Substrate Selectivity of a 3-Nitrophenol-Induced Metabolic System in Pseudomonas putida 2NP8 Transforming Nitroaromatic Compounds into Ammonia under Aerobic Conditions

The 3-nitrophenol-induced enzyme system in cells of Pseudomonas putida 2NP8 manifested a wide substrate range in transforming nitroaromatic compounds through to ammonia production. All of the 30 mono- or dinitroaromatic substrates except 4-nitrophenol, 2,4-dinitrophenol, 2,4,6-trinitrophenol, 3-nitroaniline, 2-nitrobenzoic acid, and 2-nitrofuran were quickly transformed. Ammonia production from most nitroaromatic substrates appeared to be stoichiometric.     

Kinetic and structural basis of reactivity of pentaerythritol tetranitrate reductase with NADPH, 2-cyclohexenone, nitroesters, and nitroaromatic explosives

The reaction of pentaerythritol tetranitrate reductase with reducing and oxidizing substrates has been studied by stopped-flow spectrophotometry, redox potentiometry, and X-ray crystallography. We show in the reductive half-reaction of pentaerythritol tetranitrate (PETN) reductase that NADPH binds to form an enzyme-NADPH charge transfer intermediate prior to hydride transfer from the nicotinamide coenzyme to FMN. In the oxidative half-reaction, the two-electron-reduced enzyme reacts with several substrates including nitroester explosives (glycerol trinitrate and PETN), nitroaromatic explosives (trinitrotoluene (TNT) and picric acid), and alpha,beta-unsaturated carbonyl compounds (2-cyclohexenone). Oxidation of the flavin by the nitroaromatic substrate TNT is kinetically indistinguishable from formation of its hydride-Meisenheimer complex, consistent with a mechanism involving direct nucleophilic attack by hydride from the flavin N5 atom at the electron-deficient aromatic nucleus of the substrate. The crystal structures of complexes of the oxidized enzyme bound to picric acid and TNT are consistent with direct hydride transfer from the reduced flavin to nitroaromatic substrates. The mode of binding the inhibitor 2,4-dinitrophenol (2,4-DNP) is similar to that observed with picric acid and TNT. In this position, however, the aromatic nucleus is not activated for hydride transfer from the flavin N5 atom, thus accounting for the lack of reactivity with 2,4-DNP. Our work with PETN reductase establishes further a close relationship to the Old Yellow Enzyme family of proteins but at the same time highlights important differences compared with the reactivity of Old Yellow Enzyme. Our studies provide a structural and mechanistic rationale for the ability of PETN reductase to react with the nitroaromatic explosive compounds TNT and picric acid and for the inhibition of enzyme activity with 2,4-DNP.     

Interactions of nitroaromatic compounds with the mammalian selenoprotein thioredoxin reductase and the relation to induction of apoptosis in human cancer cells

Here we described novel interactions of the mammalian selenoprotein thioredoxin reductase (TrxR) with nitroaromatic environmental pollutants and drugs. We found that TrxR could catalyze nitroreductase reactions with either one- or two-electron reduction, using its selenocysteine-containing active site and another redox active center, presumably the FAD. Tetryl and p-dinitrobenzene were the most efficient nitroaromatic substrates with a k(cat) of 1.8 and 2.8 s(-1), respectively, at pH 7.0 and 25 degrees C using 50 muM NADPH. As a nitroreductase, TrxR cycled between four- and two-electron-reduced states. The one-electron reactions led to superoxide formation as detected by cytochrome c reduction and, interestingly, reductive N-denitration of tetryl or 2,4-dinitrophenyl-N-methylnitramine, resulting in the release of nitrite. Most nitroaromatics were uncompetitive and noncompetitive inhibitors with regard to NADPH and the disulfide substrate 5,5'-dithiobis(2-nitrobenzoic acid), respectively. Tetryl and 4,6-dinitrobenzofuroxan were, however, competitive inhibitors with respect to 5,5'-dithiobis(2-nitrobenzoic acid) and were clearly substrates for the selenolthiol motif of the enzyme. Furthermore, tetryl and 4,6-dinitrobenzofuroxan efficiently inactivated TrxR, likely by alkylation of the selenolthiol motif as in the inhibition of TrxR by 1-chloro-2,4-dinitrobenzene/dinitrochlorobenzene (DNCB) or juglone. The latter compounds were the most efficient inhibitors of TrxR activity in a cellular context. DNCB, juglone, and tetryl were highly cytotoxic and induced caspase-3/7 activation in HeLa cells. Furthermore, DNCB and juglone were potent inducers of apoptosis also in Bcl2 overexpressing HeLa cells or in A549 cells. Based on these findings, we suggested that targeting of intracellular TrxR by alkylating nitroaromatic or quinone compounds may contribute to the induction of apoptosis in exposed human cancer cells.     

