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.     

Hydride-mediated reduction of 2,4,6-trinitrotoluene by yeasts as the way to its deep degradation

Broad screening of microorganisms from natural and anthropogenic ecological niches has revealed strains Candida sp. AN-L15 and Geotrichum sp. AN-Z4 which transform, 2,4,6-trinitrotoluene (TNT) via alternative pathways (with the domination of hydride ion-mediated reduction of the aromatic ring) and produce relatively high amounts of nitrites. According to the spectrophotometry data, the hydride attack of TNT by Candida sp. AN-L15 and Geotrichum sp. AN-Z4 grown at pH 5.0–8.0 leads to the mono-and dihydride complexes of TNT (H^?-TNT and 2H^?-TNT, respectively) and to protonated forms of the latter. Analysis by HPLC, GC-mass spectrometry, and ion chromatography revealed the products of deep conversion of TNT. The growth of the yeast strains in a weakly acidic medium with TNT (440 μM) is accompanied by formation of 2,4-dinitrotoluene (2,4-DNT, up to 18.2 μM). Together with accumulation of nitrites (up to 76.0 μM, depending on pH of the medium), these findings demonstrate the capacity of both strains for TNT denitration. Formation of 2,4-DNT reflects the realization of one of the possible mechanisms of TNT ortho-nitro group elimination and switching over to the pathways of metabolism of dinitrotoluenes, which are much more easily biodegradable than TNT. Simultaneously with the dominating TNT hydride attack, the mechanism of 4-and 6-electron reduction of the nitro group also functions in Candida sp. AN-L15 and Geotrichum sp. AN-Z4. Realization of the studied mechanisms of TNT transformation under growth of Candida sp. AN-L15 on n-alkane is important for bioremediation in the cases of combined pollution by oil products and explosives.     

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.     

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.     

Alternative Pathways of the Initial Transformation of 2,4,6-Trinitrotoluene by Yeasts

A new model for the initial transformation of 2,4,6-trinitrotoluene (TNT) by facultatively anaerobic and aerobic yeasts is presented. The model is based on the data that Saccharomyces sp. ZS-A1 was able to reduce the nitrogroups of TNT with the formation of 2- and 4-hydroxyaminodinitrotoluenes (2-HADNT and 4-HADNT) as the major early TNT metabolites (the molar HADNT/TNT ratio reached 0.81), whereas aminodinitrotoluenes (ADNTs) and the hydride-Meisenheimer complex of TNT (H-TNT) were the minor products. Candidasp. AN-L13 almost completely transformed TNT into H-TNT through the reduction of the aromatic ring. Candida sp. AN-L14 transformed TNT through a combination of the two mechanisms described. Aeration stimulated the production of HADNT from TNT, whereas yeast incubation under stationary conditions promoted the formation of HADNT. The transformation of TNT into HADNT led to a tenfold increase in the acute toxicity of the TNT preparation with respect to Paramecium caudatum, whereas the increase in the toxicity was about twofold in the case of the alternative attack at the aromatic ring.     

2,4,6-Trinitrotoluene Reduction by an Fe-Only Hydrogenase in Clostridium acetobutylicum

The role of hydrogenase on the reduction of 2,4,6-trinitrotoluene (TNT) in Clostridium acetobutylicum was evaluated. An Fe-only hydrogenase was isolated and identified by using TNT reduction activity as the selection basis. The formation of hydroxylamino intermediates by the purified enzyme corresponded to expected products for this reaction, and saturation kinetics were determined with a K_m of 152 μM. Comparisons between the wild type and a mutant strain lacking the region encoding an alternative Fe-Ni hydrogenase determined that Fe-Ni hydrogenase activity did not significantly contribute to TNT reduction. Hydrogenase expression levels were altered in various strains, allowing study of the role of the enzyme in TNT reduction rates. The level of hydrogenase activity in a cell system correlated (R² = 0.89) with the organism's ability to reduce TNT. A strain that overexpressed the hydrogenase activity resulted in maintained TNT reduction during late growth phases, which it is not typically observed in wild type strains. Strains exhibiting underexpression of hydrogenase produced slower TNT rates of reduction correlating with the determined level of expression. The isolated Fe-only hydrogenase is the primary catalyst for reducing TNT nitro substituents to the corresponding hydroxylamines in C. acetobutylicum in whole-cell systems. A mechanism for the reaction is proposed. Due to the prevalence of hydrogenase in soil microbes, this research may enhance the understanding of nitroaromatic compound transformation by common microbial communities.     

Subfunctionality of Hydride Transferases of the Old Yellow Enzyme Family of Flavoproteins of Pseudomonas putida

To investigate potential complementary activities of multiple enzymes belonging to the same family within a single microorganism, we chose a set of Old Yellow Enzyme (OYE) homologs of Pseudomonas putida. The physiological function of these enzymes is not well established; however, an activity associated with OYE family members from different microorganisms is their ability to reduce nitroaromatic compounds. Using an in silico approach, we identified six OYE homologs in P. putida KT2440. Each gene was subcloned into an expression vector, and each corresponding gene product was purified to homogeneity prior to in vitro analysis for its catalytic activity against 2,4,6-trinitrotoluene (TNT). One of the enzymes, called XenD, lacked in vitro activity, whereas the other five enzymes demonstrated type I hydride transferase activity and reduced the nitro groups of TNT to hydroxylaminodinitrotoluene derivatives. XenB has the additional ability to reduce the aromatic ring of TNT to produce Meisenheimer complexes, defined as type II hydride transferase activity. The condensations of the primary products of type I and type II hydride transferases react with each other to yield diarylamines and nitrite; the latter can be further reduced to ammonium and serves as a nitrogen source for microorganisms in vivo.     

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.     

Oxidative transformation of aminodinitrotoluene isomers by multicomponent dioxygenases.

The electron-withdrawing nitro substituents of 2,4,6-trinitrotoluene (TNT) make the aromatic ring highly resistant to oxidative transformation. The typical biological transformation of TNT involves reduction of one or more of the nitro groups of the ring to produce the corresponding amine. Reduction of a single nitro substituent of TNT to an amino substituent increases the electron density of the aromatic nucleus considerably. The comparatively electron-dense nuclei of the aminodinitrotoluene (ADNT) isomers would be expected to be more susceptible to oxygenase attack than TNT. The hypothesis was tested by evaluating three nitroarene dioxygenases for the ability to hydroxylate the ADNT isomers. The predominant reaction was dioxygenation of the ring to yield nitrite and the corresponding aminomethylnitrocatechol. A secondary reaction was benzylic monooxygenation to form aminodinitrobenzyl alcohol. The substrate preferences and catalytic specificities of the three enzymes differed considerably. The discovery that the ADNT isomers are substrates for the nitroarene dioxygenases reveals the potential for extensive bacterial transformation of TNT under aerobic conditions.     

Oxidative Transformation of Aminodinitrotoluene Isomers by Multicomponent Dioxygenases

The electron-withdrawing nitro substituents of 2,4,6-trinitrotoluene (TNT) make the aromatic ring highly resistant to oxidative transformation. The typical biological transformation of TNT involves reduction of one or more of the nitro groups of the ring to produce the corresponding amine. Reduction of a single nitro substituent of TNT to an amino substituent increases the electron density of the aromatic nucleus considerably. The comparatively electron-dense nuclei of the aminodinitrotoluene (ADNT) isomers would be expected to be more susceptible to oxygenase attack than TNT. The hypothesis was tested by evaluating three nitroarene dioxygenases for the ability to hydroxylate the ADNT isomers. The predominant reaction was dioxygenation of the ring to yield nitrite and the corresponding aminomethylnitrocatechol. A secondary reaction was benzylic monooxygenation to form aminodinitrobenzyl alcohol. The substrate preferences and catalytic specificities of the three enzymes differed considerably. The discovery that the ADNT isomers are substrates for the nitroarene dioxygenases reveals the potential for extensive bacterial transformation of TNT under aerobic conditions.     

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.     

Biotransformation Patterns of 2,4,6-Trinitrotoluene by Aerobic Bacteria

2,4,6-Trinitrotoluene (TNT), a toxic nitroaromatic explosive, accumulates in the environment, making necessary the remediation of contaminated areas and unused materials. Although bioremediation has been utilized to detoxify TNT, the metabolic processes involved in the metabolism of TNT have proven to be complex. The three aerobic bacterial strains reported here (Pseudomonas aeruginosa, Bacillus sp., and Staphylococcus sp.) differ in their ability to biotransform TNT and in their growth characteristics in the presence of TNT. In addition, enzymatic activities have been identified that differ in the reduction of nitro groups, cofactor preferences, and the ability to eliminate-NO₂ from the ring. The Bacillus sp. has the most diverse bioremediation potential owing to its growth in the presence of TNT, high level of reductive ability, and capability of removing-NO₂ from the nitroaromatic ring. Received: 16 May 1997 / Accepted: 19 July 1997     

