Transformation of 2,4,6-Trinitrotoluene by Purified Xenobiotic Reductase B from Pseudomonas fluorescens I-C

The enzymatic transformation of 2,4,6-trinitrotoluene (TNT) by purified XenB, an NADPH-dependent flavoprotein oxidoreductase from Pseudomonas fluorescens I-C, was evaluated by using natural abundance and [U-¹⁴C]TNT preparations. XenB catalyzed the reduction of TNT either by hydride addition to the aromatic ring or by nitro group reduction, with the accumulation of various tautomers of the protonated dihydride-Meisenheimer complex of TNT, 2-hydroxylamino-4,6-dinitrotoluene, and 4-hydroxylamino-2,6-dinitrotoluene. Subsequent reactions of these metabolites were nonenzymatic and resulted in predominant formation of at least three dimers with an anionic m/z of 376 as determined by negative-mode electrospray ionization mass spectrometry and the release of ~0.5 mol of nitrite per mol of TNT consumed. The extents of the initial enzymatic reactions were similar in the presence and in the absence of O₂, but the dimerization reaction and the release of nitrite were favored under aerobic conditions or under anaerobic conditions in the presence of NADP⁺. Reactions of chemically and enzymatically synthesized and high-pressure liquid chromatography-purified TNT metabolites showed that both a hydroxylamino-dinitrotoluene isomer and a tautomer of the protonated dihydride-Meisenheimer complex of TNT were required precursors for the dimerization and nitrite release reactions. The m/z 376 dimers also reacted with either dansyl chloride or N-1-naphthylethylenediamine HCl, providing evidence for an aryl amine functional group. In combination, the experimental results are consistent with assigning the chemical structures of the m/z 376 species to various isomers of amino-dimethyl-tetranitrobiphenyl. A mechanism for the formation of these proposed TNT metabolites is presented, and the potential enzymatic and environmental significance of their formation is discussed.     

Transformation of 2,4,6-Trinitrotoluene by Pseudomonas pseudoalcaligenes JS52

Pseudomonas pseudoalcaligenes JS52 grows on nitrobenzene via partial reduction of the nitro group and enzymatic rearrangement of the resultant hydroxylamine. Cells and cell extracts of nitrobenzene-grown JS52 catalyzed the transient formation of 4-hydroxylamino-2,6-dinitrotoluene (4HADNT), 4-amino-2,6-dinitrotoluene (4ADNT), and four previously unidentified metabolites from 2,4,6-trinitrotoluene (TNT). Two of the novel metabolites were identified by liquid chromatography/mass spectrometry and (sup1)H-nuclear magnetic resonance spectroscopy as 2,4-dihydroxylamino-6-nitrotoluene (DHANT) and 2-hydroxylamino-4-amino-6-nitrotoluene (2HA4ANT). A polar yellow metabolite also accumulated during transformation of TNT by cells and cell extracts. Under anaerobic conditions, extracts of strain JS52 did not catalyze the production of the yellow metabolite or release nitrite from TNT; moreover, DHANT and 2HA4ANT accumulated under anaerobic conditions, which indicated that their further metabolism was oxygen dependent. Small amounts of nitrite were released during transformation of TNT by strain JS52. Sustained transformation of TNT by cells required nitrobenzene, which indicated that TNT transformation does not provide energy. Transformation of TNT catalyzed by enzymes in cell extracts required NADPH. Transformation experiments with (sup14)C-TNT indicated that TNT was not mineralized; however, carbon derived from TNT became associated with cells. Nitrobenzene nitroreductase purified from strain JS52 transformed TNT to DHANT via 4HADNT, which indicated that the nitroreductase could catalyze the first two steps in the transformation of TNT. The unusual ability of the nitrobenzene nitroreductase to catalyze the stoichiometric reduction of aromatic nitro compounds to the corresponding hydroxylamine provides the basis for the novel pathway for metabolism of TNT.     