Mitochondrial permeability transition as a source of superoxide anion induced by the nitroaromatic drug nimesulide in vitro

Nimesulide, a widely used nonsteroidal anti-inflammatory drug containing a nitroaromatic moiety, has been associated with rare but serious hepatic adverse effects. The mechanisms underlying this idiosyncratic hepatotoxicity are unknown; however, both mitochondrial injury and oxidative stress have been implicated in contributing to liver injury in susceptible patients. The aim of this study was, first, to explore whether membrane permeability transition (MPT) could contribute to nimesulide's mitochondrial toxicity and, second, whether metabolism-derived reactive oxygen species (ROS) were responsible for MPT. We found that isolated mouse liver mitochondria readily underwent Ca2+-dependent, cyclosporin A-sensitive MPT upon exposure to nimesulide (at >or=3 microM). Net increases in mitochondrial superoxide anion levels, determined with the fluorescent probe dihydroethidium, were induced by nimesulide only in the presence of Ca2+ and were cyclosporin A-sensitive, indicating that superoxide production was a consequence, rather than the cause, of MPT. In addition, nimesulide caused a rapid dissipation of the inner mitochondrial transmembrane potential (at >or=3 microM), followed by a concentration-dependent decrease in ATP biosynthesis. Because nimesulide, unlike the related nitroaromatic drug nilutamide, did not produce any detectable ROS during incubation with mouse hepatic microsomes, we conclude that mitochondrial uncoupling causes MPT and that ROS production is a secondary effect.     

Construction of Escherichia coli strains for conversion of nitroacetophenones to ortho-aminophenols

The predominant bacterial pathway for nitrobenzene (NB) degradation uses an NB nitroreductase and hydroxylaminobenzene (HAB) mutase to form the ring-fission substrate ortho-aminophenol. We tested the hypothesis that constructed strains might accumulate the aminophenols from nitroacetophenones and other nitroaromatic compounds. We constructed a recombinant plasmid carrying NB nitroreductase (nbzA) and HAB mutase A (habA) genes, both from Pseudomonas pseudoalcaligenes JS45, and expressed the enzymes in Escherichia coli JS995. IPTG (isopropyl-beta-D-thiogalactopyranoside)-induced cells of strain JS995 rapidly and stoichiometrically converted NB to 2-aminophenol, 2-nitroacetophenone (2NAP) to 2-amino-3-hydroxyacetophenone (2AHAP), and 3-nitroacetophenone (3NAP) to 3-amino-2-hydroxyacetophenone (3AHAP). We constructed another recombinant plasmid containing the nitroreductase gene (nfs1) from Enterobacter cloacae and habA from strain JS45 and expressed the enzymes in E. coli JS996. Strain JS996 converted NB to 2-aminophenol, 2-nitrotoluene to 2-amino-3-methylphenol, 3-nitrotoluene to 2-amino-4-methylphenol, 4-nitrobiphenyl ether to 4-amino-5-phenoxyphenol, and 1-nitronaphthalene to 2-amino-1-naphthol. In larger-scale biotransformations catalyzed by strain JS995, 75% of the 2NAP transformed was converted to 2AHAP, whereas 3AHAP was produced stoichiometrically from 3NAP. The final yields of the aminophenols after extraction and recovery were >64%. The biocatalytic synthesis of ortho-aminophenols from nitroacetophenones suggests that strain JS995 may be useful in the biocatalytic production of a variety of substituted ortho-aminophenols from the corresponding nitroaromatic compounds.     

