Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1993 Oct;175(20):6467–6475. doi: 10.1128/jb.175.20.6467-6475.1993

Dibenzofuran 4,4a-dioxygenase from Sphingomonas sp. strain RW1: angular dioxygenation by a three-component enzyme system.

P V Bünz 1, A M Cook 1
PMCID: PMC206755  PMID: 8407823

Abstract

Sphingomonas sp. strain RW1 synthesized a constitutive enzyme system that oxygenated dibenzofuran (DBF) to 2,2',3-trihydroxybiphenyl (THB). We purified this dibenzofuran 4,4a-dioxygenase system (DBFDOS) and found it to consist of four components which catalyzed three activities. Two isofunctional, monomeric flavoproteins (components A1 and A2; M(r) of about 44,000) transferred electrons from NADH to the second component (B; M(r) of about 12,000), a ferredoxin, which transported electrons to the heteromultimeric (alpha 2 beta 2) oxygenase component (C; M(r) of alpha, 45,000; M(r) of beta, 23,000). DBFDOS consumed 1 mol each of NADH, O2, and DBF, which was dioxygenated to about 1 mol of THB; no intermediate was observed. The reaction was thus the dioxygenation of DBF at the 4 and 4a positions to give a diene-diol-hemiacetal which rearomatized by spontaneous loss of a phenolate group to form THB. Components A1 and A2 each reduced dichlorophenolindophenol but had negligible activity with cytochrome c; each lost the yellow color, observed to be flavin adenine dinucleotide, upon purification. Component B, which transported electrons to the oxygenase or cytochrome c, had an N-terminal amino acid sequence with high homology to the putidaredoxin of cytochrome P-450cam. The oxygenase had the UV spectrum of a Rieske iron-sulfur center. We presume DBFDOS to be a class IIA dioxygenase system (EC 1.14.12.-), functionally similar to pyrazon dioxygenase.

Full text

PDF
6467

Images in this article

6472Image
on p.6472

Selected References

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

  1. 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]
  2. Correll C. C., Batie C. J., Ballou D. P., Ludwig M. L. Phthalate dioxygenase reductase: a modular structure for electron transfer from pyridine nucleotides to [2Fe-2S]. Science. 1992 Dec 4;258(5088):1604–1610. doi: 10.1126/science.1280857. [DOI] [PubMed] [Google Scholar]
  3. Engesser K. H., Strubel V., Christoglou K., Fischer P., Rast H. G. Dioxygenolytic cleavage of aryl ether bonds: 1,10-dihydro-1,10-dihydroxyfluoren-9-one, a novel arene dihydrodiol as evidence for angular dioxygenation of dibenzofuran. FEMS Microbiol Lett. 1989 Nov;53(1-2):205–209. doi: 10.1016/0378-1097(89)90392-3. [DOI] [PubMed] [Google Scholar]
  4. Enzymatic -oxidation. VI. Isolation of homogeneous reduced diphosphopyridine nucleotide-rubredoxin reductase. J Biol Chem. 1972 Apr 10;247(7):2109–2116. [PubMed] [Google Scholar]
  5. 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]
  6. Fortnagel P., Harms H., Wittich R. M., Krohn S., Meyer H., Sinnwell V., Wilkes H., Francke W. Metabolism of Dibenzofuran by Pseudomonas sp. Strain HH69 and the Mixed Culture HH27. Appl Environ Microbiol. 1990 Apr;56(4):1148–1156. doi: 10.1128/aem.56.4.1148-1156.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Harayama S., Kok M., Neidle E. L. Functional and evolutionary relationships among diverse oxygenases. Annu Rev Microbiol. 1992;46:565–601. doi: 10.1146/annurev.mi.46.100192.003025. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. Locher H. H., Leisinger T., Cook A. M. 4-Toluene sulfonate methyl-monooxygenase from Comamonas testosteroni T-2: purification and some properties of the oxygenase component. J Bacteriol. 1991 Jun;173(12):3741–3748. doi: 10.1128/jb.173.12.3741-3748.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Locher H. H., Leisinger T., Cook A. M. Degradation of p-toluenesulphonic acid via sidechain oxidation, desulphonation and meta ring cleavage in Pseudomonas (Comamonas) testosteroni T-2. J Gen Microbiol. 1989 Jul;135(7):1969–1978. doi: 10.1099/00221287-135-7-1969. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. 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]
  14. Merril C. R., Goldman D., Van Keuren M. L. Silver staining methods for polyacrylamide gel electrophoresis. Methods Enzymol. 1983;96:230–239. doi: 10.1016/s0076-6879(83)96021-4. [DOI] [PubMed] [Google Scholar]
  15. Peterson J. A., Lorence M. C., Amarneh B. Putidaredoxin reductase and putidaredoxin. Cloning, sequence determination, and heterologous expression of the proteins. J Biol Chem. 1990 Apr 15;265(11):6066–6073. [PubMed] [Google Scholar]
  16. STEYN-PARVE E. P., BEINERT H. On the mechanism of dehydrogenation of fatty acyl derivatives of coenzyme A. VI. Isolation and properties of stable enzyme-substrate complexes. J Biol Chem. 1958 Oct;233(4):843–852. [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. Strubel V., Engesser K. H., Fischer P., Knackmuss H. J. 3-(2-hydroxyphenyl)catechol as substrate for proximal meta ring cleavage in dibenzofuran degradation by Brevibacterium sp. strain DPO 1361. J Bacteriol. 1991 Mar;173(6):1932–1937. doi: 10.1128/jb.173.6.1932-1937.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Thurnheer T., Zürrer D., Höglinger O., Leisinger T., Cook A. M. Initial steps in the degradation of benzene sulfonic acid, 4-toluene sulfonic acids, and orthanilic acid in Alcaligenes sp. strain O-1. Biodegradation. 1990;1(1):55–64. doi: 10.1007/BF00117051. [DOI] [PubMed] [Google Scholar]
  21. Wittich R. M., Wilkes H., Sinnwell V., Francke W., Fortnagel P. Metabolism of dibenzo-p-dioxin by Sphingomonas sp. strain RW1. Appl Environ Microbiol. 1992 Mar;58(3):1005–1010. doi: 10.1128/aem.58.3.1005-1010.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES