Bacterial detoxification of organophosphate nerve agents

Bacterial enzymes have been isolated that catalyze the hydrolysis of organophosphate nerve agents with high-rate enhancements and broad substrate specificity. Mutant forms of these enzymes have been constructed through rational redesign of the active-site binding pockets and random mutagenesis to create protein variants that are optimized for the detoxification of agricultural insecticides and chemical warfare agents. In this review, the catalytic properties of two bacterial enzymes, phosphotriesterase and organophosphorus anhdrolase, are examined for their ability to hydrolyze organophosphate nerve agents.     

Enzymes for the homeland defense: optimizing phosphotriesterase for the hydrolysis of organophosphate nerve agents

Phosphotriesterase (PTE) from soil bacteria is known for its ability to catalyze the detoxification of organophosphate pesticides and chemical warfare agents. Most of the organophosphate chemical warfare agents are a mixture of two stereoisomers at the phosphorus center, and the S(P)-enantiomers are significantly more toxic than the R(P)-enantiomers. In previous investigations, PTE variants were created through the manipulation of the substrate binding pockets and these mutants were shown to have greater catalytic activities for the detoxification of the more toxic S(P)-enantiomers of nerve agent analogues for GB, GD, GF, VX, and VR than the less toxic R(P)-enantiomers. In this investigation, alternate strategies were employed to discover additional PTE variants with significant improvements in catalytic activities relative to that of the wild-type enzyme. Screening and selection techniques were utilized to isolate PTE variants from randomized libraries and site specific modifications. The catalytic activities of these newly identified PTE variants toward the S(P)-enantiomers of chromophoric analogues of GB, GD, GF, VX, and VR have been improved up to 15000-fold relative to that of the wild-type enzyme. The X-ray crystal structures of the best PTE variants were determined. Characterization of these mutants with the authentic G-type nerve agents has confirmed the expected improvements in catalytic activity against the most toxic enantiomers of GB, GD, and GF. The values of k(cat)/K(m) for the H257Y/L303T (YT) mutant for the hydrolysis of GB, GD, and GF were determined to be 2 × 10(6), 5 × 10(5), and 8 × 10(5) M(-1) s(-1), respectively. The YT mutant is the most proficient enzyme reported thus far for the detoxification of G-type nerve agents. These results support a combinatorial strategy of rational design and directed evolution as a powerful tool for the discovery of more efficient enzymes for the detoxification of organophosphate nerve agents.     

Microbial degradation of organophosphorus compounds

Synthetic organophosphorus compounds are used as pesticides, plasticizers, air fuel ingredients and chemical warfare agents. Organophosphorus compounds are the most widely used insecticides, accounting for an estimated 34% of world-wide insecticide sales. Contamination of soil from pesticides as a result of their bulk handling at the farmyard or following application in the field or accidental release may lead occasionally to contamination of surface and ground water. Several reports suggest that a wide range of water and terrestrial ecosystems may be contaminated with organophosphorus compounds. These compounds possess high mammalian toxicity and it is therefore essential to remove them from the environments. In addition, about 200,000 metric tons of nerve (chemical warfare) agents have to be destroyed world-wide under Chemical Weapons Convention (1993). Bioremediation can offer an efficient and cheap option for decontamination of polluted ecosystems and destruction of nerve agents. The first micro-organism that could degrade organophosphorus compounds was isolated in 1973 and identified as Flavobacterium sp. Since then several bacterial and a few fungal species have been isolated which can degrade a wide range of organophosphorus compounds in liquid cultures and soil systems. The biochemistry of organophosphorus compound degradation by most of the bacteria seems to be identical, in which a structurally similar enzyme called organophosphate hydrolase or phosphotriesterase catalyzes the first step of the degradation. organophosphate hydrolase encoding gene opd (organophosphate degrading) gene has been isolated from geographically different regions and taxonomically different species. This gene has been sequenced, cloned in different organisms, and altered for better activity and stability. Recently, genes with similar function but different sequences have also been isolated and characterized. Engineered microorganisms have been tested for their ability to degrade different organophosphorus pollutants, including nerve agents. In this article, we review and propose pathways for degradation of some organophosphorus compounds by microorganisms. Isolation, characterization, utilization and manipulation of the major detoxifying enzymes and the molecular basis of degradation are discussed. The major achievements and technological advancements towards bioremediation of organophosphorus compounds, limitations of available technologies and future challenge are also discussed.     

