Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1992 Jan;174(1):279–290. doi: 10.1128/jb.174.1.279-290.1992

Purification and some properties of 2-halobenzoate 1,2-dioxygenase, a two-component enzyme system from Pseudomonas cepacia 2CBS.

S Fetzner 1, R Müller 1, F Lingens 1
PMCID: PMC205706  PMID: 1370284

Abstract

The two components of the inducible 2-halobenzoate 1,2-dioxygenase from Pseudomonas cepacia 2CBS were purified to homogeneity. Yellow component B is a monomer (Mr, 37,500) with NADH-acceptor reductase activity. Ferricyanide, 2,6-dichlorophenol indophenol, and cytochrome c acted as electron acceptors. Component B was identified as an iron-sulfur flavoprotein containing 0.8 mol of flavin adenine dinucleotide, 1.7 mol of iron, and 1.7 mol of acid-labile sulfide per mol of enzyme. The isoelectric point was estimated to be pH 4.2. Component B was reduced by the addition of NADH. Red-brown component A (Mr, 200,000 to 220,000) is an iron-sulfur protein containing 5.8 mol of iron and 6.0 mol of acid-labile sulfide. The isoelectric point was within the range of pH 4.5 to 5.3. Component A could be reduced by dithionite or by NADH plus catalytic amounts of component B. Component A consisted of nonidentical subunits alpha (Mr, 52,000) and beta (Mr, 20,000). It contained approximately equimolar amounts of alpha and beta, and cross-linking studies suggested an alpha 3 beta 3 subunit structure of component A. The NADH- and Fe(2+)-dependent enzyme system was named 2-halobenzoate 1,2-dioxygenase, because it catalyzes the conversion of 2-fluoro-, 2-bromo-, 2-chloro-, and 2-iodobenzoate to catechol. 2-Halobenzoate 1,2-dioxygenase exhibited a very broad substrate specificity, but benzoate analogs with electron-withdrawing substituents at the ortho position were transformed preferentially.

