title: Chlorocatechols substituted at positions 4 and 5 are substrates of the broad-spectrum chlorocatechol 1,2-dioxygenase of Pseudomonas chlororaphis RW71.
abstract: The nucleotide sequence of a 10,528-bp region comprising the chlorocatechol pathway gene cluster tetRtetCDEF of the 1,2,3,4-tetrachlorobenzene via the tetrachlorocatechol-mineralizing bacterium Pseudomonas chlororaphis RW71 (T. Potrawfke, K. N. Timmis, and R.-M. Wittich, Appl. Environ. Microbiol. 64:3798-3806, 1998) was analyzed. The chlorocatechol 1,2-dioxygenase gene tetC was cloned and overexpressed in Escherichia coli. The recombinant gene product was purified, and the alpha,alpha-homodimeric TetC was characterized. Electron paramagnetic resonance measurements confirmed the presence of a high-spin-state Fe(III) atom per monomer in the holoprotein. The productive transformation by purified TetC of chlorocatechols bearing chlorine atoms in positions 4 and 5 provided strong evidence for a significantly broadened substrate spectrum of this dioxygenase compared with other chlorocatechol dioxygenases. The conversion of 4,5-dichloro- or tetrachlorocatechol, in the presence of catechol, displayed strong competitive inhibition of catechol turnover. 3-Chlorocatechol, however, was simultaneously transformed, with a rate similar to that of the 4,5-halogenated catechols, indicating similar specificity constants. These novel characteristics of TetC thus differ significantly from results obtained from hitherto analyzed catechol 1,2-dioxygenases and chlorocatechol 1,2-dioxygenases.
authors.affiliation: Division of Microbiology, GBF-German Research Centre for Biotechnology, D-38124 Braunschweig, Germany.
authors.forename: T
authors.initials: T
authors.lastname: Potrawfke
authors.forename: J
authors.initials: J
authors.lastname: Armengaud
authors.forename: R M
authors.initials: RM
authors.lastname: Wittich
mesh_terms: D001051:Apoenzymes; D001673:Biodegradation, Environmental; D002396:Catechols / Q000378:metabolism; D002722:Chlorobenzenes / Q000378:metabolism; D049308:Dioxygenases; D004578:Electron Spin Resonance Spectroscopy; D019143:Evolution, Molecular; D005798:Genes, Bacterial; D020035:Holoenzymes; D007501:Iron / Q000032:analysis; D007700:Kinetics; D008954:Models, Biological; D005810:Multigene Family; D010105:Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011549:Pseudomonas / Q000201:enzymology*; D011994:Recombinant Proteins / Q000378:metabolism; D013053:Spectrophotometry; D013379:Substrate Specificity
chemical_list: D001051:Apoenzymes; D002396:Catechols; D002722:Chlorobenzenes; D020035:Holoenzymes; D011994:Recombinant Proteins; C423629:3,4,5,6-tetrachlorocatechol; C074311:3,4,5-trichlorocatechol; D007501:Iron; D010105:Oxygenases; D049308:Dioxygenases; C044173:chlorocatechol 1,2-dioxygenase; C030386:1,2,3,4-tetrachlorobenzene; C423630:4,5-dichlorocatechol