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J R Wild

Publications and source records attributed to J R Wild.

At least 37 records · Page 2Linked to original sources
PubMed1995

Characterization of P-S bond hydrolysis in organophosphorothioate pesticides by organophosphorus hydrolase.

The extensive use of organophosphorothioate insecticides in agriculture has resulted in the risk of environmental contamination with a variety of broadly based neurotoxins that inhibit the acetylcholinesterases of many different animal species. Organophosphorus hydrolase (OPH, EC 3.1.8.1) is a broad-spectrum phosphotriesterase that is capable of detoxifying a variety of organophosphorus neurotoxins by hydrolyzing various phosphorus-ester bonds (P-O, P-F, P-CN, and P-S) between the phosphorus center and an electrophilic leaving group. OPH is capable of hydrolyzing the P-X bond of various organophosphorus compounds at quite different catalytic rates: P-O bonds (kcat = 67-5000 s-1), P-F bonds (kcat = 0.01-500 s-1), and P-S bonds (kcat = 0.0067 to 167 s-1). P-S bond cleavage was readily demonstrated and characterized in these studies by quantifying the released free thiol groups using 5,5'-dithio-bis-2-nitrobenzoic acid or by monitoring an upfield shift of approximately 31 ppm by 31P NMR. A decrease in the toxicity of hydrolyzed products was demonstrated by directly quantifying the loss of inhibition of acetylcholinesterase activity. Phosphorothiolate esters, such as demeton-S, provided noncompetitive inhibition for paraoxon (a P-O triester) hydrolysis, suggesting that the binding of these two different classes of substrates was not identical.

Aryldialkylphosphatase↗
PubMed1995

Hydrolysis of tetriso by an enzyme derived from Pseudomonas diminuta as a model for the detoxication of O-ethyl S-(2-diisopropylaminoethyl) methylphosphonothiolate (VX).

An enzyme termed organophosphorus hydrolase (OPH), derived from Pseudomonas diminuta, had been found previously to hydrolyze the powerful acetylcholinesterase (AChE) inhibitor O-ethyl S-(2-diisopropylaminoethyl) methylphosphonothiolate (VX). This enzyme has now been shown to be correlated with the loss of AChE inhibitory potency (detoxication). OPH also hydrolyzed and detoxified the VX analogue, O,O-diisopropyl S-(2-diisopropylaminoethyl) phosphorothiolate (Tetriso), also a potent AChE inhibitor, about five times faster than VX. The Km for the hydrolysis of the P-S bond of Tetriso was 6.7 x 10(-3) M. OPH also hydrolyzed diisopropylphosphorofluoridate (DFP) 50-60 times faster than Tetriso, and 1,2,2-trimethylpropyl methylphosphonofluoridate (Soman) about seven times faster than Tetriso. DFP was a non-competitive inhibitor of Tetriso hydrolysis, Ki = 8.7 x 10(-4) M. The DFP hydrolysis product, diisopropyl phosphate, was a competitive inhibitor, Ki = 2.3 x 10(-4) M. The rate of detoxication of Tetriso compared with the rate of hydrolysis suggests that OPH may not be totally specific for P-S bond cleavage. OPH was inhibited completely by 1.5 x 10(-4) M 8-hydroxyquinoline-5-sulfonate or 1,10-phenanthroline, both transition element chelators, but inhibited only partially by EDTA, a much more potent chelator.

Aryldialkylphosphatase↗
PubMed1994

Bimetallic binding motifs in organophosphorus hydrolase are important for catalysis and structural organization.

Organophosphorus hydrolase is a broad spectrum phosphoric acid hydrolase (EC 3.1.8.1) which appears to contain a binuclear metal center with two metals interactively involved in catalysis and/or structural functions. Site-directed mutagenesis has been employed to evaluate the participation of the various histidine and cysteine residues in metal coordination. The kinetic characteristics and metal binding stoichiometries of the purified site-directed substitutions of each of the histidine and cysteine residues in the catalytic domain of the protein to asparagine and serine residues, respectively, were determined. These data support the hypothesis that the histidines at positions 55, 57, and 201 are coordinated to a metal ion (M1) at the active center of the enzyme and that His254 and His257 are involved in the formation of a second structural metal center (M2). These and other unidentified amino acids may participate in a co-catalytic center. Although previous solution chemical studies concluded that cysteines are not involved in metal coordination, serine substitutions for Cys59 and Cys227 do affect metal content and catalytic activity. In contrast, substitution of asparagine for His230 does not affect the metal stoichiometry, but does reduce the kcat by 10(-4), indicating that it may be directly involved in the reaction chemistry. The H201N substitution eliminates activity but maintains one molar equivalent of metal and may function as a bridging ligand.

