Biodegradation of 4-methyl-5-nitrocatechol by Pseudomonas sp. strain DNT

Abstract

Pseudomonas sp. strain DNT degrades 2,4-dinitrotoluene (DNT) by a dioxygenase attack at the 4,5 position with concomitant removal of the nitro group to yield 4-methyl-5-nitrocatechol (MNC). Here we describe the mechanism of removal of the nitro group from MNC and subsequent reactions leading to ring fission. Washed suspensions of DNT-grown cells oxidized MNC and 2,4,5-trihydroxytoluene (THT). Extracts prepared from DNT-induced cells catalyzed the disappearance of MNC in the presence of oxygen and NADPH. Partially purified MNC oxygenase oxidized MNC in a reaction requiring 1 mol of NADPH and 1 mol of oxygen per mol of substrate. The enzyme converted MNC to 2-hydroxy-5-methylquinone (HMQ), which was identified by gas chromatography-mass spectrometry. HMQ was also detected transiently in culture fluids of cells grown on DNT. A quinone reductase was partially purified and shown to convert HMQ to THT in a reaction requiring NADH. A partially purified THT oxygenase catalyzed ring fission of THT and accumulation of a compound tentatively identified as 3-hydroxy-5-(1-formylethylidene)-2-furanone. Preliminary results indicate that this compound is an artifact of the isolation procedure and suggest that 2,4-dihydroxy-5-methyl-6-oxo-2,4-hexadienoic acid is the actual ring fission product.Microorganisms can directly remove nitro groups from nitroaromatic compounds by either oxidative routes (6,12,16,17,22) or reductive routes (7,11). Both monooxygenase (14,22), and dioxygenase (6, 12, 17) enzyme systems can catalyze the elimination of nitrite from nitroaromatic compounds. A Rhodococcus strain (11) reductively eliminates nitrite from picric acid by adding a hydride ion to the aromatic ring to form a Meisenheimer complex. The Meisenheimer complex regains aromaticity upon nitrite elimination to form 2,4-dinitrophenol.In a previous report, we described the initial steps of 2,4-dinitrotoluene (DNT) degradation by a Pseudomonas strain able to use DNT as a sole carbon and energy source (17). Identification of 4-methyl-5-nitrocatechol (MNC) as an early metabolite and 1802 incorporation experiments indicated an initial dioxygenation at the 4,5 position of DNT to yield MNC with concomitant release of nitrite. The subsequent metabolism of MNC was not determined. In the present investigation, we describe the biochemical evidence for the oxidation of MNC and subsequent reactions leading to ring fission. A preliminary report of this work (8) has been presented previously. On the basis of that report and a genetic analysis of the reactions involved (20), a pathway for the degradation of DNT has been previously proposed (20) (904) 283-6090. Cassette System (Millipore Corp., Bedford, Mass.) and pelleted by centrifugation. Cell pellets were stored at -20°C until used. Growth on 2,4,5-trihydroxytoluene (THT) was tested by auxanography (13).Cell extracts were prepared in 0.02 M KH2PO4 buffer as previously described (15). Cell extracts for the partial purification of enzymes were prepared from frozen cells suspended in an equal volume (wt...

Federal Integrated Biotreatment Research Consortium (FIBRC): Flask to Field Initiative

“…The cluster in the upper left (7,8,9,10,15,16,17) consisted entirely of phenanthrene degraders (predominantly Pseudomonas species). The cluster in the lower right (1,4,5,6,13,24,25,26,29) was fluoranthene degraders, predominantly Sphingomonas species. These results suggest that certain catabolic characteristics are associated with specific genera.…”

“…Only traces of 3-nitrotoluene, 2,3-dinitrotoluene and 3,4-dinitrotoluene were generated during the manufacture of TNT, and such compounds are rare at contaminated sites. Bacteria able to utilize 2-NT Haigler et al, 1994b), 4-NT (Haigler andHe and Spain, 2000b;, 2,4-DNT Haigler et al, 1994a;Spanggord et al, 1991), and 2,6-DNT as sole carbon and nitrogen sources have been isolated and the degradative pathways determined. Multiple pathways for 4-NT degradation, all involving an initial reduction, have been identified (Haigler and Spain, 1993;Rhys-Williams et al, 1993; in three different bacteria (Fig.…”

“…When the partial reductive pathway in JS45 was described, only 4-nitrobenzoate ) and 4-NT (Haigler and Spain, 1993;RhysWilliams et al, 1993) were known to be degraded via partially reductive pathways, but whereas those pathways involved eventual conversion to a catechol/protocatechuate, the NB pathway utilized a novel mutase to convert hydroxylaminobenzene to orrÄo-aminophenol. Since then partial reduction has been found in pathways for several other nitroaromatic compounds .…”

Bioremediation of 3,5-dinitrobenzoic acid and aniline by a Corynebacterium sp.

“…However, some bacteria such as Pseudomonas sp. strain JS42 (Haigler et al, 1994), Pseudomonas putida OU83 (Walia et al, 2003) and Micrococcus sp. (Mulla et al, 2011) could use 2-nitrotoluene as a sole source of carbon.…”

Reactions Involved in the Lower Pathway for Degradation of 4-Nitrotoluene by Mycobacterium Strain HL 4-NT-1

“…One of the strategies involves partial reduction of the nitro group to a hydroxylamino group. The hydroxylamino intermediates can be transformed to catechols which enter the ring cleavage pathways of aerobic degradation of aromatic compounds, as observed during degradation of 4-nitrobenzoate (6,33), 3-nitrophenol (19), and 4-nitrotoluene (7,26). Alternatively, the hydroxylamino intermediates can be rearranged, by an intramolecular transfer of the hydroxyl group (10; L. J. Nadeau, Z.…”