Effect of organophosphorus hydrolysing enzymes on obidoxime-induced reactivation of organophosphate-inhibited human acetylcholinesterase.

The reactivation of organophosphate (OP)-inhibited acetylcholinesterase (AChE) by oximes results inevitably in the formation of highly reactive phosphyloximes (POX), which may re-inhibit the enzyme. An impairment of net reactivation by stable POX was found with 4-pyridinium aldoximes, e.g. obidoxime, and a variety of OP compounds. In this study the effect of organophosphorus hydrolase (OPH), organophosphorus acid anhydrolase (OPAA) and diisopropylfluorophosphatase (DFPase) on obidoxime-induced reactivation of human acetylcholinesterase (AChE) inhibited by different OPs was investigated. Reactivation of paraoxon-, sarin-, soman- and VX-inhibited AChE by obidoxime was impaired by POX-induced re-inhibition whereas no deviation of pseudo first-order kinetics was observed with tabun, cyclosarin and VR. OPH prevented (paraoxon) or markedly reduced the POX-induced re-inhibition (VX, sarin, soman), whereas OPAA and DFPase were without effect. Additional experiments with sarin-inhibited AChE indicate that the POX hydrolysis by OPH was concentration-dependent. The activity of OP-inhibited AChE was not affected by OPH in the absence of obidoxime. In conclusion, OPH may be a valuable contribution to the therapeutic regimen against OP poisoning by accelerating the degradation of both the parent compound, OP, and the reaction product, POX.

Stereochemical specificity of organophosphorus acid anhydrolase toward p-nitrophenyl analogs of soman and sarin.

Organophosphorus acid anhydrolase (OPAA) catalyzes the hydrolysis of p-nitrophenyl analogs of the organophosphonate nerve agents, sarin and soman. The enzyme is stereoselective toward the chiral phosphorus center by displaying a preference for the R(P)-configuration of these analogs. OPAA also exhibits an additional preference for the stereochemical configuration at the chiral carbon center of the soman analog. The preferred configuration of the chiral carbon center is dependent upon the configuration at the phosphorus center. The enzyme displays a two- to four-fold preference for the R(P)-enantiomer of the sarin analog. The k(cat)/K(m) of the R(P)-enantiomer is 250 M(-1) s(-1), while that of the S(P)-enantiomer is 110 M(-1) s(-1). The order of preference for the stereoisomers of the soman analog is R(P)S(C) > R(P)R(C) > S(P)R(C) > S(P)S(C). The k(cat)/K(m) values are 36,300 M(-1)s(-1), 1250 M(-1) s(-1), 80 M(-1) s(-1) and 5 M(-1) s(-1), respectively. The R(P)S(C)-isomer of the soman analog is therefore preferred by a factor of 7000 over the S(P)S(C)-isomer.

Interaction between organophosphorous hydrolase and paraoxon studied by surface chemistry in situ at air-water interface.

Subphase conditions have been optimized to obtain stable organophosphorous hydrolase (OPH-EC 3.1.8.1) as Langmuir films. The Langmuir film was characterized by surface pressure and surface potential-area isotherms and UV-Vis spectroscopy in situ. The interaction of an organophosphorous compound, namely Paraoxon, with the OPH film was investigated for various surface pressures. The stability of the monolayer and the evidence of the enzyme activity at air-water interface support the use of enzyme LB films as biosensor.

Long circulating liposomes encapsulating organophosphorus acid anhydrolase in diisopropylfluorophosphate antagonism.

These studies are focused on antagonizing organophosphorous (OP) intoxications by a new conceptual approach using recombinant enzymes encapsulated within sterically stabilized liposomes to enhance diisopropylfluorophosphate (DFP) degradation. The OP hydrolyzing enzyme, organophosphorous acid anhydrolase (OPAA), encapsulated within the liposomes, was employed either alone or in combination with pralidoxime (2-PAM) and/or atropine. The recombinant OPAA enzyme, from the ALTEROMONAS: strain JD6, has high substrate specificity toward a wide range of OP compounds, e.g., DFP, soman, and sarin. The rate of DFP hydrolysis by liposomes containing OPAA (SL)* was measured by determining the changes in fluoride-ion concentration using a fluoride ion-selective electrode. This enzyme carrier system serves as a biodegradable protective environment for the OP-metabolizing enzyme (OPAA), resulting in an enhanced antidotal protection against the lethal effects of DFP. Free OPAA alone showed some antidotal protection; however, the protection with 2-PAM and/or atropine was greatly enhanced when combined with (SL)*.

