Our understanding of the genetics and biochemistry of microbial 2,4,6-trinitrotoluene (TNT) biotransformation has advanced significantly during the past 10 years, and biotreatment technologies have developed. In this review, we summarize this new knowledge. A number of enzyme classes involved in TNT biotransformation include the type I nitroreductases, the old yellow enzyme family, a respiration-associated nitroreductase, and possibly ring hydroxylating dioxygenases. Several strains harbor dual pathways: nitroreduction (reduction of the nitro group in TNT to a hydroxylamino and/or amino group) and denitration (reduction of the aromatic ring of TNT to Meisenheimer complexes with nitrite release). TNT can serve as a nitrogen source for some strains, and the postulated mechanism involves ammonia release from hydroxylamino intermediates. Field biotreatment technologies indicate that both stimulation of microbial nitroreduction and phytoremediation result in significant and permanent immobilization of TNT via its metabolites. While the possibility for TNT mineralization was rekindled with the discovery of TNT denitration and oxygenolytic and respiration-associated pathways, further characterization of responsible enzymes and their reaction mechanisms are required.

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.

2,4-Dinitrotoluene (2,4-DNT) and 2,6-DNT are priority pollutants, and 2,4-DNT dioxygenase of Burkholderia sp. strain DNT (DDO) catalyzes the initial oxidation of 2,4-DNT to form 4-methyl-5-nitrocatechol and nitrite but has significantly less activity on other dinitrotoluenes and nitrotoluenes (NT). Hence, oxidation of 2,3-DNT, 2,4-DNT, 2,5-DNT, 2,6-DNT, 2NT, and 4NT were enhanced here by performing saturation mutagenesis on codon I204 of the alpha subunit (DntAc) of DDO and by using a membrane agar plate assay to detect catechol formation. Rates of degradation were quantified both by the formation of nitrite and by the formation of the intermediates with high performance liquid chromatography. The degradation of both 2,3-DNT and 2,5-DNT were achieved for the first time (no detectable activity with the wild-type enzyme) using whole Escherichia coli TG1 cells expressing DDO variants DntAc I204L and I204Y (0.70 +/- 0.03 and 0.22 +/- 0.02 nmol/min/mg protein for 2,5-DNT transformation, respectively). DDO DntAc variant I204L also transformed both 2,6-DNT and 2,4-DNT 2-fold faster than wild-type DDO (0.8 +/- 0.6 nmol/min/mg protein and 4.7 +/- 0.5 nmol/min/mg protein, respectively). Moreover, the activities of DDO for 2NT and 4NT were also enhanced 3.5-fold and 8-fold, respectively. Further, DntAc variant I204Y was also discovered with comparable rate enhancements for the substrates 2,4-DNT, 2,6-DNT, and 2NT but not 4NT. Sequencing information obtained during this study indicated that the 2,4-DNT dioxygenases of Burkholderia sp. strain DNT and B. cepacia R34 are more closely related than originally reported. This is the first report of engineering an enzyme for enhanced degradation of nitroaromatic compounds and the first report of degrading 2,5-DNT.

Microbial 2,4,6-trinitrotoluene (TNT) biotransformation via sequential nitro-reduction appears a ubiquitous process, but the kinetics of these transformations have been poorly understood or described. TNT transformation by Escherichia coli was monitored and a kinetic model for reductive TNT depletion was developed and experimentally calibrated in this report. Using resting cells of aerobically pre-grown E. coli, TNT was quickly reduced to hydroxylaminodinitrotoluenes. The standard Michaelis-Menten model was modified to include three additional parameters: product toxicity (T (c)), substrate inhibition (K (i)), and intracellular reducing power (RH) limitation. Experimentally measured product toxicity (5.2 micromol TNT/mg cellular protein) closely matched the best-fit model value (2.84 micromol TNT/mg cellular protein). Parameter identifiability and reliability (k (m), K (s), T (c), and K (i)) was evaluated and confirmed through sensitivity analyses and via Monte Carlo simulations. The resulting kinetic model adequately described TNT reduction kinetics by E. coli resting cells in the absence or presence of reducing power limitation.

FR171456 is a natural product with cholesterol-lowering properties in animal models, but its molecular target is unknown, which hinders further drug development. Here we show that FR171456 specifically targets the sterol-4-alpha-carboxylate-3-dehydrogenase (Saccharomyces cerevisiae—Erg26p, Homo sapiens—NSDHL (NAD(P) dependent steroid dehydrogenase-like)), an essential enzyme in the ergosterol/cholesterol biosynthesis pathway. FR171456 significantly alters the levels of cholesterol pathway intermediates in human and yeast cells. Genome-wide yeast haploinsufficiency profiling experiments highlight the erg26/ERG26 strain, and multiple mutations in ERG26 confer resistance to FR171456 in growth and enzyme assays. Some of these ERG26 mutations likely alter Erg26 binding to FR171456, based on a model of Erg26. Finally, we show that FR171456 inhibits an artificial Hepatitis C viral replicon, and has broad antifungal activity, suggesting potential additional utility as an anti-infective. The discovery of the target and binding site of FR171456 within the target will aid further development of this compound.