Full text

PDF
279

Images in this article

282Image
on p.282
282Image
on p.282
283Image
on p.283
283Image
on p.283

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. ARMSTRONG J. M. THE MOLAR EXTINCTION COEFFICIENT OF 2,6-DICHLOROPHENOL INDOPHENOL. Biochim Biophys Acta. 1964 Apr 4;86:194–197. doi: 10.1016/0304-4165(64)90180-1. [DOI] [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. Babson A. L., Babson S. R. Kinetic colorimetric measurement of serum lactate dehydrogenase activity. Clin Chem. 1973 Jul;19(7):766–769. [PubMed] [Google Scholar]
  4. 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]
  5. Beinert H. Semi-micro methods for analysis of labile sulfide and of labile sulfide plus sulfane sulfur in unusually stable iron-sulfur proteins. Anal Biochem. 1983 Jun;131(2):373–378. doi: 10.1016/0003-2697(83)90186-0. [DOI] [PubMed] [Google Scholar]
  6. Bergmeyer H. U. Neue Werte für die molaren Extinktions-Koeffizienten von NADH und NADPH zum Gebrauch im Routine-Laboratorium. Z Klin Chem Klin Biochem. 1975 Nov;13(11):507–508. [PubMed] [Google Scholar]
  7. Bernhardt F. H., Erdin N., Staudinger H., Ullrich V. Interactions of substrates with a purified 4-methoxybenzoate monooxygenase system (O-demethylating) from Pseudomonas putida. Eur J Biochem. 1973 May;35(1):126–134. doi: 10.1111/j.1432-1033.1973.tb02818.x. [DOI] [PubMed] [Google Scholar]
  8. Bernhardt F. H., Heymann E., Traylor P. S. Chemical and spectral properties of putidamonooxin, the iron-containing and acid-labile-sulfur-containing monooxygenase of a 4-methoxybenzoate O-demethylase from Pseudomonas putida. Eur J Biochem. 1978 Dec 1;92(1):209–223. doi: 10.1111/j.1432-1033.1978.tb12739.x. [DOI] [PubMed] [Google Scholar]
  9. Bernhardt F. H., Pachowsky H., Staudinger H. A 4-methoxybenzoate O-demethylase from Pseudomonas putida. A new type of monooxygenase system. Eur J Biochem. 1975 Sep 1;57(1):241–256. doi: 10.1111/j.1432-1033.1975.tb02296.x. [DOI] [PubMed] [Google Scholar]
  10. Bill E., Bernhardt F. H., Trautwein A. X. Mössbauer studies on the active Fe ... [2Fe-2S] site of putidamonooxin, its electron transport and dioxygen activation mechanism. Eur J Biochem. 1981 Dec;121(1):39–46. doi: 10.1111/j.1432-1033.1981.tb06426.x. [DOI] [PubMed] [Google Scholar]
  11. Bill E., Bernhardt F. H., Trautwein A. X., Winkler H. Mössbauer investigation of the cofactor iron of putidamonooxin. Eur J Biochem. 1985 Feb 15;147(1):177–182. doi: 10.1111/j.1432-1033.1985.tb08734.x. [DOI] [PubMed] [Google Scholar]
  12. Cann J. R. Multibanded isoelectric focusing patterns produced by macromolecular interactions. Methods Enzymol. 1979;61:142–147. doi: 10.1016/0076-6879(79)61012-1. [DOI] [PubMed] [Google Scholar]
  13. Crutcher S. E., Geary P. J. Properties of the iron--sulphur proteins of the benzene dioxygenase system from Pseudomonas putida. Biochem J. 1979 Feb 1;177(2):393–400. doi: 10.1042/bj1770393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Davies G. E., Stark G. R. Use of dimethyl suberimidate, a cross-linking reagent, in studying the subunit structure of oligomeric proteins. Proc Natl Acad Sci U S A. 1970 Jul;66(3):651–656. doi: 10.1073/pnas.66.3.651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Engesser K. H., Schulte P. Degradation of 2-bromo-, 2-chloro- and 2-fluorobenzoate by Pseudomonas putida CLB 250. FEMS Microbiol Lett. 1989 Jul 15;51(1):143–147. doi: 10.1016/0378-1097(89)90497-7. [DOI] [PubMed] [Google Scholar]
  16. Ensley B. D., Gibson D. T. Naphthalene dioxygenase: purification and properties of a terminal oxygenase component. J Bacteriol. 1983 Aug;155(2):505–511. doi: 10.1128/jb.155.2.505-511.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Fetzner S., Müller R., Lingens F. A novel metabolite in the microbial degradation of 2-chlorobenzoate. Biochem Biophys Res Commun. 1989 Jun 15;161(2):700–705. doi: 10.1016/0006-291x(89)92656-9. [DOI] [PubMed] [Google Scholar]
  18. Fetzner S., Müller R., Lingens F. Degradation of 2-chlorobenzoate by Pseudomonas cepacia 2CBS. Biol Chem Hoppe Seyler. 1989 Nov;370(11):1173–1182. doi: 10.1515/bchm3.1989.370.2.1173. [DOI] [PubMed] [Google Scholar]
