Reductive transformation of TNT by Escherichia coli: pathway description

Abstract

The reductive transformation of 2,4,6-trinitrotoluene (TNT) was studied using aerobically grown Escherichia coli cultures. In the absence of an external carbon or energy source, E. coli resting cells transformed TNT to hydroxylaminodinitrotoluenes (2HADNT, 4HADNT, with 4HADNT as the dominant isomer), aminodinitrotoluenes (4ADNT, with sporadic detection of 2ADNT), 2,4-di(hydroxylamino)-6-nitrotoluene (24D(HA)6NT), 2,4-diamino-6-nitrotoluene (24DA6NT), and an additional compound which was tentatively identified as a (hydroxylamino)aminonitrotoluene isomer via gas chromatography/mass spectroscopy and spectral analysis. The resting cell assay, performed in an oxygen-free atmosphere, avoided formation of azoxy dimers and provided good mass balances. Significant preference for reduction in the para versus ortho position was detected. The formation of 24D(HA)6NT, but not ADNT, appeared inhibited by the presence of TNT. The rate and extent of TNT reduction were significantly enhanced at higher cell densities, or by supplying an exogenous reducing power source, revealing the importance of enzyme concentration and reducing power. Whether the oxygen-insensitive E. coli nitroreductases, encoded by nfsA and nfsB, directly catalyze the TNT reduction or account for the complete TNT transformation pathway, remains to be determined.

Study of the role played by NfsA, NfsB nitroreductase and NemA flavin reductase from Escherichia coli in the conversion of ethyl 2-(2′-nitrophenoxy)acetate to 4-hydroxy-(2H)-1,4-benzoxazin-3(4H)-one (D-DIBOA), a benzohydroxamic acid with interesting biological properties

“…These three enzymes, NfsA, NfsB and NemA, which contain the flavin mononucleotide as prostetic group, can reduce up to two nitro groups (-NO 2 − ) from different nitroaromatics compounds as TNT (Esteve-Nuñez et al 2001;Yin et al 2005) to the corresponding amines (-NH 2 ) via hydroxylamino (-NHOH) intermediates. The reduction is catalysed through the addition of one or two electrons from the cofactors NADH and/or NADPH (Roldán et al 2008).…”

Aerobic Growth of Escherichia coli with 2,4,6-Trinitrotoluene (TNT) as the Sole Nitrogen Source and Evidence of TNT Denitration by Whole Cells and Cell-Free Extracts

“…In vitro denitration of TNT with purified NEM reductase was described by Williams et al (21), but the authors did not provide quantitative data with E. coli cells. Also, several reports have described the reduction of TNT by E. coli but not its denitration (6,14,22,23). Only one recent study has mentioned denitration of TNT by E. coli, but no quantitative data were provided and TNT was not the sole nitrogen source (13).…”

“…Next, ammonium was likely used as a nitrogen source by E. coli via the glutamine synthetase/glutamate synthase pathway (16). The formation of 4-hydroxylamino-2,6-dinitrotoluene as a major intermediate was observed in the enzymatic reduction of TNT with extracts from E. coli (20) and with several enzymes of E. coli (21,22). We observed a greater bacterial growth in the presence of glucose, and we found that more 4-A-2,6-DNT was produced than in the presence of glycerol (Table 1).…”

The Uncharacterized Transcription Factor YdhM Is the Regulator of the nemA Gene, Encoding N -Ethylmaleimide Reductase

“…For instance, some E. coli laboratory strains have multiple enzymes that degrade 2,4,6-trinitrotoluene (TNT) for release of nitrite from the nitroaromatic ring, yielding 2-hydroxylamino-6-nitrotoluene (5). In addition to NemA, both NfsA and NfsB nitroreductases catalyze TNT reduction, converting the nitro groups on the aromatic ring to the corresponding hydroxylamino derivatives (1,17,29). All these nitroreductases degrade toxic nitrous compounds, ultimately releasing ammonium ions that can, in turn, be used as a nitrogen source by E. coli for growth.…”