2,4-Dinitrotoluene (DNT) dioxygenase from Burkholderia sp. strain DNT catalyzes the initial oxidation of DNT to form 4-methyl-5-nitrocatechol (MNC) and nitrite. The displacement of the aromatic nitro group by dioxygenases has only recently been described, and nothing is known about the evolutionary origin of the enzyme systems that catalyze these reactions. We have shown previously that the gene encoding DNT dioxygenase is localized on a degradative plasmid within a 6.8-kb NsiI DNA fragment (W.-C. Suen and J. C. Spain, J. Bacteriol. 175:1831-1837, 1993). We describe here the sequence analysis and the substrate range of the enzyme system encoded by this fragment. Five open reading frames were identified, four of which have a high degree of similarity (59 to 78% identity) to the components of naphthalene dioxygenase (NDO) from Pseudomonas strains. The conserved amino acid residues within NDO that are involved in cofactor binding were also identified in the gene encoding DNT dioxygenase. An Escherichia coli clone that expressed DNT dioxygenase converted DNT to MNC and also converted naphthalene to (؉)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. In contrast, the E. coli clone that expressed NDO did not oxidize DNT. Furthermore, the enzyme systems exhibit similar broad substrate specificities and can oxidize such compounds as indole, indan, indene, phenetole, and acenaphthene. These results suggest that DNT dioxygenase and the NDO enzyme system share a common ancestor.Biodegradation of aromatic compounds by aerobic bacteria often begins with the initial oxidation of the substrate by dioxygenases which catalyze the incorporation of both atoms of molecular oxygen into the substrates. These dioxygenases are multicomponent enzyme systems. Two-component dioxygenases such as 4-sulfobenzoate 3,4-dioxygenase (23), 4-chlorophenyl acetate-3,4-dioxygenase (24), o-phthalate dioxygenase (3), benzoate 1,2-dioxygenase (45), and 2-halobenzoate 1,2-dioxygenase (10) consist of an iron-sulfur flavoprotein reductase and an iron-sulfur oxygenase (25). Three-component systems such as benzene dioxygenase (2), toluene 2,3-dioxygenase (46), pyrazon dioxygenase (32), biphenyl dioxygenase (13), ortho-halobenzoate 1,2-dioxygenase (31), dibenzofuran 4,4a-dioxygenase (5), and naphthalene dioxygenase (NDO) (7, 14-16) comprise a flavoprotein reductase, an iron-sulfur ferredoxin, and an iron-sulfur oxygenase (25). Comparison of the nucleotide sequences of the terminal oxygenase components of the benzoate, toluate, toluene, benzene, and naphthalene 1,2-dioxygenases suggests that they are evolutionarily related (27). This conclusion is based on the presence of highly conserved regions for the presumed cofactor binding sites. NDO is unique among the three-component dioxygenase systems in that it contains an additional plant-type iron-sulfur cluster in its reductase component (14,15) and it can catalyze desaturation reactions with a variety of substrates (12,14,43).We have reported previously that the initial reaction of 2,4-dinitrotoluene (DNT) degra...

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...

Cells of Pseudomonas sp. strain NCIB 9816, after growth with naphthalene or salicylate, contain a multicomponent enzyme system that oxidizes naphthalene to cis-(lR,2S)-dihydroxy-1,2-dihydronaphthalene. We purified one of these components to homogeneity and found it to be an iron-sulfur flavoprotein that loses the flavin cofactor during purification. Dialysis against flavin adenine dinucleotide (FAD) showed that the enzyme bound 1 mol of FAD per mol of enzyme protein. The enzyme consisted of a single polypeptide with an apparent molecular weight of 36,300. The purified protein contained 1.8 g-atoms of iron and 2.0 g-atoms of acid-labile sulfur and showed absorption maxima at 278, 340, 420, and 460 nm, with a broad shoulder at 540 nm. The purified enzyme catalyzed the reduction of cytochrome c, dichlorophenolindophenol, Nitro Blue Tetrazolium, and ferricyanide. These activities were enhanced in the presence of added FAD. The ability of the enzyme to catalyze the reduction of the ferredoxin involved in naphthalene reduction and other electron acceptors indicates that it functions as an NAD(P)H-oxidoreductase in the naphthalene dioxygenase system. The results suggest that naphthalene dioxygenase requires two proteins with three redox groups to transfer electrons from NADH to the terminal oxygenase.Naphthalene dioxygenase, a multicomponent enzyme system which oxidizes naphthalene to (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene, is induced in Pseudomonas sp. strain NCIB 9816 during growth on naphthalene or salicylate (2,12,20). After growth on naphthalene, the organism also oxidizes acenaphthalene (31), indole (13), and indan (39). Indan is apparently oxidized by naphthalene dioxygenase in a monooxygenation reaction similar to that described for toluene dioxygenase (39). This suggests that naphthalene dioxygenase can function as either a monooxygenase or a dioxygenase, depending on the substrate.Naphthalene dioxygenase consists of a terminal ironsulfur-containing oxygenase (ISPNAP) and two other proteins which function as a short electron transfer chain (11,12). Thus, naphthalene dioxygenase is similar to the threecomponent enzyme systems involved in the oxidation of benzene (1, 16), toluene (32-34), and pyrazon (29