Amino Acid Sequence↗
PubMed1994

Encapsulation of phosphotriesterase within murine erythrocytes.

A new conceptual approach was employed to antagonize organophosphorus intoxication by using resealed carrier erythrocytes containing a recombinant phosphotriesterase. This enzyme has been reported to hydrolyze many organophosphorus compounds, including paraoxon, a potent cholinesterase inhibitor. Paraoxon is rapidly hydrolyzed by this enzyme to p-nitrophenol and diethylphosphate. Incorporation of phosphotriesterase within resealed murine erythrocytes was accomplished by hypotonic dialysis. The properties of this enzyme within these resealed erythrocytes were investigated. Addition of paraoxon to reaction mixtures containing these resealed erythrocytes loaded with phosphotriesterase resulted in the rapid hydrolysis of paraoxon. Hydrolysis of paraoxon did not occur when these carrier erythrocytes contained no phosphotriesterase. These in vitro studies suggest that carrier erythrocytes may be developed as an approach for the prophylactic and therapeutic antagonism of organophosphorus intoxication.

Animals↗
PubMed1994

Expression of organophosphate hydrolase in the filamentous fungus Gliocladium virens.

The broad-spectrum organophosphate hydrolase (OPH; EC 3.1.8.1) encoded by the organophosphate-degrading gene (opd) from Pseudomonas diminuta MG and Flavobacterium sp. ATCC 27551 possesses capabilities of both P-O bond hydrolysis (e.g. paraoxon) and P-F bond hydrolysis [e.g. sarin and diisopropylfluorophosphate (DFP)]. In the present study a 9.4-kb plasmid, pCL1, was used to transform the saprophytic fungus Gliocladium virens. pCL1 was derived from pJS294 by placing the fungal promoter (prom1) from Cochliobolus heterostrophus upstream and the trpC terminator from Aspergillus nidulans down-stream of the opd gene. Southern analysis of restricted genomic DNA from various transformants indicated that integration occurred non-specifically at multiple sites. Western blot analysis of mycelial extracts from transformants confirmed the production of a processed form of the enzyme in the fungus. Maximal levels of OPH activity (rate of p-nitrophenol production from paraoxon) were observed after 168 h of culture and activity levels correlated with biomass production in mature vegetative growth.

Blotting, Southern↗
PubMed1994

Expression and post-translational processing of a broad-spectrum organophosphorus-neurotoxin-degrading enzyme in insect tissue culture.

A recombinant baculovirus, Autographa californica nuclear polyhedrosis virus (AcNPV), has been utilized to express the opd (organophosphate-degrading) gene from Pseudomonas diminuta in insect tissue-culture cells (Sf9) of the fall armyworm (Spodoptera frugiperda). The broad-spectrum organophosphate hydrolase (EC 3.1.8.1) encoded by this gene is a member of a general class of enzymes [organophosphate (OP) anhydrolases] that include parathion hydrolases, di-isopropyl-fluorophosphatases (DFPases), somanases, and OP phosphotriesterases. This particular enzyme possesses the ability to hydrolyse paraoxon (P-O bond), DFP, sarin (P-F bond), VX (P-S bond) and tabun (P-CN bond), as well as a number of other extensively used organophosphorus pesticides. The enzyme produced in infected Sf9 cells is post-translationally processed and resembles the mature form of the enzyme expressed in various bacterial cells as identified by immunoprecipitation on Western blots. N-terminal sequence analysis of enzyme expressed in insect cells revealed Gly-29 as the terminal residue, whereas expression in Escherichia coli removes this residue, exposing Ser-30 at the N-terminus. Conditions for optimal expression of the enzyme in this system are described. Furthermore, hydrolytic efficiency of some OPs with purified enzyme from this system is discussed in relation to the in situ activity of Pseudomonas diminuta MG cells.

