Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1995 Nov;177(22):6610–6618. doi: 10.1128/jb.177.22.6610-6618.1995

Purification and characterization of the Comamonas testosteroni B-356 biphenyl dioxygenase components.

Y Hurtubise 1, D Barriault 1, J Powlowski 1, M Sylvestre 1
PMCID: PMC177515  PMID: 7592440

Abstract

In this report, we describe some of the characteristics of the Comamonas testosteroni B-356 biphenyl (BPH)-chlorobiphenyl dioxygenase system, which includes the terminal oxygenase, an iron-sulfur protein (ISPBPH) made up of an alpha subunit (51 kDa) and a beta subunit (22 kDa) encoded by bphA and bphE, respectively; a ferredoxin (FERBPH; 12 kDa) encoded by bphF; and a ferredoxin reductase (REDBPH; 43 kDa) encoded by bphG. ISPBPH subunits were purified from B-356 cells grown on BPH. Since highly purified FERBPH and REDBPH were difficult to obtain from strain B-356, these two components were purified from recombinant Escherichia coli strains by using the His tag purification system. These His-tagged fusion proteins were shown to support BPH 2,3-dioxygenase activity in vitro when added to preparations of ISPBPH in the presence of NADH. FERBPH and REDBPH are thought to pass electrons from NADH to ISPBPH, which then activates molecular oxygen for insertion into the aromatic substrate. The reductase was found to contain approximately 1 mol of flavin adenine dinucleotide per mol of protein and was specific for NADH as an electron donor. The ferredoxin was found to contain a Rieske-type [2Fe-2S] center (epsilon 460, 7,455 M-1 cm-1) which was readily lost from the protein during purification and storage. In the presence of REDBPH and FERBPH, ISPBPH was able to convert BPH into both 2,3-dihydro-2,3-dihydroxybiphenyl and 3,4-dihydro-3,4-dihydroxybiphenyl. The significance of this observation is discussed.

Full Text

The Full Text of this article is available as a PDF (352.4 KB).