PnrA, a new nitroreductase-family enzyme in the TNT-degrading strain Pseudomonas putida JLR11

Nitroreductases are a group of proteins that catalyse pyridine nucleotide-dependent reduction of nitroaromatics compounds, showing significant human health and environmental implications. In this study we have identified the nitroreductase-family enzymes PnrA and PnrB from the TNT-degrading strain Pseudomonas putida. The enzyme encoded by the pnrA gene was expressed in Escherichia coli, purified to homogeneity and shown to be a flavoprotein that used 2 mol of NADPH to reduce 1 mol of 2,4,6-trinitrotoluene (TNT) to 4-hydroxylamine-2,6-dinitrotoluene, using a ping-pong bi-bi mechanism. The PnrA enzyme also recognized as substrates as a number of other nitroaromatic compounds, i.e. 2,4-dinitrotoluene, 3-nitrotoluene, 3- and 4-nitrobenzoate, 3,5-dinitrobenzamide and 3,5-dinitroaniline expanding the substrates profile from previously described nitroreductases. However, TNT resulted to be the most efficient substrate examined according to the Vmax/Km parameter. Expression analysis of pnrA- and pnrB-mRNA isolated from cells growing on different nitrogen sources suggested that expression of both genes was constitutive and that its level of expression was relatively constant regardless of the growth substrate. This is in agreement with enzyme-specific activity determined with cells growing with different N-sources.     

Synthesis of substituted catechols using nitroarene dioxygenases

The nitroarene dioxygenases are in the class of Rieske iron-containing oxygenases that incorporate atmospheric oxygen into substrates via electrophilic attack on the substrate. In their native role, the nitroarene dioxygenases start degradative pathways by hydroxylating nitro-substituted, and adjacent unsubstituted carbons of nitroaromatic compounds. The reaction yields the corresponding nitro-cis-cyclohexadienediol, which is unstable and spontaneously re-aromatizes to form a catechol and nitrite. In bacterial metabolism, the specificity of the hydroxylation determines subsequent steps in degradation pathways. Experiments were done to find whether the specificity could be exploited to direct the hydroxylation of multiply substituted aromatic substrates and thereby produce novel catechols. Recombinant strains carrying genes for nitroarene dioxygenases were used for transformation of various substituted nitroaromatic compounds. The reactions were analyzed using HPLC to track substrate consumption and product formation, then GC–MS and NMR to identify the reaction products. A number of substituted catechols were obtained using the recombinant biocatalysts. The nitro-substituted carbon was the primary site for dioxygenase hydroxylation. When substrates included nitro and halogen substituents, the halogen-substituted positions were also targeted, but less frequently than the nitro-substituted site. The production of catechols was limited in batch fermentations, likely due to toxicity of the quinones that result from air oxidation of catechols. The nitroarene dioxygenases will serve as catalysts for direct synthesis of highly substituted catechols, however, the reaction conditions must be engineered to overcome product toxicity and allow sustained accumulation of catecholic products.     