Animals↗
PubMed1993

Site-directed alterations to the geometry of the aspartate transcarbamoylase zinc domain: selective alteration to regulation by heterotropic ligands, isoelectric point, and stability in urea.

Structural aspects requisite for allosteric function in the regulatory chain of aspartate transcarbamoylase were explored by site-specific amino acid insertion or substitution within the zinc domain in the region of contact between the catalytic and regulatory chains. Amino acid substitution at two positions yielded enzymes which retained a maximum velocity similar to that of the wild-type enzyme but responded differently from the native enzyme in the presence of regulatory nucleoside triphosphates. A change of zinc coordinate amino acid C109 to histidine and a change of E119 to aspartic acid resulted in enzymes which demonstrated synergistic inhibition by CTP and UTP but not inhibition by CTP in either phosphate buffer or a morpholino-based tri-partate (TP) buffer at pH 7. At pH 8.3, where there is a higher proportion of T-state conformers in the native enzyme, the mutants diverged from their similar kinetic behavior. C109H remained an enzyme which was not inhibited by CTP but was still inhibited by CTP+UTP. E119D was inhibited by both CTP and CTP+UTP. Activation of the mutants by ATP was found to vary either with pH or with phosphate as a buffer component. C109H was activated by ATP in phosphate, while in TP at either pH 7 or 8.3 its activation by ATP was diminished or absent. E119D was activated by ATP in phosphate at pH 7 or in TP at pH 8.3, but not in TP at pH 7. In TP at pH 7, where neither mutant was activated by ATP, the S values and Hill coefficients of the unliganded mutant enzymes resembled those of the ATP-liganded wild-type enzyme. While neither mutation would be predicted to alter the net charge of the holoenzyme, differences in the isoelectric point of the mutants were observed if phosphate was present. This result suggests that the isoelectric point of aspartate transcarbamoylase is conformationally dependent and that the mutants exist in an altered conformation. In addition, the stabilities of both mutant holoenzymes were reduced substantially from those of the wild-type enzyme in 4 M urea. C109H was more stable at pH 8.25 in a Tris buffer; E119D was more stable at pH 7 in the phosphate buffer. Potential effects of these mutations on the active site chemistry and geometry are discussed.

Allosteric Regulation↗
PubMed1993

Characterization of organophosphorus hydrolases and the genetic manipulation of the phosphotriesterase from Pseudomonas diminuta.

There are a variety of enzymes which are specifically capable of hydrolyzing organophosphorus esters with different phosphoryl bonds from the typical phosphotriester bonds of common insecticidal neurotoxins (e.g. paraoxon or coumaphos) to the phosphonate-fluoride bonds of chemical warfare agents (e.g. soman or sarin). These enzymes comprise a diverse set of enzymes whose basic architecture and substrate specificities vary dramatically, yet they appear to be ubiquitous throughout nature. The most thoroughly studied of these enzymes is the organophosphate hydrolase (opd gene product) of Pseudomonas diminuta and Flavobacterium sp. ATCC 27551, and the heterologous expression, post-translational modification, and genetic engineering studies undertaken with this enzyme are described.

Animals↗
PubMed1991

'All is not well'.

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Diffusion of Innovation↗
PubMed1990

Expression of Pseudomonas phosphotriesterase activity in the fall armyworm confers resistance to insecticides.

The gene encoding for the phosphotriesterase (opd) from Pseudomonas diminuta has been subcloned into a baculovirus expression system. Functional enzyme is produced when the recombinant baculovirus is used to infect either cultured Spodoptera frugiperda sf9 cells or the larval stage of the fall armyworm. The LD50 for paraoxon toxicity was found to increase 280-fold in the larvae after infection with the recombinant baculovirus and expression of the functional phosphotriesterase.

Animals↗
PubMed1990

Inactivation of organophosphorus nerve agents by the phosphotriesterase from Pseudomonas diminuta.

