Nitrite Elimination and Hydrolytic Ring Cleavage in 2,4,6-Trinitrophenol (Picric Acid) Degradation

Abstract

Two hydrogenation reactions in the initial steps of degradation of 2,4,6-trinitrophenol produce the dihydride Meisenheimer complex of 2,4,6-trinitrophenol. The npdH gene (contained in the npd gene cluster of the 2,4,6-trinitrophenol-degrading strain Rhodococcus opacus HL PM-1) was shown here to encode a tautomerase, catalyzing a proton shift between the aci-nitro and the nitro forms of the dihydride Meisenheimer complex of 2,4,6-trinitrophenol. An enzyme (which eliminated nitrite from the aci-nitro form but not the nitro form of the dihydride complex of 2,4,6-trinitrophenol) was purified from the 2,4,6-trinitrophenol-degrading strain Nocardioides simplex FJ2-1A. The product of nitrite release was the hydride Meisenheimer complex of 2,4-dinitrophenol, which was hydrogenated to the dihydride Meisenheimer complex of 2,4-dinitrophenol by the hydride transferase I and the NADPH-dependent F 420 reductase from strain HL PM-1. At pH 7.5, the dihydride complex of 2,4-dinitrophenol is protonated to 2,4-dinitrocyclohexanone. A hydrolase was purified from strain FJ2-1A and shown to cleave 2,4-dinitrocyclohexanone hydrolytically to 4,6-dinitrohexanoate.2,4,6-Trinitrophenol (TNP) and 2,4-dinitrophenol (DNP) are nitroaromatic compounds of versatile use in chemical synthesis. They occur as off-stream chemicals during the production of aniline, which is one of the most important starting materials in chemical synthesis. Furthermore, TNP and its salts have been used as explosives. Large quantities of TNP in waste streams of aniline production necessitate remediation. Several bacteria of the Actinomycetales family (notably of the genera Rhodococcus and Nocardioides) grow aerobically on TNP and/or DNP and utilize the compounds as sole nitrogen, carbon, and energy sources (3,4,10,19). This capacity can be harnessed for bioremediation once we understand the underlying mechanisms of these processes.It was previously established that two hydrogenations take place in the initial attack on TNP (6,7,8,11) (Fig. 1A, panel 1). In Rhodococcus opacus HL PM-1, TNP is hydrogenated at the aromatic nucleus by the hydride transferase II (HTII) encoded by npdI (Fig. 1B) and the NADPH-dependent F 420 reductase (NDFR) encoded by npdG. The hydride Meisenheimer complex of TNP (H Ϫ -TNP) (Fig. 1A, panel 2) thereby formed is further hydrogenated by the hydride transferase I (HTI) encoded by npdC and the NDFR, producing the dihydride Meisenheimer complex of TNP (2H Ϫ -TNP) (panel 3a). In Nocardioides simplex FJ2-1A, the same reactions take place except that a single hydride transferase performs both hydrogenations (7).More than a decade ago, 4,6-dinitrohexanoate was identified as a dead-end metabolite of TNP degradation resulting from two hydride transfers to TNP. This observation coincided with the hypothesis that 2HϪ -TNP was a dead-end metabolite of TNP degradation (11). Much later it was suggested that nitrite is eliminated from 2H Ϫ -TNP to produce the hydride Meisen-Ϫ -TNP is a metabolite of productive TNP degradation. Detection of H Ϫ -DNP suggest...

Physiology, Biochemistry, and Applications of F420- and Fo-Dependent Redox Reactions

“…The results of comparative genomics suggest that homologs of these proteins are exclusively encoded by the genera Nocardioides, Rhodococcus, and Nocardia among presently sequenced organisms. Empirical studies consistently indicate that equivalent enzymatic pathways can degrade nitroaromatic compounds in five additional Rhodococcus species (398,(436)(437)(438) and three Nocardioides species (54,396,397,432,439). Beyond picrate and 2,4-dinitrophenol, LLHTs are involved in the biodegradation of other nitroaromatic compounds.…”

“…The best-characterized F 420 H 2 -dependent reductases of this superfamily are the hydride transferases involved in the biodegradation of the explosive picrate and related compounds (54,155). In Rhodococcus opacus, two LLHTs known as hydride transferase I (HTI) and hydride transferase II (HTII) catalyze the reduction of picrate into hydride-Meisenheimer and dihydride-Meisenheimer complexes (430)(431)(432). Subsequent tautomerization, nitrite elimination, reduction, and hydrolysis steps lead to the production of 4,6-dinitrohexanoate, which can then be oxidatively degraded (432).…”

“…In Rhodococcus opacus, two LLHTs known as hydride transferase I (HTI) and hydride transferase II (HTII) catalyze the reduction of picrate into hydride-Meisenheimer and dihydride-Meisenheimer complexes (430)(431)(432). Subsequent tautomerization, nitrite elimination, reduction, and hydrolysis steps lead to the production of 4,6-dinitrohexanoate, which can then be oxidatively degraded (432). The complete pathway involved is shown in Fig.…”

Nitroaromatic Compounds, from Synthesis to Biodegradation

“…A second reduction and hydride-Meisenheimer complex eventually results in hydrolytic cleavage of 2,4-dinitrophenol to 4,6-dinitrohexanoate, which may be metabolized through ␤-oxidation, using enzymes specialized in removing the remaining nitro groups. Some of the genes and enzymes of this pathway have been identified and characterized with respect to their function and regulation (75,78,127,201).…”

Diversity of ‘benzenetriol dioxygenase’ involved in p-nitrophenol degradation in soil bacteria

“…Several bacterial strains belonging to diverse taxonomic groups have been isolated and characterized for degradation of the above compounds [5,6]. The molecular regulation of some of these degradation pathways have also been elucidated in detail [7][8][9]. Aromatic ring hydroxylating dioxygenases (RHDOs) are one of the most important classes of enzymes that catalyze aromatic ring cleavage for complete degradation of these compounds.…”