2,4-Dinitrotoluene dioxygenase from Burkholderia sp. strain DNT: similarity to naphthalene dioxygenase

Abstract

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...

Biodegradation of Aromatic Compounds by Escherichia coli

“…13) should be taken into consideration when cloning and expressing heterologous aromatic catabolic clusters in this enterobacterium. In this sense, several reports on the cloning and expression of aromatic ring-hydroxylating dioxygenases in E. coli claim that equivalent enzymes from the host could explain the unexpected activities observed when some of the subunits of the cloned dioxygenases were lacking in the recombinant bacteria (82,167,186,305). The described PP-dioxygenase activity of E. coli (see above) could explain some of these unexpected findings.…”

Nitroaromatic Compounds, from Synthesis to Biodegradation

“…In Burkholderia sp. strain DNT (58,59,61,184,186,187) and Burkholderia cepacia R34 (85,86,131), the 4-methyl-5-nitrocatechol produced from 2,4-dinitrotoluene is oxidized by a monooxygenase to remove the second nitro group, forming 2,4,5-trihydroxytoluene, which is a substrate for meta ring cleavage (85). In contrast, metabolism of 2,6-dinitrotoluene by B. cepacia JS850 and Hydrogenophaga palleronii JS863 yields 3-methyl-4-nitrocatechol (Fig.…”

“…strains R34 and DNT that their pathways have evolutionary origins in a naphthalene degradation pathway like that present in Ralstonia sp. strain U2 (87,111,112,186,221). In all of these strains, the genes for the dioxygenase system are organized in very similar operons, and the deduced protein sequences share Ͼ85% identity (Fig.…”

Enhanced bioremediation of soil containing 2,4-dinitrotoluene by a genetically modified Sinorhizobium meliloti

“…Most of this research focused in using microorganisms other than nitrogen fixing bacteria for the bioremediation of contaminated soils with nitroaromatic and polychlorinated aromatic compounds (Dutta et al, 1998;Hou and Dutta, 2000;Suen and Spain, 1993;Suen et al, 1996). Therefore, this study documents for the first time that a recombinant nitrogen fixing soil bacterium containing introduced genes for degrading 2,4-DNT can enhance bioremediation of 2,4-DNT contaminated soil.…”

“…strain DNT, containing genes for 2,4-DNT degradation (pJS1) was supplied by Dr J. C. Spain, Air Force Civil Engineering Support Agency, Tyndall Air Force Base, Florida (Suen and Spain, 1993;Suen et al, 1996) …”