In vitro studies on sterically stabilized liposomes (SL) as enzyme carriers in organophosphorus (OP) antagonism.

This study describes a new approach for organophosphorous (OP) antidotal treatment by encapsulating an OP hydrolyzing enzyme, OPA anhydrolase (OPAA), within sterically stabilized liposomes. The recombinant OPAA enzyme was derived from Alteromonas strain JD6. It has broad substrate specificity to a wide range of OP compounds: DFP and the nerve agents, soman and sarin. Liposomes encapsulating OPAA (SL)* were made by mechanical dispersion method. Hydrolysis of DFP by (SL)* was measured by following an increase of fluoride ion concentration using a fluoride ion selective electrode. OPAA entrapped in the carrier liposomes rapidly hydrolyze DFP, with the rate of DFP hydrolysis directly proportional to the amount of (SL)* added to the solution. Liposomal carriers containing no enzyme did not hydrolyze DFP. The reaction was linear and the rate of hydrolysis was first order in the substrate. This enzyme carrier system serves as a biodegradable protective environment for the recombinant OP-metabolizing enzyme, OPAA, resulting in prolongation of enzymatic concentration in the body. These studies suggest that the protection of OP intoxication can be strikingly enhanced by adding OPAA encapsulated within (SL)* to pralidoxime and atropine.

Substrate and stereochemical specificity of the organophosphorus acid anhydrolase from Alteromonas sp. JD6.5 toward p-nitrophenyl phosphotriesters.

The enzyme OPAA hydrolyzes p-nitrophenyl phosphotriesters bearing substituents at the phosphorus center ranging in size from methyl to phenyl. The enzyme exhibits stereoselectivity toward the hydrolysis of chiral substrates with a preference for the Sp enantiomer.

Alteromonas prolidase for organophosphorus G-agent decontamination.

Enzymes catalyzing the hydrolysis of highly toxic organophosphorus compounds (OPs) are classified as organophosphorus acid anhydrolases (OPAA; EC 3.1.8.2). Recently, the genes encoding OPAA from two species of Alteromonas were cloned and sequenced. Sequence and biochemical analyses of the cloned genes and enzymes have established Alteromonas OPAAs to be prolidases (E.C. 3.4.13.9), a type of dipeptidase hydrolyzing dipeptides with a prolyl residue in the carboxyl-terminal position (X-Pro). Alteromonas prolidases hydrolyze a broad range of G-type chemical warfare (CW) nerve agents. Efforts to over-produce a prolidase from A. sp.JD6.5 with the goal of developing strategies for long-term storage and decontamination have been successfully achieved. Large-scale production of this G-agent degrading enzyme is now feasible with the availability of an over-producing recombinant cell line. Use of this enzyme for development of a safe and non-corrosive decontamination system is discussed.

phnE and glpT genes enhance utilization of organophosphates in Escherichia coli K-12.

Wild-type Escherichia coli K-12 strain JA221 grows poorly on low concentrations (< or = 1 mM) of diisopropyl fluorophosphate and its hydrolysis product, diisopropyl phosphate (DIPP), as sole phosphorus sources. Spontaneous organophosphate utilization (OPU) mutants were isolated that efficiently utilized these alternate sources of phosphate. A genomic library was constructed from one such OPU mutant, and two genes were isolated that conferred the OPU phenotype to strain JA221 upon transformation. These genes were identified as phnE and glpT. The original OPU mutation represented phnE gene activation and corresponded to the same 8-bp unit deletion from the cryptic wild-type E. coli K-12 phnE gene that has been shown previously to result in phnE activation. In comparison, sequence analysis revealed that the observed OPU phenotype conferred by the glpT gene was not the result of a mutation. PCR clones of glpT from both the mutant and the wild type were found to confer the OPU phenotype to JA221 when they were present on the high-copy-number pUC19 plasmid but not when they were present on the low-copy-number pWSK29 plasmid. This suggests that the OPU phenotype associated with the glpT gene is the result of amplification and overproduction of the glpT gene product. Both the active phnE and multicopy glpT genes facilitated effective metabolism of low concentrations of DIPP, whereas only the active phnE gene could confer the ability to break down a chromogenic substrate, 5-bromo-4-chloro-3-indoxyl phosphate-p-toluidine (X-Pi). This result indicates that in E. coli, X-Pi is transported exclusively by the Phn system, whereas DIPP (or its metabolite) may be transported by both Phn and Glp systems.