Development of a sensitive long-wavelength fluorogenic probe for nitroreductase: a new fluorimetric indictor for analyte determination by dehydrogenase-coupled biosensors

Nitroreductase (NTR) is a flavin-containing enzyme that uses NADH as the electron source to reduce nitroaromatic compounds to the corresponding amines. Previous studies have shown that nitroreductase-targeted latent fluorophores exhibit low solubility in the aqueous media and fluoresce at lower wavelengths upon uncloaking, thus limiting their effective applications. Here, we have prepared a new switch-on long-wavelength latent fluorogenic substrate, NTRLF (4), for NTR. In the presence of NADH, NTR catalyzes the reduction of the nitroaromatic moiety in NTRLF (4), followed by the cascade reaction, 1,6-rearrangement-elimination reaction, cyclic urea formation, and concomitant ejects a long-wavelength fluorescence coumarin (8). However, this reaction was inhibited in the presence of nitroaromatic analogues. The fluorescence signal generated by the cascade reaction was specific and insensitive to various reductants. Accordingly, we propose that NTRLF and NTR in the presences of NADH constitute a useful switch-off high-throughput fluorescence sensor for screening nitroaromatic compounds. Furthermore, NTRLF in the NTR-coupled 3-hydroxybutyrate dehydrogenase and aldehyde dehydrogenase assay reactions was a sensitive fluorimetric indicator for the quantitatively measurement of 3-hydroxybutyrate and propionaldehyde, respectively within micromolar range. Our novel NTRLF and NTR-coupled dehydrogenase assay platform may thus be effectively applied for the quantitative estimation of a broad range of analytes.     

Localization of superoxide anion production to mitochondrial electron transport chain in 3-NPA-treated cells

3-Nitropropionic acid (3-NPA), an inhibitor of succinate dehydrogenase (SDH) at complex II of the mitochondrial electron transport chain induces cellular energy deficit and oxidative stress-related neurotoxicity. In the present study, we identified the site of reactive oxygen species production in mitochondria. 3-NPA increased O2- generation in mitochondria respiring on the complex I substrates pyruvate+malate, an effect fully inhibited by rotenone. Antimycin A increased O2- production in the presence of complex I and/or II substrates. Addition of 3-NPA markedly increased antimycin A-induced O2- production by mitochondria incubated with complex I substrates, but 3-NPA inhibited O2- formation driven with the complex II substrate succinate. At 0.6 microM, myxothiazol inhibits complex III, but only partially decreases complex I activity, and allowed 3-NPA-induced O2- formation; however, at 40 microM myxothiazol (which completely inhibits both complexes I and III) eliminated O2- production from mitochondria respiring via complex I substrates. These results indicate that in the presence of 3-NPA, mitochondria generate O2- from a site between the ubiquinol pool and the 3-NPA block in the respiratory complex II.     

Antigenic properties of nitro aromatic derivatives of polyethylene glycol

The immunogenic properties of the dihapten and monohapten derivatives of polyethylene glycol with different nitroaromatic groupings were studied. 2,4-dinitrophenyl, 2, 4, 6-trinitrophenyl and trinitrophenyl-ethyl groupings were used as hapten groups. The injection of monohapten compounds was found to induce the accumulation of antibody-forming cells secreting antibodies to trinitrophenyl in the spleen of normal and athymic nude mice. As early as on day 3 the number of antibody-forming cells considerably exceeded their background level, the process of B-cell activation being, to a certain extent, thymus-independent. Dihapten compounds were not immunologically active. The effect rendered by the nitroaromatic derivatives of polyethylene glycol, revealed in this study, is linked with the known capacity of polyethylene glycol to adsorb on the surface of cell membranes.     

Cytotoxicity of TNT and its metabolites

The production and storage of explosives has resulted in the environmental accumulation of 2,4,6-trinitrotoluene (TNT). The biotransformation products of the nitroaromatic compound TNT and metabolites in mammalian cells in culture and their cytotoxicity are studied. We report after our analysis by reverse phase high performance liquid chromatography (HPLC) that the most prevalent biotransformation product of TNT in the NG108 neuroblastoma cells is primarily monoamino-dinitrotoluene (2Am-DNT). It causes toxic effects based on trypan blue exclusion and LDH-release colorimetric assays.     

