Abstract
Pseudomonas cepacia AC1100 degrades 2,4,5-trichlorophenoxyacetate (2,4,5-T), an herbicide and chlorinated aromatic compound. Although some progress has been made in understanding 2,4,5-T degradation by AC1100 by molecular analysis, little is known about the biochemistry involved. Enzymatic activity converting 2,4,5-T to 2,4,5-trichlorophenol in the presence of NADH and O(inf2) was detected in cell extracts of AC1100. Phenyl agarose chromatography of the ammonium sulfate-fractionated cell extracts yielded no active single fractions, but the mixing of two fractions, named component A and component B, resulted in the recovery of enzyme activity. Component B was further purified to homogeneity by hydroxyapatite and DEAE chromatographies. Component B had a native molecular weight of 140,000, and it was composed of two 49-kDa (alpha)-subunits and two 24-kDa (beta)-subunits. Component B was red, and its spectrum in the visible region had maxima at 430 and 560 nm (shoulder), whereas upon reduction it had maxima at 420 (shoulder) and 530 nm. Each mole of (alpha)(beta) heterodimer contained 2.9 mol of iron and 2.1 mol of labile sulfide. These properties suggest strong similarities between component B and the terminal oxygenase components of the aromatic ring-hydroxylating dioxygenases. Component A was highly purified but not to homogeneity. The reconstituted 2,4,5-T oxygenase, consisting of components A and B, converted 2,4,5-T quantitatively into 2,4,5-trichlorophenol and glyoxylate with the coconsumption of NADH and O(inf2).
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- 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]
- Chaudhry G. R., Chapalamadugu S. Biodegradation of halogenated organic compounds. Microbiol Rev. 1991 Mar;55(1):59–79. doi: 10.1128/mr.55.1.59-79.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Danganan C. E., Ye R. W., Daubaras D. L., Xun L., Chakrabarty A. M. Nucleotide sequence and functional analysis of the genes encoding 2,4,5-trichlorophenoxyacetic acid oxygenase in Pseudomonas cepacia AC1100. Appl Environ Microbiol. 1994 Nov;60(11):4100–4106. doi: 10.1128/aem.60.11.4100-4106.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fukumori F., Hausinger R. P. Alcaligenes eutrophus JMP134 "2,4-dichlorophenoxyacetate monooxygenase" is an alpha-ketoglutarate-dependent dioxygenase. J Bacteriol. 1993 Apr;175(7):2083–2086. doi: 10.1128/jb.175.7.2083-2086.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Haugland R. A., Sangodkar U. M., Chakrabarty A. M. Repeated sequences including RS1100 from Pseudomonas cepacia AC1100 function as IS elements. Mol Gen Genet. 1990 Jan;220(2):222–228. doi: 10.1007/BF00260485. [DOI] [PubMed] [Google Scholar]
- Haugland R. A., Sangodkar U. M., Sferra P. R., Chakrabarty A. M. Cloning and characterization of a chromosomal DNA region required for growth on 2,4,5-T by Pseudomonas cepacia AC1100. Gene. 1991 Apr;100:65–73. doi: 10.1016/0378-1119(91)90351-b. [DOI] [PubMed] [Google Scholar]
- Karns J. S., Duttagupta S., Chakrabarty A. M. Regulation of 2,4,5-trichlorophenoxyacetic acid and chlorophenol metabolism in Pseudomonas cepacia AC1100. Appl Environ Microbiol. 1983 Nov;46(5):1182–1186. doi: 10.1128/aem.46.5.1182-1186.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kellogg S. T., Chatterjee D. K., Chakrabarty A. M. Plasmid-assisted molecular breeding: new technique for enhanced biodegradation of persistent toxic chemicals. Science. 1981 Dec 4;214(4525):1133–1135. doi: 10.1126/science.7302584. [DOI] [PubMed] [Google Scholar]
- Kilbane J. J., Chatterjee D. K., Karns J. S., Kellogg S. T., Chakrabarty A. M. Biodegradation of 2,4,5-trichlorophenoxyacetic acid by a pure culture of Pseudomonas cepacia. Appl Environ Microbiol. 1982 Jul;44(1):72–78. doi: 10.1128/aem.44.1.72-78.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- MASSEY V. Studies on succinic dehydrogenase. VII. Valency state of the iron in beef heart succinic dehydrogenase. J Biol Chem. 1957 Dec;229(2):763–770. [PubMed] [Google Scholar]
- 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]
- Sangodkar U. M., Chapman P. J., Chakrabarty A. M. Cloning, physical mapping and expression of chromosomal genes specifying degradation of the herbicide 2,4,5-T by Pseudomonas cepacia AC1100. Gene. 1988 Nov 30;71(2):267–277. doi: 10.1016/0378-1119(88)90043-1. [DOI] [PubMed] [Google Scholar]
- Suhara K., Takemori S., Katagiri M., Wada K., Kobayashi H. Estimation of labile sulfide in iron-sulfur proteins. Anal Biochem. 1975 Oct;68(2):632–636. doi: 10.1016/0003-2697(75)90659-4. [DOI] [PubMed] [Google Scholar]
- Trijbels F., Vogels G. D. Degradation of allantoin by Pseudomonas acidovorans. Biochim Biophys Acta. 1966 Feb 14;113(2):292–301. doi: 10.1016/s0926-6593(66)80068-1. [DOI] [PubMed] [Google Scholar]
- Xun L., Orser C. S. Purification and properties of pentachlorophenol hydroxylase, a flavoprotein from Flavobacterium sp. strain ATCC 39723. J Bacteriol. 1991 Jul;173(14):4447–4453. doi: 10.1128/jb.173.14.4447-4453.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]