Production of Eight Different Hydride Complexes and Nitrite Release from 2,4,6-Trinitrotoluene by Yarrowia lipolytica

2,4,6-Trinitrotoluene (TNT) transformation by the yeast strain Yarrowia lipolytica AN-L15 was shown to occur via two different pathways. Direct aromatic ring reduction was the predominant mechanism of TNT transformation, while nitro group reduction was observed to be a minor pathway. Although growth of Y. lipolytica AN-L15 was inhibited initially in the presence of TNT, TNT transformation was observed, indicating that the enzymes necessary for TNT reduction were present initially. Aromatic ring reduction resulted in the transient accumulation of eight different TNT-hydride complexes, which were characterized using high-performance liquid chromatography, UV-visible diode array detection, and negative-mode atmospheric pressure chemical ionization mass spectrometry (APCI-MS). APCI-MS analysis revealed three different groups of TNT-hydride complexes with molecular ions at m/z 227, 228, and 230, which correspond to TNT-mono- and dihydride complexes and protonated dihydride isomers, respectively. One of the three protonated dihydride complex isomers detected appears to release nitrite in the presence of strain AN-L15. This release of nitrite is of particular interest since it can provide a pathway towards complete degradation and detoxification of TNT.     

Escherichia coli has multiple enzymes that attack TNT and release nitrogen for growth

There has been a growing interest in the degradation of 2,4,6-trinitrotoluene (TNT) over the last decade, ever since its removal from polluted sites was declared an international environmental priority. Certain aerobic and anaerobic microorganisms are capable of using TNT as an N source, although very few studies have proven the mineralization of this compound. An unexpected observation in our laboratory led us to discover that certain Escherichia coli bench laboratory strains have multiple enzymes that attack TNT. One of the NemA products is responsible for the release of nitrite from the nitroaromatic ring: among the metabolites observed in vitro include Meisenheimer dihydride complexes of TNT from which 2-hydroxylamino-6-nitrotoluene is slowly formed during their rearomatization under concomitant release of nitrite. Furthermore, NemA, together with NfsA and NfsB reduce the nitro groups on the aromatic ring to the corresponding hydroxylamino derivatives, which probably results in the release of ammonium ions which can, in turn be used as a nitrogen source by E. coli for growth.     

Type II Hydride Transferases from Different Microorganisms Yield Nitrite and Diarylamines from Polynitroaromatic Compounds

Homogenous preparations of XenB of Pseudomonas putida, pentaerythritol tetranitrate reductase of Enterobacter cloacae, and N-ethylmaleimide reductase of Escherichia coli, all type II hydride transferases of the Old Yellow Enzyme family of flavoproteins, are shown to reduce the polynitroaromatic compound 2,4,6-trinitrotoluene (TNT). The reduction of this compound yields hydroxylaminodinitrotoluenes and Meisenheimer dihydride complexes, which, upon condensation, yield stoichiometric amounts of nitrite and diarylamines, implying that type II hydride transferases are responsible for TNT denitration, a process with important environmental implications for TNT remediation.     

Denitration of 2,4,6-trinitrotoluene by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> ESA-5 in the presence of ferrihydrite

Denitration of 2,4,6-trinitrotoluene (TNT) was evaluated in oxygen-depleted enrichment cultures. These cultures were established starting with an uncontaminated or a TNT-contaminated soil inoculum and contained TNT as sole nitrogen source. Incubations were carried out in the presence or absence of ferrihydrite. A significant release of nitrite was observed in the liquid culture containing TNT, ferrihydrite, and inoculum from a TNT-contaminated soil. Under these conditions, Pseudomonas aeruginosa was the predominant bacterium in the enrichment, leading to the isolation of P. aeruginosa ESA-5 as a pure strain. The isolate had TNT denitration capabilities as confirmed by nitrite release in oxygen-depleted cultures containing TNT and ferrihydrite. In addition to reduced derivatives of TNT, several unidentified metabolites were detected. Concomitant to a decrease of TNT concentration, a release of nitrite was observed. The concentration of nitrite peaked and then it slowly decreased. In the absence of TNT, the drop in the concentration of nitrite in oxygen-depleted cultures was lower when ferrihydrite was provided, suggesting that ferrihydrite inhibited the utilization of nitrite by P. aeruginosa ESA-5.     

