Mineralization of C-14-U-ring labeled 4-hydroxylamino-2,6-dinitrotoluene 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 rrmanganese- 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 transfor med to TNT and 4-amino-2,6-dinitrotoluene (4-A-2,6-DNT). In mineralization tests, 4% of the initial C-14-U-ring labeled 4-OHA-2,6-DNT was recovered as (CO2)-C-14. 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 rat e reached 27%. At 25 mM GSH, MnP was inhibited, resulting in an insignifica nt 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 b y the degree of mineralization, but corresponding to unidentified polar com pound(s) reflecting up to 65% of the initial substrate. This is the first r eport of 4-OHA-2,6-DNT mineralization by a fungal MnP and the first clear-c ut experimental observation of 4-NO-2,6-DNT, the previously postulated inte rmediate of microbial TNT metabolism. (C) 1999 Elsevier Science B.V. All ri ghts reserved.