An isofenphos-methyl hydrolase (Imh) capable of hydrolyzing the P–O–Z moiety of organophosphorus pesticides containing an aryl or heterocyclic group

Abstract

Organophosphorus pesticide (OP) hydrolases play key roles in the degradation and decontamination of agricultural and household OPs and in the detoxification of chemical warfare agents. In this study, an isofenphos-methyl hydrolase gene (imh) was cloned from the isocarbophos-degrading strain of Arthrobacter sp. scl-2 using the polymerase chain reaction method. Isofenphos-methyl hydrolase (Imh) showed 98% sequence identity with the isofenphos hydrolase from Arthrobacter sp. strain B-5. Imh was highly expressed in Escherichia coli BL21 (DE3), and the His(6)-tagged Imh was purified (1.7 mg/ml) with a specific activity of 14.35 U/mg for the substrate isofenphos-methyl. The molecular mass of the denatured Imh is about 44 kDa, and the isoelectric point (pI) value was estimated to be 3.4. The optimal pH and temperature for hydrolysis of isofenphos-methyl were pH 8.0 and 35 °C, respectively. The secondary structure of Imh shows that Imh is a metallo-dependent hydrolase, and it was found that Imh was completely inhibited by the metalloprotease inhibitor 1,10-phenanthroline (0.5 mM), and the catalytic activity was restored by the subsequent addition of Zn(2+). Interestingly, Imh had a relatively broader substrate specificity and was capable of hydrolyzing 12 of the tested oxon and thion OPs with the P-O-Z moiety instead of the P-S(C)-Z moiety. Furthermore, it was found that the existence of an aryl or heterocyclic group in the leaving group (Z) is also important in determining the substrate specificity. Among all the substrates hydrolyzed by Imh, it was assumed that Imh preferred P-O-Z substrates still with a phosphamide bond (P-N), such as isofenphos-methyl, isofenphos, isocarbophos, and butamifos. The newly characterized Imh has a great potential for use in the decontamination and detoxification of agricultural and household OPs and is a good candidate for the study of the catalytic mechanism and substrate specificity of OP hydrolases.

Combined experimental and theoretical study on the removal of pollutant compounds by peroxidases: affinity and reactivity toward a bioremediation catalyst

“…showing good performance for complex systems, such as biomolecules (Besler, Merz, & Kollman, 1990;Singh & Kollman, 1984). The calculations were based on the generalized gradient approximation functional proposed by Gustin et al 1999 All transition states, intermediates and precursors involved were calculated and characterized by infra-red calculations identifying imaginary frequencies (Giacoppo et al, 2014;Li et al, 2012). Each conformer was fully optimized at DFT level (Sost, 1990) with conjugate gradient and quasi-Newton-Raphson algorithms.…”

Theoretical Studies Applied to the Evaluation of the DFPase Bioremediation Potential against Chemical Warfare Agents Intoxication

“…All transition states, intermediates and precursors involved were calculated and characterized by calculations identifying imaginary frequencies [ 5 , 34 , 35 ]. Each conformer was fully optimized at DFT level, with conjugate gradient and quasi-Newton-Raphson algorithms.…”

Diazinon degradation by a novel strain Ralstonia sp. DI-3 and X-ray crystal structure determination of the metabolite of diazinon

“…Use of enzymes in detoxification and decontamination of organophosphorus pesticides is considered a viable and environmentfriendly approach. Several organophosphorus pesticide hydrolases that are able to hydrolyze organophosphorus pesticides have been purified and characterized from microorganisms, and the genes encoding these hydrolases have been cloned (Li et al 2012;Barman et al 2014). In addition, this compound is usually named as 2-iso-4-methyl-6-hydroxypyrimidine (IMHP), a persistent, less toxic product (Bavcon et al 2003;Karpouzas and Singh 2006), allowing a partial degradation pathway for diazinon by pure culture to be proposed (figure 5).…”