The phosphotriesterase from Pseudomonas diminuta was tested as a catalyst for the hydrolysis of phosphofluoridates. The purified enzyme has been shown to hydrolyze the phosphorus-fluorine bond of diisopropyl fluorophosphate, isopropyl methylphosphonofluoridate, and 1,2,2-trimethylpropylmethylphosphonofluoridate at pH 7.0, 25 degrees C, with turnover numbers of 41, 56, and 5 s-1, respectively. The enzymatic rate enhancement for the hydrolysis of sarin at pH 7.0 is 2.2 X 10(7). The turnover number for paraoxon hydrolysis is 2100 s-1. The enzyme does not hydrolyze methanesulfonyl fluoride, phenylmethylsulfonyl fluoride, or O-p-nitrophenyl phenylsulfonate nor do these compounds inactivate or inhibit the ability of the enzyme to hydrolyze diethyl p-nitrophenyl phosphate. The breadth of substrate utility and the efficiency of the hydrolytic reaction exceed the more limited abilities of other prokaryotic and eukaryotic enzymes that catalyze similar reactions. The substantial rate enhancement exhibited by this enzyme for the hydrolysis of a wide variety of organophosphorus nerve agents make this enzyme the prime candidate for the biological detoxification of insecticide and mammalian acetylcholinesterase inhibitors.

Animals↗
PubMed1990

Transfer and expression of an organophosphate insecticide-degrading gene from Pseudomonas in Drosophila melanogaster.

The organophosphorus acid hydrolases represent a distinct class of enzymes that catalyze the hydrolysis of a variety of organophosphate substrates, including many insecticides and their structural analogues. The plasmid-borne opd gene of Pseudomonas diminuta strain MG specifies an organophosphorus acid hydrolase, a phosphotriesterase, that has been well characterized and can hydrolyze a broad spectrum of insect and mammalian neurotoxins. The in situ functioning of this enzyme in the metabolism of organophosphates has been analyzed directly in insects by transferring the opd gene into embryos of Drosophila melanogaster by P element-mediated transformation. The chromosomal locations of this stably inherited transgenic locus differed from strain to strain and demonstrated various expressivity on the whole-insect basis. Transcriptional induction of opd in one of these strains under control of the Drosophila heat shock promoter, hsp70, resulted in the synthesis of stable active enzyme that accumulated to high levels with repeated induction. The heat shock-induced synthesis of organophosphorus acid hydrolases in transgenic flies conferred enhanced resistance to toxic paralysis by the organophosphate insecticide paraoxon.

Animals↗
PubMed1989

Purification and properties of the phosphotriesterase from Pseudomonas diminuta.

The phosphotriesterase produced from the opd cistron of Pseudomonas diminuta was purified 1500-fold to homogeneity using a combination of gel filtration, ion exchange, hydrophobic, and dye matrix chromatographic steps. This is the first organophosphate triesterase or organophosphofluoridate hydrolyzing enzyme to be purified to homogeneity. The enzyme is a monomeric, spherical protein having a molecular weight of 39,000. A single zinc atom is bound to the enzyme and is required for catalytic activity. Incubation with metal chelating compounds, o-phenanthroline, EDTA, or 2,6-pyridine dicarboxylate inactivate the enzyme. The kinetic rate constants, kcat and kcat/Km, for the hydrolysis of paraoxon are 2100 s-1 and 4 x 10(7) M-1 s-1, respectively. The enzyme is inhibited competitively by dithiothreitol, dithioerithritol, and beta-mercaptoethanol. In addition to paraoxon the phosphotriesterase was found to hydrolyze the commonly used organophosphorus insecticides, dursban, parathion, coumaphos, diazinon, fensulfothion, methyl parathion, and cyanophos.

Amino Acids↗
PubMed1989

Comparison of the aspartate transcarbamoylases from Serratia marcescens and Escherichia coli.

