2,4,6-Trinitrotoluene Pathway Map

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This pathway was contributed by Sara McFarlan, University of Minnesota.

2,4,6-Trinitrotoluene is a yellow, odorless solid that does not occur naturally in the environment. It is commonly known as TNT and is an explosive used in military shells, bombs, and grenades, in industrial uses, and in underwater blasting. TNT production in the United State occurs solely at military arsenals. Accidental release of TNT has contaminated groundwater and soil at numerous munition manufacturing sites. The U.S. EPA has listed TNT as a priority pollutant and has recommended its removal from contaminated sites. TNT is toxic to algae and invertebrates and chronic exposure to TNT by humans causes harmful health effects, including anemia and abnormal liver function, cataract development, and skin irritation. The EPA has determined that TNT is a possible human carcinogen, based on animal studies.

This page shows only aerobic TNT degradation pathway. Its anaerobic degradation is documented elsewhere in the EAWAG-BBD. Microbial transformation of TNT usually begins with reduction of one of the nitro groups. The enzymes which catalyze these reductions are "non-specific" NAD(P)H dependent nitroreductases and are largely uncharacterized. One exception is a nitrobenzene nitroreducatase, purified from P. pseudoalcaligenes JS52, which can transform TNT to the mono- and dihydroxylamino intermediates. As its name suggests, this enzyme was originally found to reduce nitrobenzene. Aerobic bacteria are able to reduce 2 of the 3 nitro groups of TNT; the reduction of the third nitro group requires anaerobic conditions.

While denitration is often a major reaction in the biodegradation of nitrosubstituted compounds, bacterial denitration of TNT or its reduction products has been demonstrated in only a few cases. One example is shown below in which 2-amino-4-nitro-toluene is produced from 2-amino-4,6-dinitrotoluene. In addition, Martin et al. (1997) showed that P. savastanoi can generate 2,4-dinitrotoluene from TNT. The formation of Meisenheimer complexes from TNT has been described in at least 3 bacterial strains, including Rhodococcus erythropolis and Mycobacterium sp. (Vorbeck et al., 1998). These complexes result from the nucleophilic attack by a hydride ion on the aromatic ring. Formation of the hydride complexes could not be identified with the recently isolated TNT-enriched strains TNT-8 and TNT-32, nor with Pseudomonas sp. strain A (2NT-). However, these strains are able to carry out nitro-group reduction, as shown below. French et al. (1998) demonstrated that the enzyme pentaerythritol tetranitrate reductase from Enterobacter cloacae catalyzes the formation of both the hydride- and dihydride-Meisenheimer complexes.

Generally, complete mineralization of TNT is only detected with bacterial consortia. Many of the products of TNT biodegradation are highly reactive and covalently bind to cellular components and any solid supports (such as soil) present in the medium. The latter prevents or prolongs the mineralization process, but also hinders a further spread of TNT contaminants. Several authors have noted the formation of, as yet, unidentified compounds during TNT biodegradation. These have not been included in the pathway map.

The following is a text-format trinitrotoluene degradation pathway. Organisms which can initiate each pathway are given, however they do not all carry out every pathway reaction. When this is the case, organisms known to carry out a particular reaction are noted. Follow the links for more information on compounds or reactions.

|--------------Graphical Map 1 (21K)-------------| |------------Graphical Map 2 (19K)------------|

Trinitrotoluene           Trinitrotoluene                  Trinitrotoluene              Trinitrotoluene
P. aeruginosa          P. pseudoalcaligenes                Pseudomonas sp.               P. aeruginosa
MAO1                           JS52                         strain A 2NT-                     MAO1
P. savastanoi             Pseudomonas sp.               strains TNT-8 & TNT-32                  |
Actinomycetes spp.         strain A 2NT-                     Bacillus sp.                       |
|                      strains TNT-8 & TNT-32             Staphylococcus sp.                    |
|                           Bacillus sp.                           |                            |
|                        Staphylococcus sp.                        |                            |
|                                |                                 |                            |
|                                |                                 |                            |
| non-specific                   | nitrobenzene                    | non-specific               | non-specific
| NAD(P)H                        | nitroreductase                  | NAD(P)H                    | NAD(P)H
| nitroreductase                 |                                 | nitroreductase             | nitroreductase
|                                |                                 |                            |
|                                v                                 v                            |
|                 H      4-Hydroxylamino-                  2-Hydroxylamino-    M                |
|   2,2',6,6'-  <----  2,6-dinitrotoluene -------+------- 4,6-dinitrotoluene ----> 4,4',6,6'-   |
|   Tetranitro-      /           |             K |                           \     Tetranitro-  |
|   4,4'-azoxy-     /  nitroben- |               v                            \    2,2'-azoxy-  |
|     toluene      / zene nitro- |          2,4',6,6'-                         \     toluene    |
|                 /    reductase |          Tetranitro-                         \               |
|                /               |       4,2'-azoxytoluene                       \              |
|  non-specific /     P. pseudo- |                                   non-specific \             |
|  NAD(P)H NO2 /     alcaligenes |                                         NAD(P)H \            |                
|   reductase /                  |                                   nitroreductase \           |
v            /                   v                                                   \          v
4-Amino-2,6-di-          2,4-Dihydroxyl-                                          2-Amino-4,6-di-
 nitrotoluene               amino-6-                                  <------------ nitrotoluene
  \          \            nitrotoluene                   Bacillus sp./              /          /
 to the       \                  |                Staphylococcus sp./              /        to the
Anaerobic TNT  \ non-specific    | non-specific                    /   P. aerugi- /    Anaerobic TNT
  Pathway       \ NAD(P)H        | NAD(P)H                      P /    nosa MAO1 /       Pathway
                 \ nitro-        | nitro-         2-Amino-4-nitro-    P. fluor- /
                  \ reductase    | reductase          toluene           escens /
                   \             |                                            /
                    \            v                              non-specific /
                      4-Amino-2-hydroxyl-                           NAD(P)H /
                     amino-6-nitrotoluene                   nitroreductase /
                                | ^                                       /
                                | | 4-amino-2-              2,6-Diamino-4-
                                | | nitroso-6-nitro-         nitrotoluene
                                | | toluene reductase
                                v |

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Page Author(s): Sara McFarlan

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