Allyl sulfur compounds and cellular detoxification system: effects and perspectives in cancer therapy

Natural organosulfur compounds (OSCs) have been shown to have chemopreventive effects and to suppress the proliferation of tumor cells in vitro through the induction of apoptosis. The biochemical mechanisms underlying the antitumorigenic and anti-proliferative effects of garlic-derived OSCs are not fully understood. Several modes of action of these compounds have been proposed, and it seems likely that the rate of clearance of allyl sulfur groups from cells is a determinant of the overall response. The aim of this review is to focus attention on the effects of natural allyl sulfur compounds on the cell detoxification system in normal and tumor cells. It has been already reported that several natural allyl sulfur compounds induce chemopreventive effects by affecting xenobiotic metabolizing enzymes and inducing their down-activation. Moreover, different effects of water- and oil-soluble allyl sulfur compounds on enzymes involved in the detoxification system of rat tissues have been observed. A direct interaction of the garlic allyl sulfur compounds with proteins involved in the detoxification system was studied in order to support the hypothesis that proteins possessing reactive thiol groups and that are involved in the detoxification system and in the cellular redox homeostasis, are likely the preferential targets of these compounds. The biochemical transformation of the OSCs in the cell and their adducts with thiol functional groups of these proteins, could be considered relevant events to uncover the anticancer properties of the allyl sulfur compounds. Although additional studies, using proteomic approaches and transgenic models, are needed to identify the molecular targets and modes of action of these natural compounds, the allyl sulfur compounds can represent potential ideal agents in anticancer therapy, either alone or in association with other antitumor drugs.     

The bioorganic chemistry of the nitroalkyl group

Nitroalkyl groups are conspicuously rare among pharmaceutical agents, and the bioactivity of substituted nitroalkanes has been described in few instances. This article examines the natural occurrence of such compounds and the reactions of nitroalkyl compounds with enzymes. The discussion is not limited to nitro hydrocarbons; the term “nitroalkyl” is intended to distinguish the compounds from nitroaromatic compounds, which are better known in pharmacology and involve different chemical considerations. Further study of the bioorganic chemistry of the nitroalkyl group may contribute to the understanding of biosynthetic strategies and enzymatic catalysis and may permit the rational design of useful bioactive molecules.     

Reduction of polynitroaromatic compounds: the bacterial nitroreductases

Most nitroaromatic compounds are toxic and mutagenic for living organisms, but some microorganisms have developed oxidative or reductive pathways to degrade or transform these compounds. Reductive pathways are based either on the reduction of the aromatic ring by hydride additions or on the reduction of the nitro groups to hydroxylamino and/or amino derivatives. Bacterial nitroreductases are flavoenzymes that catalyze the NAD(P)H-dependent reduction of the nitro groups on nitroaromatic and nitroheterocyclic compounds. Nitroreductases have raised a great interest due to their potential applications in bioremediation, biocatalysis, and biomedicine, especially in prodrug activation for chemotherapeutic cancer treatments. Different bacterial nitroreductases have been purified and their biochemical and kinetic parameters have been determined. The crystal structure of some nitroreductases have also been solved. However, the physiological role(s) of these enzymes remains unclear. Nitroreductase genes are widely spread within bacterial genomes, but are also found in archaea and some eukaryotic species. Although studies on regulation of nitroreductase gene expression are scarce, it seems that nitroreductase genes may be controlled by the MarRA and SoxRS regulatory systems that are involved in responses to several antibiotics and environmental chemical hazards and to specific oxidative stress conditions. This review covers the microbial distribution, types, biochemical properties, structure and regulation of the bacterial nitroreductases. The possible physiological functions and the biotechnological applications of these enzymes are also discussed.     