2,4,6-trinitrotoluene reduction by carbon monoxide dehydrogenase from Clostridium thermoaceticum

Purified CO dehydrogenase (CODH) from Clostridium thermoaceticum catalyzed the transformation of 2,4,6-trinitrotoluene (TNT). The intermediates and reduced products of TNT transformation were separated and appear to be identical to the compounds formed by C. acetobutylicum, namely, 2-hydroxylamino-4,6-dinitrotoluene (2HA46DNT), 4-hydroxylamino-2,6-dinitrotoluene (4HA26DNT), 2, 4-dihydroxylamino-6-nitrotoluene (24DHANT), and the Bamberger rearrangement product of 2,4-dihydroxylamino-6-nitrotoluene. In the presence of saturating CO, CODH catalyzed the conversion of TNT to two monohydroxylamino derivatives (2HA46DNT and 4HA26DNT), with 4HA26DNT as the dominant isomer. These derivatives were then converted to 24DHANT, which slowly converted to the Bamberger rearrangement product. Apparent K(m) and k(cat) values of TNT reduction were 165 +/- 43 microM for TNT and 400 +/- 94 s(-1), respectively. Cyanide, an inhibitor for the CO/CO(2) oxidation/reduction activity of CODH, inhibited the TNT degradation activity of CODH.     

Anaerobic transformation of 2,4,6-TNT by bovine ruminal microbes

Degradation of TNT by bovine rumen fluid, a novel source of anaerobic microbes, was investigated. Whole rumen fluid contents were spiked with TNT and incubated for a 24h time period. Supernatant samples taken at 0, 1, 2, 4, and 24h were analyzed by reverse-phase HPLC with diode array detection. Within 1h, TNT was not detectable and reduction products of TNT including 2-hydroxyl-amino-4,6-dinitrotoluene, 4-hydroxylamino-2,6-dinitrotoluene, and 4-amino-2,6-dinitrotoluene were present with smaller amounts of diamino-nitrotoluenes. Within 2h, only the diamino and dihydroxyamino-nitrotoluene products remained. After 4h, 2,4-diamino-6-nitrotoluene and 2,4-dihydroxyamino-6-nitrotoluene were the only known molecular species left. At 24h known UV absorbing metabolites were no longer detected, suggesting further transformation such as complete reduction to triaminotoluene or destruction of the aromatic ring of TNT may have occurred. TNT was not transformed at 24h in autoclaved and buffered controls. This study presents the first direct evidence of biodegradation of TNT by ruminal microbes.     

Initial Reductive Reactions in Aerobic Microbial Metabolism of 2,4,6-Trinitrotoluene

Because of its high electron deficiency, initial microbial transformations of 2,4,6-trinitrotoluene (TNT) are characterized by reductive rather than oxidation reactions. The reduction of the nitro groups seems to be the dominating mechanism, whereas hydrogenation of the aromatic ring, as described for picric acid, appears to be of minor importance. Thus, two bacterial strains enriched with TNT as a sole source of nitrogen under aerobic conditions, a gram-negative strain called TNT-8 and a gram-positive strain called TNT-32, carried out nitro-group reduction. In contrast, both a picric acid-utilizing Rhodococcus erythropolis strain, HL PM-1, and a 4-nitrotoluene-utilizing Mycobacterium sp. strain, HL 4-NT-1, possessed reductive enzyme systems, which catalyze ring hydrogenation, i.e., the addition of a hydride ion to the aromatic ring of TNT. The hydride-Meisenheimer complex thus formed (H⁻-TNT) was further converted to a yellow metabolite, which by electrospray mass and nuclear magnetic resonance spectral analyses was established as the protonated dihydride-Meisenheimer complex of TNT (2H⁻-TNT). Formation of hydride complexes could not be identified with the TNT-enriched strains TNT-8 and TNT-32, or with Pseudomonas sp. clone A (2NT⁻), for which such a mechanism has been proposed. Correspondingly, reductive denitration of TNT did not occur.     

