Bamberger Rearrangement during TNT Metabolism by<i>Clostridium acetobutylicum</i>

Hughes, Joseph B.; Wang, C.; Yesland, K.; Richardson, Adam D.; Bhadra, Rajiv; Bennett, George N.; Rudolph, Frederick B.

doi:10.1021/es970612s

Cited by 75 publications

(71 citation statements)

References 22 publications

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“…The 2-hydroxylamine metabolites most likely can be produced from the 2-nitroreduction of 2-NBA and 2,4-DNBA by NbaA as a specific enzyme generally shows excellent chemoselectivity and regioselectivity in metabolism of substrate. If NbaA would reduce otherwise the 4-nitro group of 2,4-DNBA to produce 4-hydroxylamino-2-nitrobenzoic acid, the 4-hydroxylamine group could be derivatized to the 4-(Nacetoxyacetamido) form, showing the molecular ion at m/z 282.2, because the described derivatization procedure completely acetylates the 4-hydroxylamine group at both N and O centers (12). However, we detected no m/z 282.2 ion signal from the base peak chromatogram of 2,4-DNBA reduction.…”

Section: Resultsmentioning

confidence: 99%

“…Metabolites derived from the reduction of 2-NBA and 2,4-DNBA by NbaA were analyzed by thin-layer chromatography (TLC) and high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS) after following a modified procedure of derivatization of the unstable hydroxylamine groups with acetic anhydride (12). Reconstituted NbaA at a final concentration of 0.1 M monomer was added to 10 ml of 50 mM sodium phosphate buffer (pH 7.4) containing 0.5 mM either of 2-NBA or 2,4-DNBA, 0.1 mM MnCl 2 , and 10 M FMN.…”

Section: Materials and Chemicalsmentioning

confidence: 99%

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2-Nitrobenzoate 2-Nitroreductase (NbaA) Switches Its Substrate Specificity from 2-Nitrobenzoic Acid to 2,4-Dinitrobenzoic Acid under Oxidizing Conditions

Kim

Song

et al. 2013

J Bacteriol

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e 2-Nitrobenzoate 2-nitroreductase (NbaA) of Pseudomonas fluorescens strain KU-7 is a unique enzyme, transforming 2-nitrobenzoic acid (2-NBA) and 2,4-dinitrobenzoic acid (2,4-DNBA) to the 2-hydroxylamine compounds. Sequence comparison reveals that NbaA contains a conserved cysteine residue at position 141 and two variable regions at amino acids 65 to 74 and 193 to 216. The truncated mutant ⌬65-74 exhibited markedly reduced activity toward 2,4-DNBA, but its 2-NBA reduction activity was unaffected; however, both activities were abolished in the ⌬193-216 mutant, suggesting that these regions are necessary for the catalysis and specificity of NbaA. NbaA showed different lag times for the reduction of 2-NBA and 2,4-DNBA with NADPH, and the reduction of 2,4-DNBA, but not 2-NBA, failed in the presence of 1 mM dithiothreitol or under anaerobic conditions, indicating oxidative modification of the enzyme for 2,4-DNBA. The enzyme was irreversibly inhibited by 5,5=-dithio-bis-(2-nitrobenzoic acid) and ZnCl 2 , which bind to reactive thiol/thiolate groups, and was eventually inactivated during the formation of higherorder oligomers at high pH, high temperature, or in the presence of H 2 O 2 . SDS-PAGE and mass spectrometry revealed the formation of intermolecular disulfide bonds by involvement of the two cysteines at positions 141 and 194. Site-directed mutagenesis indicated that the cysteines at positions 39, 103, 141, and 194 played a role in changing the enzyme activity and specificity toward 2-NBA and 2,4-DNBA. This study suggests that oxidative modifications of NbaA are responsible for the differential specificity for the two substrates and further enzyme inactivation through the formation of disulfide bonds under oxidizing conditions.

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Section: Resultsmentioning

confidence: 99%

Section: Materials and Chemicalsmentioning

confidence: 99%

2-Nitrobenzoate 2-Nitroreductase (NbaA) Switches Its Substrate Specificity from 2-Nitrobenzoic Acid to 2,4-Dinitrobenzoic Acid under Oxidizing Conditions

Kim

Song

et al. 2013

J Bacteriol

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“…Hughes and colleagues have described the partial reduction of nitro groups on the TNT ring to hydroxylamino, which may undergo a Bamberger rearrangement to produce a phenolic amine (16,32).…”

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confidence: 99%

Assimilation of Nitrogen from Nitrite and Trinitrotoluene in Pseudomonas putida JLR11

et al. 2005

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Pseudomonas putida JLR11 releases nitrogen from the 2,4,6-trinitrotoluene (TNT) ring as nitrite or ammonium. These processes can occur simultaneously, as shown by the observation that a nasB mutant impaired in the reduction of nitrite to ammonium grew at a slower rate than the parental strain. Nitrogen from TNT is assimilated via the glutamine syntethase-glutamate synthase (GS-GOGAT) pathway, as evidenced by the inability of GOGAT mutants to use TNT. This pathway is also used to assimilate ammonium from reduced nitrate and nitrite. Three mutants that had insertions in ntrC, nasT, and cnmA, which encode regulatory proteins, failed to grow on nitrite but grew on TNT, although slower than the wild type.

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“…C. acetobutylicum transformed TNT with accumulation of the hydroxylamino intermediates, specifically 4-hydroxylamino-2,6-dinitrotoluene (4HA26DNT) and 2,4-dihydroxylamino-6-nitrotoluene (24DHA6NT), without formation of commonly observed amines (8,32,36). Further reduction of these metabolites by Clostridium acetobutylicum results in the formation of a phenolic amine through a Bamberger rearrangement (19). TNT is only completely reduced to 2,4,6-triaminotoluene under strictly anaerobic conditions (32).…”

mentioning

confidence: 99%

“…TNT is only completely reduced to 2,4,6-triaminotoluene under strictly anaerobic conditions (32). The catalytic process for these systems has not been clarified to date, although evidence for biocatalysis has been presented (19) and probable key enzymes, including hydrogenases, have been implicated in reduction steps (17). Several additional findings support the possible role of hydrogenase in initial TNT transformation.…”

mentioning

confidence: 99%

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

Watrous¹,

Clark

Kutty

et al. 2003

Appl Environ Microbiol

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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 M. 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 (R 2 ‫؍‬ 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.

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Bamberger Rearrangement during TNT Metabolism byClostridium acetobutylicum

Cited by 75 publications

References 22 publications

2-Nitrobenzoate 2-Nitroreductase (NbaA) Switches Its Substrate Specificity from 2-Nitrobenzoic Acid to 2,4-Dinitrobenzoic Acid under Oxidizing Conditions

2-Nitrobenzoate 2-Nitroreductase (NbaA) Switches Its Substrate Specificity from 2-Nitrobenzoic Acid to 2,4-Dinitrobenzoic Acid under Oxidizing Conditions

Assimilation of Nitrogen from Nitrite and Trinitrotoluene in Pseudomonas putida JLR11

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

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