Microbial degradation of explosives: biotransformation versus mineralization

The nitroaromatic explosive 2,4,6-trinitrotoluene (TNT) is a reactive molecule that biotransforms readily under both aerobic and anaerobic conditions to give aminodinitrotoluenes. The resulting amines biotransform to give several other products, including azo, azoxy, acetyl and phenolic derivatives, leaving the aromatic ring intact. Although some Meisenheimer complexes, initiated by hydride ion attack on the ring, can be formed during TNT biodegradation, little or no mineralization is encountered during bacterial treatment. Also, although the ligninolytic physiological phase and manganese peroxidase system of fungi can cause some TNT mineralization in liquid cultures, little to no mineralization is observed in soil. Therefore, despite more than two decades of intensive research to biodegrade TNT, no biomineralization-based technologies have been successful to date. The non-aromatic cyclic nitramine explosives hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) lack the electronic stability enjoyed by TNT or its transformed products. Predictably, a successful enzymatic change on one of the N–NO₂ or C–H bonds of the cyclic nitramine would lead to a ring cleavage because the inner C–N bonds in RDX become very weak (<2 kcal/mol). Recently this hypothesis was tested and proved feasible, when RDX produced high amounts of carbon dioxide and nitrous oxide following its treatment with either municipal anaerobic sludge or the fungus Phanaerocheate chrysosporium. Research aimed at the discovery of new microorganisms and enzymes capable of mineralizing energetic chemicals and/or enhancing irreversible binding (immobilization) of their products to soil is presently receiving considerable attention from the scientific community. Received: 14 February 2000 / Received revision: 9 June 2000 / Accepted: 13 June 2000     

Hydride-mediated reduction of 2,4,6-trinitrotoluene by yeasts as the way to its deep degradation

Broad screening of microorganisms from natural and anthropogenic ecological niches has revealed strains Candida sp. AN-L15 and Geotrichum sp. AN-Z4 which transform 2,4,6-trinitrotoluene (TNT) via alternative pathways (with the domination of hydride ion-mediated reduction of the aromatic ring) and produce relatively high amounts of nitrites. According to the spectrophotometry data, the hydride attack of TNT by Candida sp. AN-L15 and Geotrichum sp. AN-Z4 grown at pH 5.0-8.0 leads to the mono- and dihydride complexes of TNT (H(-)-TNT and 2H(-)-TNT, respectively) and to protonated forms of the latter. Analysis by HPLC, GC-mass spectrometry, and ion chromatography revealed the products of deep conversion of TNT. The growth of the yeast strains in a weakly acidic medium with TNT (440 microM) is accompanied by formation of 2,4-dinitrotoluene (2,4-DNT, up to 18.2 microM). Together with accumulation of nitrites (up to 76.0 microM, depending on pH of the medium), these findings demonstrate the capacity of both strains for TNT denitration. Formation of 2,4-DNT reflects the realization of one of the possible mechanisms of TNT ortho-nitro group elimination and switching over to the pathways of metabolism of dinitrotoluenes, which are much more easily biodegradable than TNT. Simultaneously with the dominating TNT hydride attack, the mechanism of 4- and 6-electron reduction of the nitro group also functions in Candida sp. AN-L15 and Geotrichum sp. AN-Z4. Realization of the studied mechanisms of TNT transformation under growth of Candida sp. AN-L15 on n-alkane is important for bioremediation in the cases of combined pollution by oil products and explosives.     

Alternative pathways of the initial transformation of 2,4,6-trinitrotoluene by yeasts

A new model for the initial transformation of 2,4,6-trinitrotoluene (TNT) by facultatively anaerobic and aerobic yeasts is presented. The model is based on the data that Saccharomyces sp. ZS-A1 was able to reduce the nitrogroups of TNT with the formation of 2- and 4-hydroxyaminodinitrotoluenes (2-HADNT and 4-HADNT) as the major early TNT metabolites (the molar HADNT/TNT ratio reached 0.81), whereas aminodinitrotoluenes (ADNTs) and the hydride-Meisenheimer complex of TNT (H-TNT) were the minor products. Candida sp. AN-L13 almost completely transformed TNT into H-TNT through the reduction of the aromatic ring. Candida sp. AN-L14 transformed TNT through a combination of the two mechanisms described. Aeration stimulated the production of HADNT from TNT, whereas yeast incubation under stationary conditions promoted the formation of HADNT. The transformation of TNT into HADNT led to a tenfold increase in the acute toxicity of the TNT preparation with respect to Paramecium caudatum, whereas the increase in the toxicity was about twofold in the case of the alternative attack at the aromatic ring.     