Independent Relationship between Amyloid Precursor Protein (APP) Dimerization and γ-Secretase Processivity

Altered production of β-amyloid (Aβ) from the amyloid precursor protein (APP) is closely associated with Alzheimer’s disease (AD). APP has a number of homo- and hetero-dimerizing domains, and studies have suggested that dimerization of β-secretase derived APP carboxyl terminal fragment (CTFβ, C99) impairs processive cleavage by γ-secretase increasing production of long Aβs (e.g., Aβ1-42, 43). Other studies report that APP CTFβ dimers are not γ-secretase substrates. We revisited this issue due to observations made with an artificial APP mutant referred to as 3xK-APP, which contains three lysine residues at the border of the APP ectodomain and transmembrane domain (TMD). This mutant, which dramatically increases production of long Aβ, was found to form SDS-stable APP dimers, once again suggesting a mechanistic link between dimerization and increased production of long Aβ. To further evaluate how multimerization of substrate affects both initial γ-secretase cleavage and subsequent processivity, we generated recombinant wild type- (WT) and 3xK-C100 substrates, isolated monomeric, dimeric and trimeric forms of these proteins, and evaluated both ε-cleavage site utilization and Aβ production. These show that multimerization significantly impedes γ-secretase cleavage, irrespective of substrate sequence. Further, the monomeric form of the 3xK-C100 mutant increased long Aβ production without altering the initial ε-cleavage utilization. These data confirm and extend previous studies showing that dimeric substrates are not efficient γ-secretase substrates, and demonstrate that primary sequence determinants within APP substrate alter γ-secretase processivity.     

Mitochondrial Complex I superoxide production is attenuated by uncoupling

Complex I, i.e. proton-pumping NADH:quinone oxidoreductase, is an essential component of the mitochondrial respiratory chain but produces superoxide as a side-reaction. However, conditions for maximum superoxide production or its attenuation are not well understood. Unlike for Complex III, it has not been clear whether a Complex I-derived superoxide generation at forward electron transport is sensitive to membrane potential or protonmotive force. In order to investigate this, we used Amplex Red for H(2)O(2) monitoring, assessing the total mitochondrial superoxide production in isolated rat liver mitochondria respiring at state 4 as well as at state 3, namely with exclusive Complex I substrates or with Complex I substrates plus succinate. We have shown for the first time, that uncoupling diminishes rotenone-induced H(2)O(2) production also in state 3, while similar attenuation was observed in state 4. Moreover, we have found that 5-(N-ethyl-N-isopropyl) amiloride is a real inhibitor of Complex I H(+) pumping (IC(50) of 27 microM) without affecting respiration. It also partially prevented suppression by FCCP of rotenone-induced H(2)O(2) production with Complex I substrates alone (glutamate and malate), but nearly completely with Complexes I and II substrates. Sole 5-(N-ethyl-N-isopropyl) amiloride alone suppressed 20% and 30% of total H(2)O(2) production, respectively, under these conditions. Our data suggest that Complex I mitochondrial superoxide production can be attenuated by uncoupling, which means by acceleration of Complex I H(+) pumping due to the respiratory control. However, when this acceleration is prevented by 5-(N-ethyl-N-isopropyl) amiloride inhibition, no attenuation of superoxide production takes place.     

Volatile metabolites and other indicators of Penicillium aurantiogriseum growth on different substrates

Penicillium aurantiogriseum Dierckx was cultivated on six agar substrates (barley meal agar, oat meal agar, wheat meal agar, malt extract agar, Czapek agar, and Norkrans agar) and on oat grain for 5 days in cultivation vessels provided with an inlet and an outlet for air. Volatile metabolites produced by the cultures were collected on a porous polymer adsorbent by passing an airstream through the vessel. Volatile metabolites were collected between days 2 and 5 after inoculation. CO2 production was simultaneously measured, and after the cultivation period ergosterol contents and the numbers of CFU of the cultures were determined. Alcohols of low molecular weight and sesquiterpenes were the dominant compounds found. During growth on oat grain the production of 8-carbon alcohols and 3-methyl-1-butanol was higher and the production of terpenes was lower than during growth on agar substrates. The compositions of the volatile metabolites from oat grain were more similar to those from wheat grain, which was used as a substrate in a previous investigation, than to those produced on any of the agar substrates. Regarding the agar substrates, the production of terpenes was most pronounced on the artificial substrates (Czapek agar and Norkrans agar) whereas alcohol production was highest on substrates based on cereals. The production of volatile metabolites was highly correlated with the production of CO2 and moderately correlated with ergosterol contents, whereas no correlation with the numbers of CFU was found. Thus, the volatile metabolites formed and the ergosterol contents of fungal cultures should be good indicators of present and past fungal activity.     

