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. 1996 Aug;178(16):4926–4934. doi: 10.1128/jb.178.16.4926-4934.1996

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

W C Suen 1, B E Haigler 1, J C Spain 1
PMCID: PMC178276  PMID: 8759857

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.

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Selected References

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  1. An D., Gibson D. T., Spain J. C. Oxidative release of nitrite from 2-nitrotoluene by a three-component enzyme system from Pseudomonas sp. strain JS42. J Bacteriol. 1994 Dec;176(24):7462–7467. doi: 10.1128/jb.176.24.7462-7467.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Axcell B. C., Geary P. J. Purification and some properties of a soluble benzene-oxidizing system from a strain of Pseudomonas. Biochem J. 1975 Jan;146(1):173–183. doi: 10.1042/bj1460173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Batie C. J., LaHaie E., Ballou D. P. Purification and characterization of phthalate oxygenase and phthalate oxygenase reductase from Pseudomonas cepacia. J Biol Chem. 1987 Feb 5;262(4):1510–1518. [PubMed] [Google Scholar]
  4. Brand J. M., Cruden D. L., Zylstra G. J., Gibson D. T. Stereospecific hydroxylation of indan by Escherichia coli containing the cloned toluene dioxygenase genes from Pseudomonas putida F1. Appl Environ Microbiol. 1992 Oct;58(10):3407–3409. doi: 10.1128/aem.58.10.3407-3409.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bünz P. V., Cook A. M. Dibenzofuran 4,4a-dioxygenase from Sphingomonas sp. strain RW1: angular dioxygenation by a three-component enzyme system. J Bacteriol. 1993 Oct;175(20):6467–6475. doi: 10.1128/jb.175.20.6467-6475.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Denome S. A., Stanley D. C., Olson E. S., Young K. D. Metabolism of dibenzothiophene and naphthalene in Pseudomonas strains: complete DNA sequence of an upper naphthalene catabolic pathway. J Bacteriol. 1993 Nov;175(21):6890–6901. doi: 10.1128/jb.175.21.6890-6901.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Ensley B. D., Gibson D. T., Laborde A. L. Oxidation of naphthalene by a multicomponent enzyme system from Pseudomonas sp. strain NCIB 9816. J Bacteriol. 1982 Mar;149(3):948–954. doi: 10.1128/jb.149.3.948-954.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Ensley B. D., Ratzkin B. J., Osslund T. D., Simon M. J., Wackett L. P., Gibson D. T. Expression of naphthalene oxidation genes in Escherichia coli results in the biosynthesis of indigo. Science. 1983 Oct 14;222(4620):167–169. doi: 10.1126/science.6353574. [DOI] [PubMed] [Google Scholar]
  9. Erickson B. D., Mondello F. J. Nucleotide sequencing and transcriptional mapping of the genes encoding biphenyl dioxygenase, a multicomponent polychlorinated-biphenyl-degrading enzyme in Pseudomonas strain LB400. J Bacteriol. 1992 May;174(9):2903–2912. doi: 10.1128/jb.174.9.2903-2912.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fetzner S., Müller R., Lingens F. Purification and some properties of 2-halobenzoate 1,2-dioxygenase, a two-component enzyme system from Pseudomonas cepacia 2CBS. J Bacteriol. 1992 Jan;174(1):279–290. doi: 10.1128/jb.174.1.279-290.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gibson D. T., Resnick S. M., Lee K., Brand J. M., Torok D. S., Wackett L. P., Schocken M. J., Haigler B. E. Desaturation, dioxygenation, and monooxygenation reactions catalyzed by naphthalene dioxygenase from Pseudomonas sp. strain 9816-4. J Bacteriol. 1995 May;177(10):2615–2621. doi: 10.1128/jb.177.10.2615-2621.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Haddock J. D., Nadim L. M., Gibson D. T. Oxidation of biphenyl by a multicomponent enzyme system from Pseudomonas sp. strain LB400. J Bacteriol. 