Organophosphate-Hydrolyzing Enzymes as First-Line of Defence Against Nerve Agent-Poisoning: Perspectives and the Road Ahead

Nerve agents (NAs) are extremely neurotoxic synthetic organophosphate (OP) compounds exploited as weapons of mass destruction in terrorist attacks and chemical warfare. Considering the current world scenario, there is a persistent threat of NA-exposure to military personals and civilians. Various prophylactic and post-exposure treatments (such as atropine and oximes) available currently for NA-poisoning are inadequate and unsatisfactory and suffer from severe limitations. Hence, developing safe and effective treatment(s) against NA-poisoning is a critical necessity. With regards to counteracting NA-toxicity, the OP-hydrolyzing enzymes (OPHEs), which can hydrolyze and inactivate a variety of NAs, have emerged as promising candidates for the development of prophylactic therapy against NA-poisoning. However, there are many hurdles to be crossed before these enzymes can be brought to therapeutic use in humans. In this article, we have reviewed the various advancements in the field of development of OPHEs as prophylactic against NA-poisoning. The article majorly focuses on the toxic effects of NAs, various available therapies to counteract NA poisoning, the current status of OPHEs and attempts made to improve the various properties of these enzymes. Further, we have also briefly discussed about the prospective work that is needed to be undertaken for developing these OPHEs into those suitable for use in humans.     

The enzymatic synthesis of antiviral agents.

The majority of potential antiviral agents which are currently undergoing clinical trials are inhibitors of the replication of nucleic acids. The most common class of these inhibitors are nucleoside analogues and the elucidation of synthetic routes to these compounds has been of interest for many years as many are anticancer agents. One synthetic development has been the application of bio-transformations to nucleoside syntheses. This topic has been reviewed recently (Shirae et al., 1991) but this review is not widely available. In the present review, the application of biotechnology to the synthesis of antiviral agents including those which are not nucleoside analogues will be discussed. Enzymatic syntheses of nucleosides can be simpler and quicker than syntheses carried out by chemical methods. The most useful enzymes are those found in catabolic pathways. Nucleoside phosphorylases and N-deoxyribosyltransferases have both been widely used for nucleoside synthesis catalysing the transfer of sugar residues from a donor nucleoside to a heterocyclic base. Enzymatic methods have also been applied to the resolution of racemic mixtures and adenosine deaminase is a convenient catalyst for the hydrolysis of amino groups on purines and purine analogues. Regioselective deprotection of nucleoside esters has been achieved with lipases and these enzymes have also been applied to the synthesis of esters of sugar-like alkaloids. The latter have potential as inhibitors of the replication of HIV. Esterases have also been used in combined chemical and enzymatic syntheses of organophosphorus antiviral agents.     

Enzymatic detoxification of cyanide: clues from Pseudomonas aeruginosa Rhodanese

Cyanide is a dreaded chemical because of its toxic properties. Although cyanide acts as a general metabolic inhibitor, it is synthesized, excreted and metabolized by hundreds of organisms, including bacteria, algae, fungi, plants, and insects, as a mean to avoid predation or competition. Several cyanide compounds are also produced by industrial activities, resulting in serious environmental pollution. Bioremediation has been exploited as a possible alternative to chemical detoxification of cyanide compounds, and various microbial systems allowing cyanide degradation have been described. Enzymatic pathways involving hydrolytic, oxidative, reductive, and substitution/transfer reactions are implicated in detoxification of cyanide by bacteria and fungi. Amongst enzymes involved in transfer reactions, rhodanese catalyzes sulfane sulfur transfer from thiosulfate to cyanide, leading to the formation of the less toxic thiocyanate. Mitochondrial rhodanese has been associated with protection of aerobic respiration from cyanide poisoning. Here, the biochemical and physiological properties of microbial sulfurtransferases are reviewed in the light of the importance of rhodanese in cyanide detoxification by the cyanogenic bacterium Pseudomonas aeruginosa. Critical issues limiting the application of a rhodanese-based cellular system to cyanide bioremediation are also discussed.     