Nucleotide sequence of a gene encoding an organophosphorus nerve agent degrading enzyme from Alteromonas haloplanktis.

Organophosphorus acid anhydrolases (OPAA) catalyzing the hydrolysis of a variety of toxic organophosphorus cholinesterase inhibitors offer potential for decontamination of G-type nerve agents and pesticides. The gene (opa) encoding an OPAA was cloned from the chromosomal DNA of Alteromonas haloplanktis ATCC 23821. The nucleotide sequence of the 1.7 -kb DNA fragment contained the opa gene (1.3 kb) and its flanking region. We report structural and functional similarity of OPAAs from A. haloplanktis and Alteromonas sp JD6.5 with the enzyme prolidase that hydrolyzes dipeptides with a prolyl residue in the carboxyl-terminal position. These results corroborate the earlier conclusion that the OPAA is a type of X-Pro dipeptidase, and that X-Pro could be the native substrate for such an enzyme in Alteromonas cells.

Enzymatic hydrolysis of Russian-VX by organophosphorus hydrolase.

The Russian-VX (R-VX) is the principle V-type nerve agent in the chemical warfare (CW) arsenal of the Former Soviet Union. We here report the enzymatic hydrolysis of the P-S bond of Russian-VX by organophosphorus hydrolase (OPH) from Pseudomonas diminuta. While the Michaelis constant, K(m) for R-VX (474 microM), was similar to that for VX (434 microM), the Vmax for R-VX (2.1 mumoles/mg/min) was about four-fold higher compared to that for VX (0.56 mumoles/mg/min). A 50% inhibition in the rate of the enzymatic hydrolysis of R-VX was observed in the presence of 0.5% ethanol, isoamyl-alcohol, or isopropanol. The presence of acetonitrile, diethylene glycol, or methanol had marginal effects. These results comprise the first demonstration of enzymatic detoxification of R-VX.

Screening of halophilic bacteria and Alteromonas species for organophosphorus hydrolyzing enzyme activity.

Previously, a G-type nerve agent degrading enzyme activity was found in a halophilic bacterial isolate designated JD6.5. This organism was tentatively identified as an unknown species of the genus Alteromonas. In order to determine whether this type of enzyme activity was common in other species of Alteromonas, a screening program was initiated. A number of Alteromonas species and five halophilic bacterial isolates were cultured and their crude cell extracts screened for hydrolytic activity against several organophosphorus chemical agents and other related compounds. The samples were also screened for cross-reactivity with a monoclonal antibody raised against the purified enzyme from JD6.5 and for hybridization with a DNA probe based on its N-terminal amino acid sequence A wide spectrum of activities and reactivities were seen, suggesting a significant heterogeneity between the functionally similar enzymes that are present in these bacterial species. Enzymes of the type described here have considerable potential for the decontamination and demilitarization of chemical warfare agents.

Purification and properties of an organophosphorus acid anhydrase from a halophilic bacterial isolate.

A moderately halophilic bacterial isolate has been found to possess high levels of enzymatic activity against several highly toxic organophosphorus compounds. The predominant enzyme, designated organophosphorus acid anhydrase 2, has been purified 1,000-fold to homogeneity and characterized. The enzyme is a single polypeptide with a molecular weight of 60,000. With diisopropylfluorophosphate as a substrate, the enzyme has optimum activity at pH 8.5 and 50 degrees C, and it is stimulated by manganese and cobalt.

Soman-hydrolyzing and -detoxifying properties of an enzyme from a thermophilic bacterium.