Selected References

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

  1. Ahmad D., Massé R., Sylvestre M. Cloning and expression of genes involved in 4-chlorobiphenyl transformation by Pseudomonas testosteroni: homology to polychlorobiphenyl-degrading genes in other bacteria. Gene. 1990 Jan 31;86(1):53–61. doi: 10.1016/0378-1119(90)90113-6. [DOI] [PubMed] [Google Scholar]
  2. Asturias J. A., Díaz E., Timmis K. N. The evolutionary relationship of biphenyl dioxygenase from gram-positive Rhodococcus globerulus P6 to multicomponent dioxygenases from gram-negative bacteria. Gene. 1995 Apr 14;156(1):11–18. doi: 10.1016/0378-1119(94)00530-6. [DOI] [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. Bergeron J., Ahmad D., Barriault D., Larose A., Sylvestre M., Powlowski J. Identification and mapping of the gene translation products involved in the first steps of the Comamonas testosteroni B-356 biphenyl/chlorobiphenyl biodegradation pathway. Can J Microbiol. 1994 Sep;40(9):743–753. doi: 10.1139/m94-118. [DOI] [PubMed] [Google Scholar]
  5. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  6. Brown R. E., Jarvis K. L., Hyland K. J. Protein measurement using bicinchoninic acid: elimination of interfering substances. Anal Biochem. 1989 Jul;180(1):136–139. doi: 10.1016/0003-2697(89)90101-2. [DOI] [PubMed] [Google Scholar]
  7. Erickson B. D., Mondello F. J. Enhanced biodegradation of polychlorinated biphenyls after site-directed mutagenesis of a biphenyl dioxygenase gene. Appl Environ Microbiol. 1993 Nov;59(11):3858–3862. doi: 10.1128/aem.59.11.3858-3862.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Fukuda M., Yasukochi Y., Kikuchi Y., Nagata Y., Kimbara K., Horiuchi H., Takagi M., Yano K. Identification of the bphA and bphB genes of Pseudomonas sp. strains KKS102 involved in degradation of biphenyl and polychlorinated biphenyls. Biochem Biophys Res Commun. 1994 Jul 29;202(2):850–856. doi: 10.1006/bbrc.1994.2008. [DOI] [PubMed] [Google Scholar]
  10. Geary P. J., Mason J. R., Joannou C. L. Benzene dioxygenase from Pseudomonas putida, ML2 (NCIB 12190). Methods Enzymol. 1990;188:52–60. doi: 10.1016/0076-6879(90)88012-y. [DOI] [PubMed] [Google Scholar]
  11. Gnanaiah W., Omdahl J. L. Isolation and characterization of pig kidney mitochondrial ferredoxin:NADP+ oxidoreductase. J Biol Chem. 1986 Sep 25;261(27):12649–12654. [PubMed] [Google Scholar]
  12. Gunsalus I. C., Wagner G. C. Bacterial P-450cam methylene monooxygenase components: cytochrome m, putidaredoxin, and putidaredoxin reductase. Methods Enzymol. 1978;52:166–188. doi: 10.1016/s0076-6879(78)52019-3. [DOI] [PubMed] [Google Scholar]
  13. Haddock J. D., Horton J. R., Gibson D. T. Dihydroxylation and dechlorination of chlorinated biphenyls by purified biphenyl 2,3-dioxygenase from Pseudomonas sp. strain LB400. J Bacteriol. 1995 Jan;177(1):20–26. doi: 10.1128/jb.177.1.20-26.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. 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]
  17. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  18. Hirose J., Suyama A., Hayashida S., Furukawa K. Construction of hybrid biphenyl (bph) and toluene (tod) genes for functional analysis of aromatic ring dioxygenases. Gene. 1994 Jan 28;138(1-2):27–33. doi: 10.1016/0378-1119(94)90779-x. [DOI] [PubMed] [Google Scholar]
  19. Khan A., Walia S. Cloning of bacterial genes specifying degradation of 4-chlorobiphenyl from Pseudomonas putida OU83. Appl Environ Microbiol. 1989 Apr;55(4):798–805. doi: 10.1128/aem.55.4.798-805.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kikuchi Y., Nagata Y., Hinata M., Kimbara K., Fukuda M., Yano K., Takagi M. Identification of the bphA4 gene encoding ferredoxin reductase involved in biphenyl and polychlorinated biphenyl degradation in Pseudomonas sp. strain KKS102. J Bacteriol. 1994 Mar;176(6):1689–1694. doi: 10.1128/jb.176.6.1689-1694.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  23. Massé R., Messier F., Péloquin L., Ayotte C., Sylvestre M. Microbial biodegradation of 4-chlorobiphenyl, a model compound of chlorinated biphenyls. Appl Environ Microbiol. 1984 May;47(5):947–951. doi: 10.1128/aem.47.5.947-951.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. NOZAKI M., KAGAMIYAMA H., HAYAISHI O. METAPYROCATECHASE. I. PURIFICATION, CRYSTALLIZATION AND SOME PROPERTIES. Biochem Z. 1963;338:582–590. [PubMed] [Google Scholar]
  25. 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]
  26. Stuehr D. J., Cho H. J., Kwon N. S., Weise M. F., Nathan C. F. Purification and characterization of the cytokine-induced macrophage nitric oxide synthase: an FAD- and FMN-containing flavoprotein. Proc Natl Acad Sci U S A. 1991 Sep 1;88(17):7773–7777. doi: 10.1073/pnas.88.17.7773. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. 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]
  29. 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]
  30. Taira K., Hirose J., Hayashida S., Furukawa K. Analysis of bph operon from the polychlorinated biphenyl-degrading strain of Pseudomonas pseudoalcaligenes KF707. J Biol Chem. 1992 Mar 5;267(7):4844–4853. [PubMed] [Google Scholar]
  31. Tan H. M., Cheong C. M. Substitution of the ISP alpha subunit of biphenyl dioxygenase from Pseudomonas results in a modification of the enzyme activity. Biochem Biophys Res Commun. 1994 Oct 28;204(2):912–917. doi: 10.1006/bbrc.1994.2546. [DOI] [PubMed] [Google Scholar]
  32. Tang C., Henry H. L. Overexpression in Escherichia coli and affinity purification of chick kidney ferredoxin. J Biol Chem. 1993 Mar 5;268(7):5069–5076. [PubMed] [Google Scholar]
  33. 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]
  34. 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