Chemical induction of streptomycin-resistant mutations in Escherichia coli. Dose and mutagenic effects of dichlorvos and methyl methanesulfonate

A procedure for the quantitative determination of induced streptomycin-resistant mutants in E. coli was applied to study and compare mutation induction by the organophosphate dichlorvos and by methyl methanesulfonate (MMS). Both compounds increased the frequency of mutants even under conditions where no inactivation of cell was observed. Mutation induction by these agents as a function of both concentration and exposure time was measured. The dose-response curves found with both mutagens were non-linear; atp higher doses more mutants were induced per unit dose than at lower doses. Possible relationships between dose-effect curves and the chemical nature of alkylating mutagenic agents are discussed.     

Generation of a mutagenized organophosphorus hydrolase for the biodegradation of the organophosphate pesticides malathion and demeton‐S

Aims: The bacterial organophosphorus hydrolase (OPH) enzyme hydrolyses and detoxifies a broad range of toxic organophosphate pesticides and warfare nerve agents by cleaving the various phosphorus‐ester bonds (P–O, P–F, P–CN, P–S); however, OPH hydrolyses these bonds with varying efficiencies. The aim of this study was to generate a variant OPH enzyme with improved hydrolytic efficiency against the poorly hydrolysed P–S class of organophosphates. Methods and Results: The gene encoding OPH was sequentially mutated at specific codons by saturation mutagenesis and screened for improved activity against the P–S substrates demeton‐S methyl and malathion. Escherichia coli lysates harbouring the variants displayed up to 177‐ and 1800‐fold improvement in specific activity against demeton‐S methyl and malathion, respectively, compared to the wild‐type lysates. The specificity constants of the purified variant proteins were improved up to 25‐fold for demeton‐S methyl and malathion compared to the wild‐type. Activity was associated with organophosphate detoxification as the hydrolysed substrate lost the ability to inhibit acetylcholinesterase. The improved hydrolytic efficiency against demeton‐S translated to the improved ability to hydrolyse the warfare agent VX. Conclusions: OPH variant enzymes were generated that displayed significantly improved ability to hydrolyse and detoxify organophosphates harbouring the P–S bond. Significance and Impact of the Study: The long‐term goal is to generate an environmentally‐friendly enzyme‐mediated bioremediation approach for the removal of toxic organophosphate compounds in the environment.     

Formation and detoxification of reactive intermediates in the metabolism of chlorinated ethenes

Short-chain halogenated aliphatics, such as chlorinated ethenes, constitute a large group of priority pollutants. This paper gives an overview on the chemical and physical properties of chlorinated aliphatics that are critical in determining their toxicological characteristics and recalcitrance to biodegradation. The toxic effects and principle metabolic pathways of halogenated ethenes in mammals are briefly discussed. Furthermore, the bacterial degradation of halogenated compounds is reviewed and it is described how product toxicity may explain why most chlorinated ethenes are only degraded cometabolically under aerobic conditions. The cometabolic degradation of chlorinated ethenes by oxygenase-producing microorganisms has been extensively studied. The physiology and bioremediation potential of methanotrophs has been well characterized and an overview of the available data on these organisms is presented. The sensitivity of methanotrophs to product toxicity is a major limitation for the transformation of chlorinated ethenes by these organisms. Most toxic effects arise from the inability to detoxify the reactive chlorinated epoxyethanes occurring as primary metabolites. Therefore, the last part of this review focuses on the metabolic reactions and enzymes that are involved in the detoxification of epoxides in mammals. A key role is played by glutathione S-transferases. Furthermore, an overview is presented on the current knowledge about bacterial enzymes involved in the metabolism of epoxides. Such enzymes might be useful for detoxifying chlorinated ethene epoxides and an example of a glutathione S-transferase with activity for dichloroepoxyethane is highlighted.     

Conjugating Enzymes Involved in Xenobiotic Metabolism of Organic Xenobiotics in Plants

Phytoremediation of organic pollutants has become a topic of great interest in many countries due to the increasing number of recorded spill sites. When applying plant remediation techniques to unknown pollutant mixtures, information on the uptake rates as well as on the final fate of the compounds is generally lacking. A range of compounds are easily taken up by plants, whereas others may stay motionless and recalcitrant in the soil or sediment. Uptake is a necessary prerequisite for close contact between the pollutant and the detoxifying enzymes of plants that are localized in the cytosol of living cells. The presence and activity of these enzymes is crucial for a potential metabolization and further degradation of the chemicals under consideration. Conjugation to biomolecules is regarded as a beneficial detoxification reaction. The present review summarizes several prerequisites for pollutant uptake and discusses information on conjugating detoxification reactions. The final fate of compounds is critically discussed and perspectives for phytoremediation are given.     