1993 Jan;175(2):395–400. doi: 10.1128/jb.175.2.395-400.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Haigler B. E., Gibson D. T. Purification and properties of NADH-ferredoxinNAP reductase, a component of naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816. J Bacteriol. 1990 Jan;172(1):457–464. doi: 10.1128/jb.172.1.457-464.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Haigler B. E., Gibson D. T. Purification and properties of ferredoxinNAP, a component of naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816. J Bacteriol. 1990 Jan;172(1):465–468. doi: 10.1128/jb.172.1.465-468.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Haigler B. E., Nishino S. F., Spain J. C. Biodegradation of 4-methyl-5-nitrocatechol by Pseudomonas sp. strain DNT. J Bacteriol. 1994 Jun;176(11):3433–3437. doi: 10.1128/jb.176.11.3433-3437.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Haigler B. E., Wallace W. H., Spain J. C. Biodegradation of 2-nitrotoluene by Pseudomonas sp. strain JS42. Appl Environ Microbiol. 1994 Sep;60(9):3466–3469. doi: 10.1128/aem.60.9.3466-3469.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jeffrey A. M., Yeh H. J., Jerina D. M., Patel T. R., Davey J. F., Gibson D. T. Initial reactions in the oxidation of naphthalene by Pseudomonas putida. Biochemistry. 1975 Feb 11;14(3):575–584. doi: 10.1021/bi00674a018. [DOI] [PubMed] [Google Scholar]
  18. Jerina D. M., Daly J. W., Jeffrey A. M., Gibson D. T. Cis-1,2-dihydroxy-1,2-dihydronaphthalene: a bacterial metabolite from naphthalene. Arch Biochem Biophys. 1971 Jan;142(1):394–396. doi: 10.1016/0003-9861(71)90298-0. [DOI] [PubMed] [Google Scholar]
  19. Kurkela S., Lehväslaiho H., Palva E. T., Teeri T. H. Cloning, nucleotide sequence and characterization of genes encoding naphthalene dioxygenase of Pseudomonas putida strain NCIB9816. Gene. 1988 Dec 20;73(2):355–362. doi: 10.1016/0378-1119(88)90500-8. [DOI] [PubMed] [Google Scholar]
  20. Lipman D. J., Pearson W. R. Rapid and sensitive protein similarity searches. Science. 1985 Mar 22;227(4693):1435–1441. doi: 10.1126/science.2983426. [DOI] [PubMed] [Google Scholar]
  21. Markus A., Klages U., Krauss S., Lingens F. Oxidation and dehalogenation of 4-chlorophenylacetate by a two-component enzyme system from Pseudomonas sp. strain CBS3. J Bacteriol. 1984 Nov;160(2):618–621. doi: 10.1128/jb.160.2.618-621.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Mason J. R., Cammack R. The electron-transport proteins of hydroxylating bacterial dioxygenases. Annu Rev Microbiol. 1992;46:277–305. doi: 10.1146/annurev.mi.46.100192.001425. [DOI] [PubMed] [Google Scholar]
  23. Mermod N., Lehrbach P. R., Reineke W., Timmis K. N. Transcription of the TOL plasmid toluate catabolic pathway operon of Pseudomonas putida is determined by a pair of co-ordinately and positively regulated overlapping promoters. EMBO J. 1984 Nov;3(11):2461–2466. doi: 10.1002/j.1460-2075.1984.tb02156.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Neidle E. L., Hartnett C., Ornston L. N., Bairoch A., Rekik M., Harayama S. Nucleotide sequences of the Acinetobacter calcoaceticus benABC genes for benzoate 1,2-dioxygenase reveal evolutionary relationships among multicomponent oxygenases. J Bacteriol. 1991 Sep;173(17):5385–5395. doi: 10.1128/jb.173.17.5385-5395.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Resnick S. M., Torok D. S., Gibson D. T. Oxidation of carbazole to 3-hydroxycarbazole by naphthalene 1,2-dioxygenase and biphenyl 2,3-dioxygenase. FEMS Microbiol Lett. 1993 Nov 1;113(3):297–302. doi: 10.1111/j.1574-6968.1993.tb06530.x. [DOI] [PubMed] [Google Scholar]
  26. Robertson J. B., Spain J. C., Haddock J. D., Gibson D. T. Oxidation of nitrotoluenes by toluene dioxygenase: evidence for a monooxygenase reaction. Appl Environ Microbiol. 1992 Aug;58(8):2643–2648. doi: 10.1128/aem.58.8.2643-2648.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Romanov V., Hausinger R. P. Pseudomonas aeruginosa 142 uses a three-component ortho-halobenzoate 1,2-dioxygenase for metabolism of 2,4-dichloro- and 2-chlorobenzoate. J Bacteriol. 1994 Jun;176(11):3368–3374. doi: 10.1128/jb.176.11.3368-3374.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sauber K., Fröhner C., Rosenberg G., Eberspächer J., Lingens F. Purification and properties of pyrazon dioxygenase from pyrazon-degrading bacteria. Eur J Biochem. 1977 Mar 15;74(1):89–97. doi: 10.1111/j.1432-1033.1977.tb11370.x. [DOI] [PubMed] [Google Scholar]
  29. Simon M. J., Osslund T. D., Saunders R., Ensley B. D., Suggs S., Harcourt A., Suen W. C., Cruden D. L., Gibson D. T., Zylstra G. J. Sequences of genes encoding naphthalene dioxygenase in Pseudomonas putida strains G7 and NCIB 9816-4. Gene. 1993 May 15;127(1):31–37. doi: 10.1016/0378-1119(93)90613-8. [DOI] [PubMed] [Google Scholar]
  30. Smith P. K., Krohn R. I., Hermanson G. T., Mallia A. K., Gartner F. H., Provenzano M. D., Fujimoto E. K., Goeke N. M., Olson B. J., Klenk D. C. Measurement of protein using bicinchoninic acid. Anal Biochem. 1985 Oct;150(1):76–85. doi: 10.1016/0003-2697(85)90442-7. [DOI] [PubMed] [Google Scholar]
  31. Spanggord R. J., Spain J. C., Nishino S. F., Mortelmans K. E. Biodegradation of 2,4-dinitrotoluene by a Pseudomonas sp. Appl Environ Microbiol. 1991 Nov;57(11):3200–3205. doi: 10.1128/aem.57.11.3200-3205.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Stanier R. Y., Palleroni N. J., Doudoroff M. The aerobic pseudomonads: a taxonomic study. J Gen Microbiol. 1966 May;43(2):159–271. doi: 10.1099/00221287-43-2-159. [DOI] [PubMed] [Google Scholar]
  33. Suen W. C., Spain J. C. Cloning and characterization of Pseudomonas sp. strain DNT genes for 2,4-dinitrotoluene degradation. J Bacteriol. 1993 Mar;175(6):1831–1837. doi: 10.1128/jb.175.6.1831-1837.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Tabor S., Richardson C. C. DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc Natl Acad Sci U S A. 1987 Jul;84(14):4767–4771. doi: 10.1073/pnas.84.14.4767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Takizawa N., Kaida N., Torigoe S., Moritani T., Sawada T., Satoh S., Kiyohara H. Identification and characterization of genes encoding polycyclic aromatic hydrocarbon dioxygenase and polycyclic aromatic hydrocarbon dihydrodiol dehydrogenase in Pseudomonas putida OUS82. J Bacteriol. 1994 Apr;176(8):2444–2449. doi: 10.1128/jb.176.8.2444-2449.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Wackett L. P., Kwart L. D., Gibson D. T. Benzylic monooxygenation catalyzed by toluene dioxygenase from Pseudomonas putida. Biochemistry. 1988 Feb 23;27(4):1360–1367. doi: 10.1021/bi00404a041. [DOI] [PubMed] [Google Scholar]
  37. Yamaguchi M., Yamauchi T., Fujisawa H. Studies on mechanism of double hydroxylation. I. Evidence for participation of NADH-cytochrome c reductase in the reaction of benzoate 1,2-dioxygenase (benzoate hydroxylase). Biochem Biophys Res Commun. 1975 Nov 3;67(1):264–271. doi: 10.1016/0006-291x(75)90311-3. [DOI] [PubMed] [Google Scholar]
  38. Yeh W. K., Gibson D. T., Liu T. N. Toluene dioxygenase: a multicomponent enzyme system. Biochem Biophys Res Commun. 1977 Sep 9;78(1):401–410. doi: 10.1016/0006-291x(77)91268-2. [DOI] [PubMed] [Google Scholar]
  39. Zylstra G. J., Gibson D. T. Toluene degradation by Pseudomonas putida F1. Nucleotide sequence of the todC1C2BADE genes and their expression in Escherichia coli. J Biol Chem. 1989 Sep 5;264(25):14940–14946. [PubMed] [Google Scholar]

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