Pseudomonas sp. strain 9816-4 grows with naphthalene as the sole source of carbon and energy (9). The initial reaction is catalyzed by a multicomponent enzyme system designated naphthalene dioxygenase (NDO) (11,12,23,24). NDO catalyzes the NAD(P)H-dependent enantiospecific incorporation of dioxygen into naphthalene to form (ϩ)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene (cis-naphthalene dihydrodiol) (26, 27) ( Fig. 1). An analogous reaction is catalyzed by toluene dioxygenase (TDO) from Pseudomonas putida F1, where enantiomerically pure (ϩ)-cis-(1S,2R)-dihydroxy-3-methylcyclohexa-3,5-diene (cis-toluene dihydrodiol) is the first detectable oxidation product (17,31,60). TDO also catalyzes the enantiospecific oxidation of naphthalene to (ϩ)-cis-naphthalene dihydrodiol (18,39).In addition to the enantiospecific oxidation of naphthalene and toluene, NDO and TDO from the above strains oxidize many related aromatic compounds to optically active dihydrodiols (10,18,28,30). Other bacterial dioxygenases show similar properties, and more than 130 chiral arene cis-dihydrodiols have been produced from a small number of strains (7,35,48). The high enantiomeric purity of these compounds has led to their use as chiral synthons in the enantiospecific synthesis of a wide variety of biologically active natural products (7,8,46,57). The present studies focus on another facet of this interesting group of dioxygenases, that is, their ability to catalyze reactions other than the formation of arene cis-dihydrodiols. For example, the TDO expressed by P. putida F39/D oxidizes indan to (1R)-indanol and oxidizes indene to cis-(1S,2R)-indandiol and (1S)-indenol (55). Similar reactions have been reported for TDO from P. putida UV4, although the 1-indenol produced by this strain is the (1R)-enantiomer (3, 5).We now report the identification and absolute stereochemistry of the products formed from indan and indene by NDO from Pseudomonas sp. strain 9816-4 and confirm earlier observations on the desaturation of indan to indene by NDO (22). MATERIALS AND METHODSOrganisms. Pseudomonas sp. strain 9816/11 is a mutant which oxidizes naphthalene stoichiometrically to (ϩ)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene (40). This organism is a derivative of Pseudomonas sp. strain 9816-4 (9, 59), which harbors the genes for naphthalene catabolism on an 83-kb NAH plasmid designated pDTG1 (45). Pseudomonas sp. strain 9816/C84, a cured strain, was used as a control in experiments with strain 9816/11. Escherichia coli strain JM109 (DE3)[pDTG141] contains the structural genes (nahAaAbAcAd) for NDO in plasmid pT7-5 (50). Expression of NDO in this strain is inducible by the addition of isopropylthiogalactopyranoside (IPTG). E. coli JM109(DE3)[pT7-5] was used as a control in experiments with strain JM109(DE3) [pDTG141].Biotransformation experiments. Strain 9816/11 was grown at 30ЊC in mineral salts basal medium (MSB) (49) with 0.2% (wt/vol) pyruvate as a carbon source in the presence of 0.05% (wt/vol) salicylate or anthranilate. These aromatic acids induce the s...

A Pseudomonas sp. that was capable of growth on 1,2-dichlorobenzene (o-DCB) or chlorobenzene as a sole source of carbon and energy was isolated by selective enrichment from activated sludge. The initial steps involved in the degradation of o-DCB were investigated by isolation of metabolites, respirometry, and assay of enzymes in cell extracts. Extracts of o-DCB-grown cells converted radiolabeled o-DCB to 3,4-dichloro-cis-1,2-dihydroxycyclohexa-3,5-diene (o-DCB dihydrodiol). 3,4-Dichlorocatechol and o-DCB dihydrodiol accumulated in culture fluids of cells exposed to o-DCB. The results suggest that o-DCB is initially converted by a dioxygenase to a dihydrodiol, which is converted to 3,4-dichlorocatechol by an NAD+-dependent dehydrogenase. Ring cleavage of 3,4-dichlorocatechol is by a catechol 1,2-oxygenase to form 2,3-dichloro-cis,cis-muconate. Preliminary results indicate that chloride is eliminated during subsequent lactonization of the 2,3-dichloro-cis,cis-muconate, followed by hydrolysis to form 5-chloromaleylacetic acid.