  19. Geary P. J., Saboowalla F., Patil D., Cammack R. An investigation of the iron-sulphur proteins of benzene dioxygenase from Pseudomonas putida by electron-spin-resonance spectroscopy. Biochem J. 1984 Feb 1;217(3):667–673. doi: 10.1042/bj2170667. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Ghosal D., You I. S., Chatterjee D. K., Chakrabarty A. M. Microbial degradation of halogenated compounds. Science. 1985 Apr 12;228(4696):135–142. doi: 10.1126/science.228.4696.135. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. Harayama S., Rekik M., Timmis K. N. Genetic analysis of a relaxed substrate specificity aromatic ring dioxygenase, toluate 1,2-dioxygenase, encoded by TOL plasmid pWW0 of Pseudomonas putida. Mol Gen Genet. 1986 Feb;202(2):226–234. doi: 10.1007/BF00331641. [DOI] [PubMed] [Google Scholar]
  24. Hare D. L., Stimpson D. I., Cann J. R. Multiple bands produced by interaction of a single macromolecule with carrier ampholytes during isoelectric focusing. Arch Biochem Biophys. 1978 Apr 15;187(1):274–275. doi: 10.1016/0003-9861(78)90034-6. [DOI] [PubMed] [Google Scholar]
  25. Harpel M. R., Lipscomb J. D. Gentisate 1,2-dioxygenase from pseudomonas. Purification, characterization, and comparison of the enzymes from Pseudomonas testosteroni and Pseudomonas acidovorans. J Biol Chem. 1990 Apr 15;265(11):6301–6311. [PubMed] [Google Scholar]
  26. Inouye S., Nakazawa A., Nakazawa T. Nucleotide sequence of the promoter region of the xylDEGF operon on TOL plasmid of Pseudomonas putida. Gene. 1984 Sep;29(3):323–330. doi: 10.1016/0378-1119(84)90061-1. [DOI] [PubMed] [Google Scholar]
  27. Irie S., Doi S., Yorifuji T., Takagi M., Yano K. Nucleotide sequencing and characterization of the genes encoding benzene oxidation enzymes of Pseudomonas putida. J Bacteriol. 1987 Nov;169(11):5174–5179. doi: 10.1128/jb.169.11.5174-5179.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kaplan L. J., Foster J. F. Isoelectric focusing behavior of bovine plasma albumin, mercaptalbumin, and beta-lactoglobulins A and B. Biochemistry. 1971 Feb 16;10(4):630–636. doi: 10.1021/bi00780a014. [DOI] [PubMed] [Google Scholar]
  29. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  30. Lehrbach P. R., Zeyer J., Reineke W., Knackmuss H. J., Timmis K. N. Enzyme recruitment in vitro: use of cloned genes to extend the range of haloaromatics degraded by Pseudomonas sp. strain B13. J Bacteriol. 1984 Jun;158(3):1025–1032. doi: 10.1128/jb.158.3.1025-1032.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Locher H. H., Leisinger T., Cook A. M. 4-Sulphobenzoate 3,4-dioxygenase. Purification and properties of a desulphonative two-component enzyme system from Comamonas testosteroni T-2. Biochem J. 1991 Mar 15;274(Pt 3):833–842. doi: 10.1042/bj2740833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. MASSEY V. The microestimation of succinate and the extinction coefficient of cytochrome c. Biochim Biophys Acta. 1959 Jul;34:255–256. doi: 10.1016/0006-3002(59)90259-8. [DOI] [PubMed] [Google Scholar]
  33. Markus A., Krekel D., Lingens F. Purification and some properties of component A of the 4-chlorophenylacetate 3,4-dioxygenase from Pseudomonas species strain CBS. J Biol Chem. 1986 Sep 25;261(27):12883–12888. [PubMed] [Google Scholar]
  34. Nielsen P., Rauschenbach P., Bacher A. Preparation, properties, and separation by high-performance liquid chromatography of riboflavin phosphates. Methods Enzymol. 1986;122:209–220. doi: 10.1016/0076-6879(86)22172-2. [DOI] [PubMed] [Google Scholar]
  35. Reineke W., Knackmuss H. J. Chemical structure and biodegradability of halogenate aromatic compounds. Substituent effects on 1,2-dioxygenation of benzoic acid. Biochim Biophys Acta. 1978 Sep 6;542(3):412–423. doi: 10.1016/0304-4165(78)90372-0. [DOI] [PubMed] [Google Scholar]
  36. Reineke W., Knackmuss H. J. Hybrid pathway for chlorobenzoate metabolism in Pseudomonas sp. B13 derivatives. J Bacteriol. 1980 May;142(2):467–473. doi: 10.1128/jb.142.2.467-473.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Reiner A. M. Metabolism of benzoic acid by bacteria: 3,5-cyclohexadiene-1,2-diol-1-carboxylic acid is an intermediate in the formation of catechol. J Bacteriol. 1971 Oct;108(1):89–94. doi: 10.1128/jb.108.1.89-94.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. SCHELLENBERG K. A., HELLERMAN L. Oxidation of reduced diphosphopyridine nucleotide. J Biol Chem. 1958 Mar;231(1):547–556. [PubMed] [Google Scholar]
  39. 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]
  40. Schell M. A. Homology between nucleotide sequences of promoter regions of nah and sal operons of NAH7 plasmid of Pseudomonas putida. Proc Natl Acad Sci U S A. 1986 Jan;83(2):369–373. doi: 10.1073/pnas.83.2.369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Schweizer D., Markus A., Seez M., Ruf H. H., Lingens F. Purification and some properties of component B of the 4-chlorophenylacetate 3,4-dioxygenase from Pseudomonas species strain CBS 3. J Biol Chem. 1987 Jul 5;262(19):9340–9346. [PubMed] [Google Scholar]