Purification and characterization of NAD(P)H-dependent nitroreductase I from Klebsiella sp. C1 and enzymatic transformation of 2,4,6-trinitrotoluene

Three NAD(P)H-dependent nitroreductases that can transform 2,4,6-trinitrotoluene (TNT) by two reduction pathways were detected in Klebsiella sp. C1. Among these enzymes, the protein with the highest reduction activity of TNT (nitroreductase I) was purified to homogeneity using ion-exchange, hydrophobic interaction, and size exclusion chromatographies. Nitroreductase I has a molecular mass of 27 kDa as determined by SDS-PAGE, and exhibits a broad pH optimum between 5.5 and 6.5, with a temperature optimum of 30–40°C. Flavin mononucleotide is most likely the natural flavin cofactor of this enzyme. The N-terminal amino acid sequence of this enzyme does not show a high degree of sequence similarity with nitroreductases from other enteric bacteria. This enzyme catalyzed the two-electron reduction of several nitroaromatic compounds with very high specific activities of NADPH oxidation. In the enzymatic transformation of TNT, 2-amino-4,6-dinitrotoluene and 2,2′,6,6′-tetranitro-4,4′-azoxytoluene were detected as transformation products. Although this bacterium utilizes the direct ring reduction and subsequent denitration pathway together with a nitro group reduction pathway, metabolites in direct ring reduction of TNT could not easily be detected. Unlike other nitroreductases, nitroreductase I was able to transform hydroxylaminodinitrotoluenes (HADNT) into aminodinitrotoluenes (ADNT), and could reduce ortho isomers (2-HADNT and 2-ADNT) more easily than their para isomers (4-HADNT and 4-ADNT). Only the nitro group in the ortho position of 2,4-DNT was reduced to produce 2-hydroxylamino-4-nitrotoluene by nitroreductase I; the nitro group in the para position was not reduced.     

NAD(P)H:flavin mononucleotide oxidoreductase inactivation during 2,4,6-trinitrotoluene reduction

Bacteria readily transform 2,4,6-trinitrotoluene (TNT), a contaminant frequently found at military bases and munitions production facilities, by reduction of the nitro group substituents. In this work, the kinetics of nitroreduction were investigated by using a model nitroreductase, NAD(P)H:flavin mononucleotide (FMN) oxidoreductase. Under mediation by NAD(P)H:FMN oxidoreductase, TNT rapidly reacted with NADH to form 2-hydroxylamino-4,6-dinitrotoluene and 4-hydroxylamino-2,6-dinitrotoluene, whereas 2-amino-4,6-dinitrotoluene and 4-amino-2,6-dinitrotoluene were not produced. Progressive loss of activity was observed during TNT reduction, indicating inactivation of the enzyme during transformation. It is likely that a nitrosodinitrotoluene intermediate reacted with the NAD(P)H:FMN oxidoreductase, leading to enzyme inactivation. A half-maximum constant with respect to NADH, K(N), of 394 microM was measured, indicating possible NADH limitation under typical cellular conditions. A mathematical model that describes the inactivation process and NADH limitation provided a good fit to TNT reduction profiles. This work represents the first step in developing a comprehensive enzyme level understanding of nitroarene biotransformation.     

Aerobic Degradation of 2,4,6-Trinitrotoluene by Enterobacter cloacae PB2 and by Pentaerythritol Tetranitrate Reductase

Enterobacter cloacae PB2 was originally isolated on the basis of its ability to utilize nitrate esters, such as pentaerythritol tetranitrate (PETN) and glycerol trinitrate, as the sole nitrogen source for growth. The enzyme responsible is an NADPH-dependent reductase designated PETN reductase. E. cloacae PB2 was found to be capable of slow aerobic growth with 2,4,6-trinitrotoluene (TNT) as the sole nitrogen source. Dinitrotoluenes were not produced and could not be used as nitrogen sources. Purified PETN reductase was found to reduce TNT to its hydride-Meisenheimer complex, which was further reduced to the dihydride-Meisenheimer complex. Purified PETN reductase and recombinant Escherichia coli expressing PETN reductase were able to liberate nitrogen as nitrite from TNT. The ability to remove nitrogen from TNT suggests that PB2 or recombinant organisms expressing PETN reductase may be useful for bioremediation of TNT-contaminated soil and water.     

