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...

The degradation of 2,4-dinitrotoluene (DNT) by Pseudomonas sp. strain DNT is initiated by a dioxygenase attack to yield 4-methyl-5-nitrocatechol (MNC) and nitrite. Subsequent oxidation of MNC by a monooxygenase results in the removal of the second molecule of nitrite, and further enzymatic reactions lead to ring fission. Initial studies on the molecular basis of DNT degradation in this strain revealed the presence of three plasmids. Mitomycin-derived mutants deficient in either DNT dioxygenase only or DNT dioxygenase and MNC monooxygenase were isolated. Plasmid profiles of mutant strains suggested that the mutations resulted from deletions in the largest plasmid. Total plasmid DNA partially digested by EcoRI was cloned into a broad-host-range cosmid vector, pCP13. Recombinant clones containing genes encoding DNT dioxygenase, MNC monooxygenase, and 2,4,5-trihydroxytoluene oxygenase were characterized by identification of reaction products and the ability to complement mutants. Subcloning analysis suggests that the DNT dioxygenase is a multicomponent enzyme system and that the genes for the DNT pathway are organized in at least three different operons.

4-Methyl-5-nitrocatechol (MNC) is an intermediate in the degradation of 2,4-dinitrotoluene byBurkholderia sp. strain DNT. In the presence of NADPH and oxygen, MNC monooxygenase catalyzes the removal of the nitro group from MNC to form 2-hydroxy-5-methylquinone. The gene (dntB) encoding MNC monooxygenase has been previously cloned and characterized. In order to examine the properties of MNC monooxygenase and to compare it with other enzymes, we sequenced the gene encoding the MNC monooxygenase and purified the enzyme from strain DNT. dntB was localized within a 2.2-kb ApaI DNA fragment. Sequence analysis of this fragment revealed an open reading frame of 1,644 bp with an N-terminal amino acid sequence identical to that of purified MNC monooxygenase from strain DNT. Comparison of the derived amino acid sequences with those of other genes showed that DntB contains the highly conserved ADP and flavin adenine dinucleotide (FAD) binding motifs characteristic of flavoprotein hydroxylases. MNC monooxygenase was purified to homogeneity from strain DNT by anion exchange and gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein with a molecular weight of 60,200, which is consistent with the size determined from the gene sequence. The native molecular weight determined by gel filtration was 65,000, which indicates that the native enzyme is a monomer. It used either NADH or NADPH as electron donors, and NADPH was the preferred cofactor. The purified enzyme contained 1 mol of FAD per mol of protein, which is also consistent with the detection of an FAD binding motif in the amino acid sequence of DntB. MNC monooxygenase has a narrow substrate specificity. MNC and 4-nitrocatechol are good substrates whereas 3-methyl-4-nitrophenol, 3-methyl-4-nitrocatechol, 4-nitrophenol, 3-nitrophenol, and 4-chlorocatechol were not. These studies suggest that MNC monooxygenase is a flavoprotein that shares some properties with previously studied nitrophenol oxygenases.Microorganisms can use either monooxygenase (33,34,36,49) or dioxygenase (2,15,21,25,33,37,40) enzymes to catalyze the oxidative removal of nitro groups from nitroaromatic compounds. Although the recent literature contains numerous examples of oxygenase-catalyzed nitro group displacements (33), little is known about the enzymes involved in these reactions. Monooxygenases that oxidize 4-and 2-nitrophenol (34, 36, 49) have been described, but only the 2-nitrophenol monooxygenase from Pseudomonas putida B2 has been purified and characterized (49). Recent evidence has suggested that some of the dioxygenases involved in the removal of nitro groups from aromatic compounds are multicomponent enzyme systems similar to the naphthalene dioxygenase enzyme system (2, 40). Burkholderia sp. strain DNT (formerly Pseudomonas sp. strain DNT) uses both mono-and dioxygenase enzymes to remove nitro groups (13,37,40) in an oxidative pathway that leads to the mineralization of 2,4-dinitrotoluene (2,4-DNT) (37).The initial attack on 2,4-DNT by...

2,4,5-Trihydroxytoluene (THT) oxygenase fromBurkholderia sp. strain DNT catalyzes the conversion of THT to an unstable ring fission product. Biochemical and genetic studies of THT oxygenase were undertaken to elucidate the mechanism of the ring fission reaction. The THT oxygenase gene (dntD) was previously localized to the 1.2-kb DNA insert subcloned in the recombinant plasmid designated pJS76 (W. C. Suen and J. C. Spain, J. Bacteriol. 175:1831–1837, 1993). Analysis of the deduced amino acid sequence of DntD revealed the presence of the highly conserved residues characteristic of the catechol 2,3-dioxygenase gene family I. The deduced amino acid sequence of DntD corresponded to a molecular mass of 35 kDa. The native molecular masses for the THT oxygenase estimated by using gel filtration chromatography and nondenaturing gel electrophoresis were 67.4 and 77.8 kDa, respectively. The results suggested that the native protein consists of two identical subunits. The colorless protein contained 2 mol of iron per mol of protein. Stimulation of activity in the presence of ferrous iron and ascorbate suggested a requirement for ferrous iron in the active site. The properties of the enzyme are similar to those of the catechol 2,3-dioxygenases (meta-cleavage dioxygenases). In addition to THT, the enzyme exhibited activity towards 1,2,4-benzenetriol, catechol, 3- and 4-methylcatechol, and 3- and 4-chlorocatechol. The chemical analysis of the THT ring cleavage product showed that the product was 2,4-dihydroxy-5-methyl-6-oxo-2,4-hexadienoic acid, consistent with extradiol ring fission of THT.