Mineralization of 14-C-U-ring labeled 4-hydroxylamino-2,6-trinitrotoluene by manganese-dependent peroxidase of the white-rot basidiomycete Phlebia radiata

The in vitro biotransformation of 4-hydroxylamino-2,6-dinitrotoluene (4-OHA-2,6-DNT)—the first identified reduction product of 2,4,6-trinitrotoluene (TNT)—was studied in the presence of a cell-free preparation of manganese-dependent peroxidase (MnP) from the white-rot basidiomycete Phlebia radiata. 4-OHA-2,6-DNT was rapidly oxidized to 4-nitroso-2,6-dinitrotoluene (4-NO-2,6-DNT), part of which reacted with the remaining 4-OHA-2,6-DNT to give 4,4′-azoxy-2,2′,6,6′-tetranitrotoluene. 4-NO-2,6-DNT was also slowly transformed to TNT and 4-amino-2,6-dinitrotoluene (4-A-2,6-DNT). In mineralization tests, 4% of the initial 14C-U-ring labeled 4-OHA-2,6-DNT was recovered as 14CO2. In the presence of up to 10 mM of reduced glutathione (GSH), 4-OHA-2,6-DNT was directly reduced to 4-A-2,6-DNT and the mineralization rate reached 27%. At 25 mM GSH, MnP was inhibited, resulting in an insignificant mineralization rate. The inclusion of GSH in the in vitro system led to a 4-OHA-2,6-DNT deficit in the HPLC mass balances not fully accounted for by the degree of mineralization, but corresponding to unidentified polar compound(s) reflecting up to 65% of the initial substrate. This is the first report of 4-OHA-2,6-DNT mineralization by a fungal MnP and the first clear-cut experimental observation of 4-NO-2,6-DNT, the previously postulated intermediate of microbial TNT metabolism.