Volatile metabolites and other indicators of Penicillium aurantiogriseum growth on different substrates.

Penicillium aurantiogriseum Dierckx was cultivated on six agar substrates (barley meal agar, oat meal agar, wheat meal agar, malt extract agar, Czapek agar, and Norkrans agar) and on oat grain for 5 days in cultivation vessels provided with an inlet and an outlet for air. Volatile metabolites produced by the cultures were collected on a porous polymer adsorbent by passing an airstream through the vessel. Volatile metabolites were collected between days 2 and 5 after inoculation. CO2 production was simultaneously measured, and after the cultivation period ergosterol contents and the numbers of CFU of the cultures were determined. Alcohols of low molecular weight and sesquiterpenes were the dominant compounds found. During growth on oat grain the production of 8-carbon alcohols and 3-methyl-1-butanol was higher and the production of terpenes was lower than during growth on agar substrates. The compositions of the volatile metabolites from oat grain were more similar to those from wheat grain, which was used as a substrate in a previous investigation, than to those produced on any of the agar substrates. Regarding the agar substrates, the production of terpenes was most pronounced on the artificial substrates (Czapek agar and Norkrans agar) whereas alcohol production was highest on substrates based on cereals. The production of volatile metabolites was highly correlated with the production of CO2 and moderately correlated with ergosterol contents, whereas no correlation with the numbers of CFU was found. Thus, the volatile metabolites formed and the ergosterol contents of fungal cultures should be good indicators of present and past fungal activity.     

Conformational aspects of polypeptides. XXV. Solvent and temperature effects on the conformations of copolymers of benzyl and methyl L-aspartate with nitrobenzyl L-aspartate

A series of copolymers of β-p-nitrobenzyl L -aspartate with β-benzyl L -aspartate and with β-mcthyl L -aspartatc in helix-supporting and helix-breaking conditions have been reexamined by using ultraviolet isotropic, absorption, optical rotatory dispersion, and circular dichroism techniques. Many different conformations are apparent, depending on solvent and temperature. Chloroform, trifluoroethanol, and methylene dichloride support the left-handed helical conformation of the copolymers containing less than about 20 mole-% nitroaromatic residues and the right-handed helical conformation of the copolymers containing more than approximately 30 mole-% nitroaromatic residues. In trifluoroacetic acid all the copolymers are in a random-coil conformation. In hexa-fluoroacetone trihydrate and in trimethyl phosphate, the copolypeptides with low nitroaromatic residues content are predominantly in a disordered conformation, while those with high nitroaromatic residues content show a right-handed helical array. Reversible helix-ramlom-coil transitions are observed with increasing temperature in trimethyl phosphate. An example of right-handed-left-handed helix reversible transition with temperature is reported in a chloroform-trimethyl phosphate (2:1) mixture. Nitrobenzyl-nilrobenzyl side-chain interactions in chloroform, but not in trifluoroacetic acid or in trimethyl phosphate, have been confirmed. For the first time we report the circular dichroism spectra in which the n-π* peptide band of a left-handed helical conformation is almost completely evident.     