Studies on the distribution of a naturally occurring nltroaliphatic acid in crownvetch (Coronilla Varia,Fabaceae)

The biosynthesis of nitro compounds and their esterification to glucose moieties are rare phenomena in the plant kingdom. Crownvetch (C. varia, Fabaceae) has not only these special biosynthetic capabilities, but also a nitro compound degrading system of interest to the energetic materials utilizing sectors. In this study we extend the observations on 3-nitropropionic acid content in crownvetch to pods and seedling tissues. The seedlings are recommended as preferred sources of enzymes capable of synthesis, and the mature plant tissues for degrading enzymes.     

General esterases of Bemisia tabaci (Hemiptera: Aleyrodidae) change in response to feed on cotton varieties

The esterase enzymes are a major component of insect detoxification systems and play a crucial role in hydrolyzing lots of xenobiotic compounds. Among insect, generalist herbivores can exhibit developed biochemical defences as a result of exposing to a wide range of plant chemical compounds. To overcome this ability, host plants may affect the level of hydrolases in herbivore insects feeding on. To examine this hypothesis, in the present study total esterase activity was investigated in a highly polyphagous whitefly, Bemisia tabaci, reared on six different varieties of cotton, Gossypium hirsutum. Results showed significant differences in esterase activity of B. tabaci feeding on the host plant varieties. The highest esterase activities were detected in whiteflies feeding on Sk-Tb and Siokra varieties, whereas those whiteflies that feed on Hopicala variety exhibited the least esterase activities. Our findings highlight the important role of host plants in detoxification ability of herbivore insects. The importance of these findings in biology of insect pests and their applications in integrated pest management programmes of B. tabaci have been discussed in detail.     

Biodegradation of neutralized sarin.

This research investigated the biotransformation of IMPA, the neutralization product of the nerve agent Sarin, by a microbial consortia. As mandated by the Chemical Weapons Convention signed by 132 countries in 1993, all chemical warfare agents are to be destroyed within ten years of ratification. Technologies must be developed to satisfy this commitment. This paper presents data from a biodegradation kinetics study and background information on the biological transformation of IMPA. Microbial transformation of organophosphate nerve agents and organophosphate pesticide intermediates can be incorporated into a treatment process for the fast and efficient destruction of these similar compounds. Sarin (isopropyl methylphosphonofluoridate), also known as GB, is one of several highly neurotoxic chemical warfare agents that have been developed over the past 50 to 60 years. Four mixed cultures were acclimated to the Sarin hydrolysis product, isopropyl methylphosphonic acid (IMPA). Two of these cultures, APG microorganisms and SX microorganisms, used IMPA as the sole phosphorus source. Extended exposure to IMPA improved the cultures' abilities to degrade IMPA to form methylphosphonic acid (MPA) and inorganic phosphate. The presence of free phosphate in the reactor suppressed the degradation of IMPA. IMPA did not inhibit either cultural consortia within the tested concentration range (0 to 1250 mg/L). The numax was 120.9 mg/L/day for the SX microorganisms and 118.3 mg/L/day for the APG microorganisms. Initial IMPA concentrations of 85 to 90 mg/L were degraded to nondetectable levels within 75 h. These results demonstrate the potential for biodegradation to serve as a complementary treatment process for the destruction of stockpiled Sarin.     