Structure of a Laccase-Mediated Product of Coupling of 2,4-Diamino-6-Nitrotoluene to Guaiacol, a Model for Coupling of 2,4,6-Trinitrotoluene Metabolites to a Humic Organic Soil Matrix

This work presents laccase-mediated model reactions for coupling of reduced 2,4,6-trinitrotoluene (TNT) metabolites to an organic soil matrix. The structure of an isolated coupling product of 2,4-diamino-6-nitrotoluene (2,4-DANT) to guaiacol as humic constituent was determined. Among several structures, the compound was identified conclusively to be the trinuclear coupling product 5-(2-amino-3-methyl-4-nitroanilino)-3,3(prm1)-dimethoxy-4,4(prm1)-diphenoqu inone. The compound has a weight of 409 g mol(sup-1) and may serve as a model reaction for the biogenic formation of bound residues in soil from TNT by coupling aminotoluenes (reduced TNT metabolites) to humic constituents. A linear correlation of the substrate consumption to the enzyme activity was detected. Based on this observation, the described reaction of 2,4-DANT coupling to guaiacol may be used for determination of laccase activity since the reaction was not inhibited by other compounds of culture supernatants. We propose a two-step mechanism for the coupling reaction because 2,4-DANT was not transformed by laccases in the absence of guaiacol and guaiacol oxidation was independent of the presence of 2,4-DANT. The first reaction step is a laccase-mediated dimerization of two guaiacol monomers with subsequent oxidation to a diphenoquinone. The second step is the nucleophilic addition of 2,4-DANT to the ortho position of the carbonyl group of the diphenoquinone structure.     

NAD(P)H:Flavin Mononucleotide Oxidoreductase Inactivation during 2,4,6-Trinitrotoluene Reduction

Bacteria readily transform 2,4,6-trinitrotoluene (TNT), a contaminant frequently found at military bases and munitions production facilities, by reduction of the nitro group substituents. In this work, the kinetics of nitroreduction were investigated by using a model nitroreductase, NAD(P)H:flavin mononucleotide (FMN) oxidoreductase. Under mediation by NAD(P)H:FMN oxidoreductase, TNT rapidly reacted with NADH to form 2-hydroxylamino-4,6-dinitrotoluene and 4-hydroxylamino-2,6-dinitrotoluene, whereas 2-amino-4,6-dinitrotoluene and 4-amino-2,6-dinitrotoluene were not produced. Progressive loss of activity was observed during TNT reduction, indicating inactivation of the enzyme during transformation. It is likely that a nitrosodinitrotoluene intermediate reacted with the NAD(P)H:FMN oxidoreductase, leading to enzyme inactivation. A half-maximum constant with respect to NADH, K_N, of 394 μM was measured, indicating possible NADH limitation under typical cellular conditions. A mathematical model that describes the inactivation process and NADH limitation provided a good fit to TNT reduction profiles. This work represents the first step in developing a comprehensive enzyme level understanding of nitroarene biotransformation.     

Biodegradation of 2,4,6-trinitrotoluene (TNT) by the white-rot basidiomycete Phlebia radiata

Phlebia radiatatransformed 2,4,6-trinitrotoluene (TNT), as well as its first reduction products, the aminodinitrotoluenes, into 4-hydroxylamino-2,6-dinitrotoluene (4-OHA-2,6-DNT) and 4-amino-2,6-dinitrotoluene (4-A-2,6-DNT). No extracellular peroxidases were involved in this step. The ligninolytic extracellular fluid, assumed to contain peroxidases, did not reduce TNT. However, ligninolytic peroxidases are implicated in the transformation of the first reduction products of TNT.     

Flavoenzyme-catalyzed redox cycling of hydroxylamino- and amino metabolites of 2,4,6-trinitrotoluene: implications for their cytotoxicity

The toxicity of 2,4,6-trinitrotoluene (TNT), a widespread environmental contaminant, is exerted through its enzymatic redox cycling and/or covalent binding of its reduction products to proteins and DNA. In this study, we examined the possibility of another cytotoxicity mechanism of the amino- and hydroxylamino metabolites of TNT, their flavoenzyme-catalyzed redox cycling. The above compounds acted as redox-cycling substrates for single-electron transferring NADPH:cytochrome P-450 reductase (P-450R) and ferredoxin:NADP(+) reductase (FNR), as well as substrates for the two-electron transferring flavoenzymes rat liver NAD(P)H:quinone oxidoreductase (NQO1) and Enterobacter cloacae NAD(P)H:nitroreductase (NR). Their reactivity in P-450R-, FNR-, and NR-catalyzed reactions increased with an increase in their single-electron reduction potential (E(1)(7)) or the decrease in the enthalpy of free radical formation. The cytotoxicity of the amino- and hydroxylamino metabolites of TNT towards bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK) was partly prevented by the antioxidant N,N'-diphenyl-p-phenylene diamine and desferrioxamine, and potentiated by 1,3-bis-(2-chloroethyl)-1-nitrosourea, thus pointing to the involvement of oxidative stress. In general, their cytotoxicity increased with an increase in their electron accepting properties, or their reactivity towards the single-electron transferring FNR and P-450R. Thus, our data imply that the flavoenzyme-catalyzed redox cycling of amino and hydroxylamino metabolites of TNT may be an important factor in their cytotoxicity.     

A double mutant of Pseudomonas putida JLR11 deficient in the synthesis of the nitroreductase PnrA and assimilatory nitrite reductase NasB is impaired for growth on 2,4,6-trinitrotoluene (TNT)

Pseudomonas putida JLR11 can grow on 2,4,6-trinitrotoluene (TNT) as the sole nitrogen source. We created nasB (nitrite reductase), pnrA (nitroaromatic reductase) and pnrA nasB mutants and tested their growth with TNT as the sole N source. The nasB and pnrA mutants grew at a reduced rate on TNT, whereas the double nasB pnrA mutant did not. This suggests that P. putida JLR11 carries out multiple enzymatic attacks on TNT-releasing nitrite and/or ammonium. The PnrA nitroreductase plays a key role in the reduction of TNT to 2,6-dinitro-4-hydroxylaminotoluene and the subsequent release of ammonium for growth.     

Biological Degradation of 2,4,6-Trinitrotoluene

Nitroaromatic compounds are xenobiotics that have found multiple applications in the synthesis of foams, pharmaceuticals, pesticides, and explosives. These compounds are toxic and recalcitrant and are degraded relatively slowly in the environment by microorganisms. 2,4,6-Trinitrotoluene (TNT) is the most widely used nitroaromatic compound. Certain strains of Pseudomonas and fungi can use TNT as a nitrogen source through the removal of nitrogen as nitrite from TNT under aerobic conditions and the further reduction of the released nitrite to ammonium, which is incorporated into carbon skeletons. Phanerochaete chrysosporium and other fungi mineralize TNT under ligninolytic conditions by converting it into reduced TNT intermediates, which are excreted to the external milieu, where they are substrates for ligninolytic enzymes. Most if not all aerobic microorganisms reduce TNT to the corresponding amino derivatives via the formation of nitroso and hydroxylamine intermediates. Condensation of the latter compounds yields highly recalcitrant azoxytetranitrotoluenes. Anaerobic microorganisms can also degrade TNT through different pathways. One pathway, found in Desulfovibrio and Clostridium, involves reduction of TNT to triaminotoluene; subsequent steps are still not known. Some Clostridium species may reduce TNT to hydroxylaminodinitrotoluenes, which are then further metabolized. Another pathway has been described in Pseudomonas sp. strain JLR11 and involves nitrite release and further reduction to ammonium, with almost 85% of the N-TNT incorporated as organic N in the cells. It was recently reported that in this strain TNT can serve as a final electron acceptor in respiratory chains and that the reduction of TNT is coupled to ATP synthesis. In this review we also discuss a number of biotechnological applications of bacteria and fungi, including slurry reactors, composting, and land farming, to remove TNT from polluted soils. These treatments have been designed to achieve mineralization or reduction of TNT and immobilization of its amino derivatives on humic material. These approaches are highly efficient in removing TNT, and increasing amounts of research into the potential usefulness of phytoremediation, rhizophytoremediation, and transgenic plants with bacterial genes for TNT removal are being done.     

