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Anaerobic transformation of the explosive RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) by microorganisms involves sequential reduction of N-NO2 to the corresponding N-NO groups resulting in the initial formation of MNX (hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine). MNX is further reduced to the dinitroso (DNX) and trinitroso (TNX) derivatives. In this paper, we describe the degradation of MNX and TNX by the unusual cytochrome P450 XplA that mediates metabolism of RDX in Rhodococcus rhodochrous strain 11Y. XplA is known to degrade RDX under aerobic and anaerobic conditions, and, in the present study, was found able to degrade MNX to give similar products distribution including NO2-, NO3-, N2O, and HCHO but with varying stoichiometric ratio, that is, 2.06, 0.33, 0.33, 1.18, and 1.52, 0.15, 1.04, 2.06, respectively. In addition, the ring cleavage product 4-nitro-2,4,-diazabutanal (NDAB) and a trace amount of another intermediate with a [M-H]- at 102 Da, identified as ONNHCH2NHCHO (NO-NDAB), were detected mostly under aerobic conditions. Interestingly, degradation of TNX was observed only under anaerobic conditions in the presence of RDX and/or MNX. When we incubated RDX and its nitroso derivatives with XplA, we found that successive replacement of N-NO2 by N-NO slowed the removal rate of the chemicals with degradation rates in the order RDX > MNX > DNX, suggesting that denitration was mainly responsible for initiating cyclic nitroamines degradation by XplA. This study revealed that XplA preferentially cleaved the N-NO2 over the N-NO linkages, but could nevertheless degrade all three nitroso derivatives, demonstrating the potential for complete RDX removal in explosives-contaminated sites. [ABSTRACT FROM AUTHOR]

Previously we demonstrated that Rhodococcus sp. strain DN22 can degrade RDX (hexahydro-1,3,5-trinitro-l,3,5-triazine) aerobically via initial denization. The present study describes the role of oxygen and water in the key denitration step leading to RDX decomposition using 18O2 and H2 18O labeling experiments. We also investigated degradation of MNX (hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine) with DN22 under similar conditions. DN22 degraded RDX and MNX giving N02-, NO3-. NDAB (4-nitro-diazabutanal), NH3, N2O, and HCHO with NO2-/NO3- molar ratio reaching 17 and ca. 2, respectively. In the presence of 18O2, DN22 degraded RDX and produced NO2- with m/z at 46 Da that subsequently oxidized to NO3- containing one 18O atom, but in the presence of H2 18O we detected NO3- without 18O. A control containing NO2-, DN22, and 18O2 gave NO3- with one 18O, confirming biotic oxidation of NO2- to NO3-. Treatment of MNX with DN22 and 18O2 produced NO3- with two mass ions, one (66 Da) incorporating two 18O atoms and another (64 Da) incorporating only one 18O atom and we attributed their formation to bio-oxidation of the initially formed NO and NO2-, respectively. In the presence of H218O we detected NO2- with two different masses, one representing NO2- (46 Da) and another representing NO2-. (48 Da) with the inclusion of one 18O atom suggesting auto-oxidation of NO to NO2-. Results indicated that denitration of either RDX or MNX and denitrosation of MNX by DN22 did not involve direct participation of either oxygen or water, but both played major roles in subsequent secondary chemical and biochemical reactions of NO and NO2-. [ABSTRACT FROM AUTHOR]