Nitroaromatic Compounds, from Synthesis to Biodegradation

Summary: Nitroaromatic compounds are relatively rare in nature and have been introduced into the environment mainly by human activities. This important class of industrial chemicals is widely used in the synthesis of many diverse products, including dyes, polymers, pesticides, and explosives. Unfortunately, their extensive use has led to environmental contamination of soil and groundwater. The nitro group, which provides chemical and functional diversity in these molecules, also contributes to the recalcitrance of these compounds to biodegradation. The electron-withdrawing nature of the nitro group, in concert with the stability of the benzene ring, makes nitroaromatic compounds resistant to oxidative degradation. Recalcitrance is further compounded by their acute toxicity, mutagenicity, and easy reduction into carcinogenic aromatic amines. Nitroaromatic compounds are hazardous to human health and are registered on the U.S. Environmental Protection Agency''s list of priority pollutants for environmental remediation. Although the majority of these compounds are synthetic in nature, microorganisms in contaminated environments have rapidly adapted to their presence by evolving new biodegradation pathways that take advantage of them as sources of carbon, nitrogen, and energy. This review provides an overview of the synthesis of both man-made and biogenic nitroaromatic compounds, the bacteria that have been identified to grow on and completely mineralize nitroaromatic compounds, and the pathways that are present in these strains. The possible evolutionary origins of the newly evolved pathways are also discussed.     

Mineralization of paraoxon and its use as a sole C and P source by a rationally designed catabolic pathway in Pseudomonas putida

Organophosphate compounds, which are widely used as pesticides and chemical warfare agents, are cholinesterase inhibitors. These synthetic compounds are resistant to natural degradation and threaten the environment. We constructed a strain of Pseudomonas putida that can efficiently degrade a model organophosphate, paraoxon, and use it as a carbon, energy, and phosphorus source. This strain was engineered with the pnp operon from Pseudomonas sp. strain ENV2030, which encodes enzymes that transform p-nitrophenol into beta-ketoadipate, and with a synthetic operon encoding an organophosphate hydrolase (encoded by opd) from Flavobacterium sp. strain ATCC 27551, a phosphodiesterase (encoded by pde) from Delftia acidovorans, and an alkaline phosphatase (encoded by phoA) from Pseudomonas aeruginosa HN854 under control of a constitutive promoter. The engineered strain can efficiently mineralize up to 1 mM (275 mg/liter) paraoxon within 48 h, using paraoxon as the sole carbon and phosphorus source and an inoculum optical density at 600 nm of 0.03. Because the organism can utilize paraoxon as a sole carbon, energy, and phosphorus source and because one of the intermediates in the pathway (p-nitrophenol) is toxic at high concentrations, there is no need for selection pressure to maintain the heterologous pathway.     

Use of NMR techniques for toxic organophosphorus compound profiling

This review presents with selected examples the versatility of nuclear magnetic resonance (NMR) spectroscopy in the analysis of toxic organophosphorus (OP) compounds, i.e. OP pesticides and chemical warfare agents (CWAs). Several NMR applications of biological importance, like studies on inhibition mechanism, metabolism, and exposure determination, are presented. The review also concerns with the environmental analysis of OP compounds by NMR spectroscopy. Residue analysis of environment and food samples as well as characterization of degradation in environment is discussed. Some of the NMR studies that have been done to support the Chemical Weapons Convention, i.e. the development of suitable CWA detoxification means and the method development of verification analysis for CWAs and their degradation products, are outlined.     

Crystal structures of human carboxylesterase 1 in covalent complexes with the chemical warfare agents soman and tabun

The organophosphorus nerve agents sarin, soman, tabun, and VX exert their toxic effects by inhibiting the action of human acetylcholinesterase, a member of the serine hydrolase superfamily of enzymes. The current treatments for nerve agent exposure must be administered quickly to be effective, and they often do not eliminate long-term toxic side effects associated with organophosphate poisoning. Thus, there is significant need for effective prophylactic methods to protect at-risk personnel from nerve agent exposure, and protein-based approaches have emerged as promising candidates. We present the 2.7 A resolution crystal structures of the serine hydrolase human carboxylesterase 1 (hCE1), a broad-spectrum drug metabolism enzyme, in covalent acyl-enzyme intermediate complexes with the chemical weapons soman and tabun. The structures reveal that hCE1 binds stereoselectively to these nerve agents; for example, hCE1 appears to react preferentially with the 10(4)-fold more lethal PS stereoisomer of soman relative to the PR form. In addition, structural features of the hCE1 active site indicate that the enzyme may be resistant to dead-end organophosphate aging reactions that permanently inactivate other serine hydrolases. Taken together, these data provide important structural details toward the goal of engineering hCE1 into an organophosphate hydrolase and protein-based therapeutic for nerve agent exposure.