  42. Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
  43. Subramanian V., Liu T. N., Yeh W. K., Gibson D. T. Toluene dioxygenase: purification of an iron-sulfur protein by affinity chromatography. Biochem Biophys Res Commun. 1979 Dec 14;91(3):1131–1139. doi: 10.1016/0006-291x(79)91998-3. [DOI] [PubMed] [Google Scholar]
  44. Subramanian V., Liu T. N., Yeh W. K., Narro M., Gibson D. T. Purification and properties of NADH-ferredoxinTOL reductase. A component of toluene dioxygenase from Pseudomonas putida. J Biol Chem. 1981 Mar 25;256(6):2723–2730. [PubMed] [Google Scholar]
  45. Subramanian V., Liu T. N., Yeh W. K., Serdar C. M., Wackett L. P., Gibson D. T. Purification and properties of ferredoxinTOL. A component of toluene dioxygenase from Pseudomonas putida F1. J Biol Chem. 1985 Feb 25;260(4):2355–2363. [PubMed] [Google Scholar]
  46. Sylvestre M., Mailhiot K., Ahmad D., Massé R. Isolation and preliminary characterization of a 2-chlorobenzoate degrading Pseudomonas. Can J Microbiol. 1989 Apr;35(4):439–443. doi: 10.1139/m89-067. [DOI] [PubMed] [Google Scholar]
  47. Takeda H., Yamamoto S., Kojima Y., Hayaishi O. Studies on monooxygenases. I. General properties of crystalline L-lysine monooxygenase. J Biol Chem. 1969 Jun 10;244(11):2935–2941. [PubMed] [Google Scholar]
  48. WHITBY L. G. A new method for preparing flavin-adenine dinucleotide. Biochem J. 1953 Jun;54(3):437–442. doi: 10.1042/bj0540437. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Wackett L. P., Brusseau G. A., Householder S. R., Hanson R. S. Survey of microbial oxygenases: trichloroethylene degradation by propane-oxidizing bacteria. Appl Environ Microbiol. 1989 Nov;55(11):2960–2964. doi: 10.1128/aem.55.11.2960-2964.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Wackett L. P., Householder S. R. Toxicity of Trichloroethylene to Pseudomonas putida F1 Is Mediated by Toluene Dioxygenase. Appl Environ Microbiol. 1989 Oct;55(10):2723–2725. doi: 10.1128/aem.55.10.2723-2725.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Wende P., Bernhardt F. H., Pfleger K. Substrate-modulated reactions of putidamonooxin. The nature of the active oxygen species formed and its reaction mechanism. Eur J Biochem. 1989 Apr 15;181(1):189–197. doi: 10.1111/j.1432-1033.1989.tb14710.x. [DOI] [PubMed] [Google Scholar]
  52. White-Stevens R. H., Kamin H. Studies of a flavoprotein, salicylate hydroxylase. I. Preparation, properties, and the uncoupling of oxygen reduction from hydroxylation. J Biol Chem. 1972 Apr 25;247(8):2358–2370. [PubMed] [Google Scholar]
  53. Whited G. M., Gibson D. T. Toluene-4-monooxygenase, a three-component enzyme system that catalyzes the oxidation of toluene to p-cresol in Pseudomonas mendocina KR1. J Bacteriol. 1991 May;173(9):3010–3016. doi: 10.1128/jb.173.9.3010-3016.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Whited G. M., McCombie W. R., Kwart L. D., Gibson D. T. Identification of cis-diols as intermediates in the oxidation of aromatic acids by a strain of Pseudomonas putida that contains a TOL plasmid. J Bacteriol. 1986 Jun;166(3):1028–1039. doi: 10.1128/jb.166.3.1028-1039.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Wubbolts M. G., Timmis K. N. Biotransformation of substituted benzoates to the corresponding cis-diols by an engineered strain of Pseudomonas oleovorans producing the TOL plasmid-specified enzyme toluate-1,2-dioxygenase. Appl Environ Microbiol. 1990 Feb;56(2):569–571. doi: 10.1128/aem.56.2.569-571.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Yamaguchi M., Fujisawa H. Characterization of NADH-cytochrome c reductase, a component of benzoate 1,2-dioxygenase system from Pseudomonas arvilla c-1. J Biol Chem. 1978 Dec 25;253(24):8848–8853. [PubMed] [Google Scholar]
  57. Yamaguchi M., Fujisawa H. Purification and characterization of an oxygenase component in benzoate 1,2-dioxygenase system from Pseudomonas arvilla C-1. J Biol Chem. 1980 Jun 10;255(11):5058–5063. [PubMed] [Google Scholar]
  58. 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]
  59. Zamanian M., Mason J. R. Benzene dioxygenase in Pseudomonas putida. Subunit composition and immuno-cross-reactivity with other aromatic dioxygenases. Biochem J. 1987 Jun 15;244(3):611–616. doi: 10.1042/bj2440611. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. 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]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES