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. 1994 Nov;176(21):6644–6652. doi: 10.1128/jb.176.21.6644-6652.1994

Terephthalate 1,2-dioxygenase system from Comamonas testosteroni T-2: purification and some properties of the oxygenase component.

H R Schläfli 1, M A Weiss 1, T Leisinger 1, A M Cook 1
PMCID: PMC197021  PMID: 7961417

Abstract

Comamonas testosteroni T-2, grown in terephthalate (TER)-salts medium, synthesizes inducible enzymes that convert TER to (1R,2S)-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylic acid (DCD) and protocatechuate (PC). Anion-exchange chromatography of cell extracts yielded two sets of fractions, R and Z, that were necessary for oxygenation of TER to DCD; we termed this activity the TER dioxygenase system (TERDOS). An NAD(+)-dependent DCD dehydrogenase, which converted DCD to PC, overlapped all fractions R. No significant purification from fraction R, which contained an NADH-dependent reductase function(s) of TERDOS, was attained. Fraction Z, at the end of the gradient, contained essentially one protein, which was further purified by hydrophobic interaction chromatography. This component, Z, had the UV-visible spectrum and electron paramagnetic resonance characteristics of a Rieske [2Fe-2S] protein and was considered to be the oxygenase. M(r)s of about 126,000 for oxygenase Z under native conditions were observed. Oxygenase Z consisted of two subunits, alpha and beta, with M(r)s of 49,000 and 18,000, respectively, under denaturing conditions. We presume that this oxygenase has an alpha 2 beta 2 structure. The sequences of the N-terminal amino acids of each subunit were determined. The activity of the purified enzyme was enhanced about fivefold by addition of Fe2+. In the presence of O2, NADH, and fraction R, component Z catalyzed the stoichiometric transformation of TER to PC, with the intermediate formation of DCD. The reaction was confirmed as a dioxygenation when we observed incorporation of two oxygen atoms from 18O2 into PC. The substrate range of TERDOS appeared to be narrow; apart from TER, only 2,5-dicarboxypyridine and 1,4-dicarboxynaphthalene (of 11 compounds tested) were converted to a product.

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Selected References

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  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. 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]
  4. 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]
  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. Busse H. J., el-Banna T., Oyaizu H., Auling G. Identification of xenobiotic-degrading isolates from the beta subclass of the Proteobacteria by a polyphasic approach including 16S rRNA partial sequencing. Int J Syst Bacteriol. 1992 Jan;42(1):19–26. doi: 10.1099/00207713-42-1-19. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. 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]
  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. Gibson D. T., Koch J. R., Kallio R. E. Oxidative degradation of aromatic hydrocarbons by microorganisms. I. Enzymatic formation of catechol from benzene. Biochemistry. 1968 Jul;7(7):2653–2662. doi: 10.1021/bi00847a031. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Junker F., Field J. A., Bangerter F., Ramsteiner K., Kohler H. P., Joannou C. L., Mason J. R., Leisinger T., Cook A. M. Oxygenation and spontaneous deamination of 2-aminobenzenesulphonic acid in Alcaligenes sp. strain O-1 with subsequent meta ring cleavage and spontaneous desulphonation to 2-hydroxymuconic acid. Biochem J. 1994 Jun 1;300(Pt 2):429–436. doi: 10.1042/bj3000429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Keyser P., Pujar B. G., Eaton R. W., Ribbons D. W. Biodegradation of the phthalates and their esters by bacteria. Environ Health Perspect. 1976 Dec;18:159–166. doi: 10.1289/ehp.7618159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. 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]
  18. 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]
  19. Locher H. H., Poolman B., Cook A. M., Konings W. N. Uptake of 4-toluene sulfonate by Comamonas testosteroni T-2. J Bacteriol. 1993 Feb;175(4):1075–1080. doi: 10.1128/jb.175.4.1075-1080.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Riester J., Zumft W. G., Kroneck P. M. Nitrous oxide reductase from Pseudomonas stutzeri. Redox properties and spectroscopic characterization of different forms of the multicopper enzyme. Eur J Biochem. 1989 Jan 2;178(3):751–762. doi: 10.1111/j.1432-1033.1989.tb14506.x. [DOI] [PubMed] [Google Scholar]
  22. Tan H. M., Joannou C. L., Cooper C. E., Butler C. S., Cammack R., Mason J. R. The effect of ferredoxin(BED) overexpression on benzene dioxygenase activity in Pseudomonas putida ML2. J Bacteriol. 1994 May;176(9):2507–2512. doi: 10.1128/jb.176.9.2507-2512.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]

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