The aspartate transcarbamoylases (ATCase, EC 2.1.3.2) of Escherichia coli and Serratia marcescens have similar dodecameric enzyme structures (2(c3):3(r2] but differ in both regulatory and catalytic characteristics. The catalytic cistrons (pyrB) of the ATCases from E. coli and S. marcescens encode polypeptides of 311 and 306 amino acids, respectively; there is a 76% identity between the DNA sequences and an overall amino acid homology of 88% (38 differences). The regulatory cistrons (pyrI) of these ATCases encode polypeptides of 153 and 154 amino acids, respectively, and there is a 75% identity between the DNA sequences and an overall amino acid homology of 77% (36 differences). In both species, the two genes are arranged as a bicistronic operon, with pyrB promoter proximal. A comparison of the deduced amino acid sequences reveals that the active site and the allosteric binding sites, as well as most of the intrasubunit interactions and intersubunit associations, are conserved in the E. coli and the S. marcescens enzymes; however, there are specific differences which undoubtedly contribute to the catalytic and regulatory differences between the enzymes of the two species. These differences include residues that have been implicated in the T-R transition, c1:r1 interface interactions, and the CTP binding site. A hybrid ATCase assembled in vivo with catalytic subunits from E. coli and regulatory subunits from S. marcescens has a 6 mM requirement for aspartate at half-maximal saturation, similar to the 5.5 mM aspartate requirement of the native E. coli holoenzyme at half-maximal saturation. However, the heterotropic response of this hybrid enzyme is characteristic of the heterotropic response of the native S. marcescens holoenzyme: ATP activation and CTP activation. Activation by both allosteric effectors indicates that the heterotropic response of this hybrid holoenzyme (Cec:Rsm) is determined by the associated S. marcescens regulatory subunits.

Amino Acid Sequence↗
PubMed1989

Discrimination between nucleotide effector responses of aspartate transcarbamoylase due to a single site substitution in the allosteric binding site.

The substitution of alanine for lysine at position 56 of the regulatory polypeptide of aspartate transcarbamoylase affected both homotropic and heterotropic characteristics. In the absence of effectors, the ALAr56-substituted holoenzyme lost the homotropic cooperativity observed for aspartate in the wild-type holoenzyme. Under conditions of allosteric inhibition in the presence of 2mM CTP, the cooperative character of ATCase was restored, and the Hill coefficient increased from 1.0 to 1.7. In contrast to the native enzyme, the altered enzyme did not respond to ATP; however, ATP could still bind to the enzyme as demonstrated by its direct competition with CTP. Furthermore, the recently observed CTP-UTP synergism of the wild-type enzyme was not detectable. The site-directed mutant enzyme could not be activated by low levels of the bisubstrate analogue, N-(phosphonacetyl)-L-aspartate, and the rate of association of pHMB with the cysteine residues located at the interface of the catalytic and regulatory chains was slightly altered. These characteristics suggested that the mutant holoenzyme assumed a relaxed (or abnormal T state) conformation. Thus, this single substitution differentially affected the heterotropic responses to the various allosteric effectors of ATCase and eliminated the homotropic characteristics in response to aspartate in the absence of CTP.

Adenosine Triphosphate↗
PubMed1989

Reconstruction of an enzyme by domain substitution effectively switches substrate specificity.

The polar domains of the two transcarbamoylases, aspartate transcarbamoylase (ATCase) and ornithine transcarbamoylase, (OTCase) from Escherichia coli bind the common substrate carbamoyl phosphate and share extensive amino-acid sequence homology. The equatorial domains of the two enzymes differ in their substrate specificity (ATCase binds aspartate, OTCase binds ornithine) and have decreased sequence identity. While addressing the conservation of specific protein interactions during the evolution of these enzymes, we were able to switch one of their amino-acid-specific equatorial domains to produce a viable chimaeric enzyme. This was achieved by the in vitro fusion of DNA encoding the polar domain of OTCase to DNA encoding the equatorial domain of ATCase. The resulting gene fusion successfully transformed an argI-pyrB deletion strain of E. coli to pyrimidine prototrophy, giving rise to Pyr+ transformants that expressed ATCase but not OTCase activity. The formation of this active chimaeric enzyme shows that by exchanging protein domains between two functionally divergent enzymes we have achieved a switching in substrate specificity.

Aspartate Carbamoyltransferase↗