Ferrihydrite-Dependent Growth of Sulfurospirillum deleyianum through Electron Transfer via Sulfur Cycling

Observations in enrichment cultures of ferric iron-reducing bacteria indicated that ferrihydrite was reduced to ferrous iron minerals via sulfur cycling with sulfide as the reductant. Ferric iron reduction via sulfur cycling was investigated in more detail with Sulfurospirillum deleyianum, which can utilize sulfur or thiosulfate as an electron acceptor. In the presence of cysteine (0.5 or 2 mM) as the sole sulfur source, no (microbial) reduction of ferrihydrite or ferric citrate was observed, indicating that S. deleyianum is unable to use ferric iron as an immediate electron acceptor. However, with thiosulfate at a low concentration (0.05 mM), growth with ferrihydrite (6 mM) was possible and sulfur was cycled up to 60 times. Also, spatially distant ferrihydrite in agar cultures was reduced via diffusible sulfur species. Due to the low concentrations of thiosulfate, S. deleyianum produced only small amounts of sulfide. Obviously, sulfide delivered electrons to ferrihydrite with no or only little precipitation of black iron sulfides. Ferrous iron and oxidized sulfur species were produced instead, and the latter served again as the electron acceptor. These oxidized sulfur species have not yet been identified. However, sulfate and sulfite cannot be major products of ferrihydrite-dependent sulfide oxidation, since neither compound can serve as an electron acceptor for S. deleyianum. Instead, sulfur (elemental S or polysulfides) and/or thiosulfate as oxidized products could complete a sulfur cycle-mediated reduction of ferrihydrite.     

Biodegradation of 2,4,6-trinitrotoluene (TNT): An enzymatic perspective

Enzymatic degradation of TNT by aerobic bacteria is mediated by oxygen insensitive (Type 1) or by oxygen sensitive nitroreductases (Type II nitroreductases). Transformation by Type I nitroreductases proceeds through two successive electron reductions either by hydride addition to the aromatic ring or by direct nitro group reduction following a ping pong kinetic mechanism. TNT is reduced to the level of hydroxylaminodinitrotoluenes and aminodinitrotoluenes by pure enzyme preparations without achieving mineralization. Interestingly, database gene and amino acid sequence comparisons of nitroreductases reveal a close relationship among all enzymes involved in TNT transformation. They are all flavoproteins which use NADPH/NADH as electron donor and reduce a wide range of electrophilic xenobiotics. TNT degradation by fungi is initiated by mycelia bound nitroreductases which reduce TNT to hydroxylaminodinitrotoluenes and aminodinitrotoluenes. Further degradation of these products and mineralization is achieved through the activity of oxidative enzymes especially lignin degrading enzymes (lignin and manganese peroxidases).     

The effect of 5-substitution on the electrochemical behavior and antitubercular activity of PA-824

Nitroimidazole PA-824 is part of an exciting new class of compounds currently undergoing clinical evaluation as novel TB therapeutics. The recently elucidated mechanism of action of PA-824 involves reduction of the nitroimidazole ring and subsequent nitric oxide release. The importance of this compound and its unique activity prompted us to explore how substitution of the nitroimidazole ring would affect electrochemical reduction and antitubercular activity. We prepared analogs of PA-824 with bromo, chloro, cyano, and amino substituents in the 5-position of the aromatic ring. We found that substitution of the imidazole ring greatly influences reduction and the stability of the corresponding nitro radical anion. Further, the antitubercular activities of the bromo and chloro analogs may indicate that an alternate nitroreductase pathway within Mycobacterium tuberculosis exists.     

Biodegradation of 2,4,6-trinitrotoluene (TNT): An enzymatic perspective

Enzymatic degradation of TNT by aerobic bacteria is mediated by oxygen insensitive (Type 1) or by oxygen sensitive nitroreductases (Type II nitroreductases). Transformation by Type I nitroreductases proceeds through two successive electron reductions either by hydride addition to the aromatic ring or by direct nitro group reduction following a ping pong kinetic mechanism. TNT is reduced to the level of hydroxylaminodinitrotoluenes and aminodinitrotoluenes by pure enzyme preparations without achieving mineralization. Interestingly, database gene and amino acid sequence comparisons of nitroreductases reveal a close relationship among all enzymes involved in TNT transformation. They are all flavoproteins which use NADPH/NADH as electron donor and reduce a wide range of electrophilic xenobiotics. TNT degradation by fungi is initiated by mycelia bound nitroreductases which reduce TNT to hydroxylaminodinitrotoluenes and aminodinitrotoluenes. Further degradation of these products and mineralization is achieved through the activity of oxidative enzymes especially lignin degrading enzymes (lignin and manganese peroxidases).     

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-(14)C]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 approximately 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 O(2), 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.