Implication of manganese (III), oxalate, and oxygen in the degradation of nitroaromatic compounds by manganese peroxidase (MnP)

The fungal ligninolytic enzyme manganese peroxidase (MnP) is known to function by oxidizing Mn(II) to Mn(III), a powerful oxidant. In this work, an abiotic system consisting of Mn(III) in oxalate buffer under aerobic conditions (Mn(III)/oxalate/O2 system) was shown to be capable of extensively transforming 2-amino-4,6-dinitrotoluene (2A46DNT)--one of the main reduction products of 2,4,6-trinitrotoluene (TNT). No significant transformation occurred in the presence of other organic acids or under anaerobic conditions. The Mn(III)/oxalate/O2 system was also able to transform other nitroaromatic compounds such as 2-nitrotoluene, 4-nitrotoluene, 2,4-dinitrotoluene, TNT - the latter to a lesser extent -, and their reduction derivatives. The Mn(III)/oxalate/O2 system mineralized 14C-U-ring labeled 2A46DNT slightly, while no significant mineralization of 14C-U-ring labeled TNT was observed. Unidentified 14C-transformation products were highly polar. Electron spin resonance experiments performed on the Mn(III)/oxalate/O2 system revealed the generation of formyl free radicals (*COO-). The oxygen requirement for the transformation of nitroaromatic compounds suggests the involvement of superoxide free radicals (O2-*). produced through autoxidation of *COO- by molecular oxygen. The implication of such a Mn(III)/oxalate/O2 system in the MnP-catalyzed degradation of nitroaromatic pollutants by white-rot fungi is further discussed.     

Transformation of nitroaromatic compounds by a methanogenic bacterium,Methanococcus sp. (strain B)

The transformation of several nitroaromatic compounds by a newly isolated methanogenic bacterium, Methanococcus sp. (strain B) was studied. The presence of nitroaromatic compounds (0.5 mM) viz., nitrobenzene, 2,4-dinitrobenzene, 2,4,6-trinitrobenzene, 2,4-dinitrophenol, 2,4-dinitrobenzene, and 2,6-dinitrotoluene in the culture medium did not inhibit growth of the isolate. The bacteria grew rapidly and reached stationary phase within seven days of incubation. All the nitroaromatic compounds tested were 80 to 100% transformed by the bacterium to amino compounds by a reduction process. The isolate did not use the nitroaromatic compounds as the sole source of carbon or nitrogen. The transformation of nitroaromatic compounds by this isolate was compared to that of other methanogenic bacteria. Out of five methanogens studied, only Methanococcus deltae and Methanococcus thermolithotrophicus could transform the nitroaromatic compounds; however, the transformation rates were significantly less than that of the new isolate Methanococcus sp. (strain B). The nitroaromatic compounds were not transformed by Methanosarcina barkeri, Methanobacterium thermoautotrophicum, and Methanobrevibacter ruminantium.Abbreviations NB Nitrobenzene - DNB 2,4-Dinitrobenzene - TNB 2,4,6-Trinitrobenzene - DNP 2,4-Dinitrophenol - 2,4-DNT 2,4-Dinitrotoluene - 2,6-DNT 2,6-Dinitrotoluene     

Biodegradation of nitroaromatics and other nitrogen-containing xenobiotics

Nitroaromatic compounds constitute a major class of widely distributed environmental contaminants. Compounds like nitrobenzene, nitrotoluenes, nitrophenols, nitrobenzoates and nitrate esters are of considerable industrial importance. They are frequently used as pesticides, explosives, dyes, and in the manufacture of polymers and pharmaceuticals. Many nitroaromatic compounds and their conversion products have been shown to have toxic or mutagenic properties. Most of them are biodegradable in nature by various microorganisms. However, most contaminated environments have combinations of nitroaromatic compounds present, which complicates the bioremediation efforts. During the last 10 years, research on the biodegradation of nitroaromatic compounds has yielded a wealth of information on the microbiological, biochemical and genetic aspects of the process. New metabolic pathways have been discovered and genes and enzymes responsible for key transformation reactions have been identified and characterized. Knowledge and advances in pathway engineering have helped further understanding of the nature of nitroaromatic biodegradation and the development of bioremediation solutions. In this paper, an overview of recent developments on the biodegradation of nitrogen-containing xenobiotics is presented.