Biological degradation of 2,4,6-trinitrotoluene.

Nitroaromatic compounds are xenobiotics that have found multiple applications in the synthesis of foams, pharmaceuticals, pesticides, and explosives. These compounds are toxic and recalcitrant and are degraded relatively slowly in the environment by microorganisms. 2,4,6-Trinitrotoluene (TNT) is the most widely used nitroaromatic compound. Certain strains of Pseudomonas and fungi can use TNT as a nitrogen source through the removal of nitrogen as nitrite from TNT under aerobic conditions and the further reduction of the released nitrite to ammonium, which is incorporated into carbon skeletons. Phanerochaete chrysosporium and other fungi mineralize TNT under ligninolytic conditions by converting it into reduced TNT intermediates, which are excreted to the external milieu, where they are substrates for ligninolytic enzymes. Most if not all aerobic microorganisms reduce TNT to the corresponding amino derivatives via the formation of nitroso and hydroxylamine intermediates. Condensation of the latter compounds yields highly recalcitrant azoxytetranitrotoluenes. Anaerobic microorganisms can also degrade TNT through different pathways. One pathway, found in Desulfovibrio and Clostridium, involves reduction of TNT to triaminotoluene; subsequent steps are still not known. Some Clostridium species may reduce TNT to hydroxylaminodinitrotoluenes, which are then further metabolized. Another pathway has been described in Pseudomonas sp. strain JLR11 and involves nitrite release and further reduction to ammonium, with almost 85% of the N-TNT incorporated as organic N in the cells. It was recently reported that in this strain TNT can serve as a final electron acceptor in respiratory chains and that the reduction of TNT is coupled to ATP synthesis. In this review we also discuss a number of biotechnological applications of bacteria and fungi, including slurry reactors, composting, and land farming, to remove TNT from polluted soils. These treatments have been designed to achieve mineralization or reduction of TNT and immobilization of its amino derivatives on humic material. These approaches are highly efficient in removing TNT, and increasing amounts of research into the potential usefulness of phytoremediation, rhizophytoremediation, and transgenic plants with bacterial genes for TNT removal are being done.     

2,4,6-Trinitrotoluene Reduction by Carbon Monoxide Dehydrogenase from Clostridium thermoaceticum

Purified CO dehydrogenase (CODH) from Clostridium thermoaceticum catalyzed the transformation of 2,4,6-trinitrotoluene (TNT). The intermediates and reduced products of TNT transformation were separated and appear to be identical to the compounds formed by C. acetobutylicum, namely, 2-hydroxylamino-4,6-dinitrotoluene (2HA46DNT), 4-hydroxylamino-2,6-dinitrotoluene (4HA26DNT), 2,4-dihydroxylamino-6-nitrotoluene (24DHANT), and the Bamberger rearrangement product of 2,4-dihydroxylamino-6-nitrotoluene. In the presence of saturating CO, CODH catalyzed the conversion of TNT to two monohydroxylamino derivatives (2HA46DNT and 4HA26DNT), with 4HA26DNT as the dominant isomer. These derivatives were then converted to 24DHANT, which slowly converted to the Bamberger rearrangement product. Apparent K_m and k_cat values of TNT reduction were 165 ± 43 μM for TNT and 400 ± 94 s⁻¹, respectively. Cyanide, an inhibitor for the CO/CO₂ oxidation/reduction activity of CODH, inhibited the TNT degradation activity of CODH.