An enzyme that hydrolyzes soman (1,2,2-trimethylpropyl methylphosphonofluoridate) and two other phosphonofluoridates, but does not hydrolyze DFP (diisopropylphosphorofluoridate), has been partially purified from a rod-shaped spore-forming gram-positive OT (obligate thermophilic) bacterium. The enzyme shows a marked Mn2+ stimulation, and in this and its substrate preference does not resemble the organophosphorus acid anhydrolase (sometimes termed DFPase) found in squid. Like the squid enzyme, it is not inhibited by mipafox (N,N'-diisopropylphosphordiamidofluoridate), is not inactivated by ammonium sulfate, and does hydrolyze the acetylcholinesterase-inhibitory pair of diastereoisomers of soman as well as the relatively noninhibitory pair, thus detoxifying soman. In these three properties the OT enzyme does not resemble the ubiquitous organophosphorus acid anhydrolase often purified from mammalian and bacterial sources by cold ethanol fractionation. Thus this phosphono-specific OT enzyme may have a natural substrate and a physiological role distinct from other organophosphorus acid anhydrolases.

p-cymene pathway in Pseudomonas putida: initial reactions.

Initial reactions of the p-cymene pathway induced in Pseudomonas putida PL have been reinvestigated. Oxidation of the methyl group attached to the nucleus occurs in three steps to give p-cumic acid. The substrate for the ring cleavage of 2,3-dihydroxy-p-cumate is formed from p-cumate in two reactions via a dihydrodiol intermediate (2,3-dihydroxy-4-isopropylcyclohexa-4,6-dienoate) and not as previously postulated via 3-hydroxy-p-cumate. There are three pieces of evidence for the physiological role of the dihydrodiol intermediate. (i) a mutant of P. putida PL-pT-11/43, which is unable to grow with p-cumate, accumulates a compound from p-cumate, which was identified as 2,3-dihydroxy-4-isopropylcyclohexa-4,6-dienoate. (II) This metabolite is enzymically oxidized by a nicotinamide adenine dinucleotide-dependent dehydrogenase that is present in crude extracts of the wild type and a revertant strain (PL-pT-11/43-R1) but not in the mutant. (iii) 3-Hydroxy-p-cumate does not support growth of P . putida PL-W, and it is not oxidized by cells or extracts. 3-Hydroxy-p-cumate was readily isolated as before from culture supernatants, due to its ready formation from the dihydrodiol in acid solution. Mass spectral analysis of the dihydrodiol accumulated in 18O2-enriched atmospheres showed that both hydroxyl atoms are derived from the same molecule of O2. The formation and absorbance maxima of dihydrodiols that accumulated during the growth of the mutant PL-pT-11/43 in the presence of various benzoates (or toluenes) that have substituents at the carbon 4 atom also is reported.

p-Cymene pathway in Pseudomonas putida: ring cleavage of 2,3-dihydroxy-p-cumate and subsequent reactions.

It was confirmed that 2,3-dihydroxy-p-cumate is a substrate for ring cleavage in Pseudomonas putida PL-W after growth with p-cymene or p-cumate. This compound was oxidized to pyruvate, acetaldehyde, isobutyrate, and carbon dioxide by extracts of cells, and these products appear in equimolar amounts. The transient appearance of compounds and 2,3-dihydroxy-p-cumate to a yellow intermediate (lambda max, 345 nm) without decarboxylation. Extracts of the benzene nucleus; this is followed by decarboxylation to give the 393-nm species, which gives rise to isobutyrate, acetaldehyde, and pyruvate by the hydrolytic route of meta cleavage of catechols, via 4-hydroxy-2-oxovalerate. This was confirmed with a mutant of P. putida PL-RF-1 that was unable to grow with p-cymene (or p-cumate) but was able to oxidize both compounds AND 2,3-DIHYDROXY-P-CUMATE TO A YELLOW INTERMEDIATE (LAMBDA MAX, 345 NM) WITHOUT DECARBOXYLATION. Extrats of P. putida PL-W (wild type) or a revertant of the mutant PL-RF-1 catalyzed the decarboxlation of the 345-nm intermediate with transient formation of the compound that absorbed at 393 nm. The substrate specificities of the 3,4-dioxygenative ring cleavage enzyme, and the decarboxylase were determined in crude extracts of P. putida PL-W and Pseudomonas fluorescens 007. It was conclude that 3,4-dioxygenative cleavage and decarboxylation are sequential enzyme-catalyzed reactions common to both P. putida and P. fluorescens for the oxidation of 2,3-dihydroxybenzoates. Unlike P. putida PL-W, which exclusively use the hydrolase branch, P. fluorescens 007 uses the dehydrogenase branch of the meta pathways that diverge after ring cleavage and later converge at oxoenate intermediates.