrogliosis or altered neurogenesis in the pathogenesis of cognitive deficits in juvenile rats acutely intoxicated with OPs.

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

Aminoalcohol-Induced Activation of Organophosphorus Hydrolase (OPH) towards Diisopropylfluorophosphate (DFP).

Publications

Aminoalcohol-Induced Activation of Organophosphorus Hydrolase (OPH) towards Diisopropylfluorophosphate (DFP).

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