Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1991 Jun;173(12):3741–3748. doi: 10.1128/jb.173.12.3741-3748.1991

4-Toluene sulfonate methyl-monooxygenase from Comamonas testosteroni T-2: purification and some properties of the oxygenase component.

H H Locher 1, T Leisinger 1, A M Cook 1
PMCID: PMC208003  PMID: 2050632

Abstract

Comamonas testosteroni T-2 synthesizes an inducible enzyme system that oxygenates 4-toluene sulfontate (TS) to 4-sulfobenzyl alcohol when grown in TS-salts medium. We purified this TS methyl-monooxygenase system (TSMOS) and found it to consist of two components. A monomeric, iron-sulfur flavoprotein (component B), which has been shown to act as a reductase in the 4-sulfobenzoate dioxygenase system of this organism (H. H. Locher, T. Leisinger, and A. M. Cook, Biochem. J. 274:833-842, 1991), carried electrons from NADH to component M, an oxygenase. This oxygenase had the UV-visible spectral characteristics of an iron-sulfur protein. Mrs of about 152,000 for the native oxygenase and of 43,000 under denaturing conditions indicated a homotri- or homotetrameric enzyme, whose N-terminal amino acids and amino acid composition were determined. The activity of the purified enzyme was enhanced about fivefold by the addition of Fe2+. In the presence of O2 and NADH, components B and M together catalyzed the stoichiometric transformation of TS or p-toluate to the corresponding alcohol. The reaction was confirmed as oxygenation of the methyl group by observation of an oxygen atom from 18O2 in carboxybenzyl alcohol. The substrate range of TSMOS included carboxylated analogs of TS (p- and m-toluates and 4-ethylbenzoate), whereas p-xylene, toluene, and p-cresol were not substrates. TSMOS also catalyzed demethylation; 4-methoxybenzoate was transformed to 4-hydroxybenzoate and formaldehyde.

Full text

PDF
3747

Images in this article

3744Image
on p.3744

Selected References

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

  1. 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]
  2. 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]
  3. 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]
  4. 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]
  5. 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]
  6. 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: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Dagley S. Catabolism of aromatic compounds by micro-organisms. Adv Microb Physiol. 1971;6(0):1–46. doi: 10.1016/s0065-2911(08)60066-1. [DOI] [PubMed] [Google Scholar]
  9. DeFrank J. J., Ribbons D. W. p-cymene pathway in Pseudomonas putida: initial reactions. J Bacteriol. 1977 Mar;129(3):1356–1364. doi: 10.1128/jb.129.3.1356-1364.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Fox B. G., Froland W. A., Dege J. E., Lipscomb J. D. Methane monooxygenase from Methylosinus trichosporium OB3b. Purification and properties of a three-component system with high specific activity from a type II methanotroph. J Biol Chem. 1989 Jun 15;264(17):10023–10033. [PubMed] [Google Scholar]
  12. Harayama S., Leppik R. A., Rekik M., Mermod N., Lehrbach P. R., Reineke W., Timmis K. N. Gene order of the TOL catabolic plasmid upper pathway operon and oxidation of both toluene and benzyl alcohol by the xylA product. J Bacteriol. 1986 Aug;167(2):455–461. doi: 10.1128/jb.167.2.455-461.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. 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]
  16. 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]
  17. Peterson J. A., Basu D., Coon M. J. Enzymatic omega-oxidation. I. Electon carriers in fatty acid and hydrocarbon hydroxylation. J Biol Chem. 1966 Nov 10;241(21):5162–5164. [PubMed] [Google Scholar]
  18. Reeve C. D., Carver M. A., Hopper D. J. Stereochemical aspects of the oxidation of 4-ethylphenol by the bacterial enzyme 4-ethylphenol methylenehydroxylase. Biochem J. 1990 Aug 1;269(3):815–819. doi: 10.1042/bj2690815. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ribbons D. W. Requirement of two protein fractions for O-demethylase activity in Pseudomonas testosteroni. FEBS Lett. 1971 Jan 12;12(3):161–165. doi: 10.1016/0014-5793(71)80058-3. [DOI] [PubMed] [Google Scholar]
  20. Ruettinger R. T., Olson S. T., Boyer R. F., Coon M. J. Identification of the omega-hydroxylase of Pseudomonas oleovorans as a nonheme iron protein requiring phospholipid for catalytic activity. Biochem Biophys Res Commun. 1974 Apr 23;57(4):1011–1017. doi: 10.1016/0006-291x(74)90797-9. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. Suzuki M., Hayakawa T., Shaw J. P., Rekik M., Harayama S. Primary structure of xylene monooxygenase: similarities to and differences from the alkane hydroxylation system. J Bacteriol. 1991 Mar;173(5):1690–1695. doi: 10.1128/jb.173.5.1690-1695.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. Worsey M. J., Williams P. A. Metabolism of toluene and xylenes by Pseudomonas (putida (arvilla) mt-2: evidence for a new function of the TOL plasmid. J Bacteriol. 1975 Oct;124(1):7–13. doi: 10.1128/jb.124.1.7-13.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Yamaguchi M., Fujisawa H. Subunit structure of oxygenase component in benzoate-1,2-dioxygenase system from Pseudomonas arvilla C-1. J Biol Chem. 1982 Nov 10;257(21):12497–12502. [PubMed] [Google Scholar]

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

RESOURCES