title: Overproduction, purification, and characterization of chlorocatechol dioxygenase, a non-heme iron dioxygenase with broad substrate tolerance.
abstract: We show here that purified chlorocatechol dioxygenase from Pseudomonas putida is able to oxygenate a wide range of substituted catechols with turnover numbers ranging from 2 to 29 s-1. This enzyme efficiently cleaves substituted catechols bearing electron-donating or multiple electron-withdrawing groups in an intradiol manner with kcat/KM values between 0.2 x 10(7) and 1.4 x 10(7) M-1 s-1. These unique catalytic properties prompted a comparison with the related but highly specific enzymes catechol 1,2-dioxygenase and protocatechuate 3,4-dioxygenase. The chlorocatechol dioxygenase gene (clcA) from the Pseudomonas plasmid pAC27 was subcloned into the expression vector pKK223-3, allowing production of chlorocatechol dioxygenase to approximately 7-8% of total cellular protein. An average of 4 mg of purified enzyme has been obtained per gram of wet cells. Protein and iron analyses indicate an iron stoichiometry of 1 iron/57.5-kDa homodimer, alpha 2Fe. The electronic absorption spectrum contains a broad tyrosinate to iron charge transfer transition centered at 430 nm (epsilon = 3095 M-1 cm-1 based on iron concentration) which shifts to 490 nm (epsilon = 3380 M-1 cm-1) upon catechol binding. The resonance Raman spectrum of the native enzyme exhibits characteristic tyrosine ring vibrations. Electron paramagnetic resonance data for the resting enzyme (g = 4.25, 9.83) is consistent with high-spin iron (III) in a rhombic environment. This similarity between the spectroscopic properties of the Fe(III) centers in chlorocatechol dioxygenase and the more specific dioxygenases suggests a highly conserved catalytic site. We infer that the unique catalytic properties of chlorocatechol dioxygenase are due to other characteristics of its substrate binding pocket.
authors.affiliation: Department of Chemistry, Northwestern University, Evanston, Illinois 60208-3113.
authors.forename: J B
authors.initials: JB
authors.lastname: Broderick
authors.forename: T V
authors.initials: TV
authors.lastname: O'Halloran
mesh_terms: D050548:Catechol 1,2-Dioxygenase; D003001:Cloning, Molecular; D049308:Dioxygenases; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D010105:Oxygenases / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D010957:Plasmids; D011520:Protocatechuate-3,4-Dioxygenase / Q000378:metabolism; D011549:Pseudomonas / Q000201:enzymology; D013059:Spectrum Analysis, Raman; D013379:Substrate Specificity
chemical_list: D007501:Iron; D010105:Oxygenases; D049308:Dioxygenases; C053101:catechol oxygenase II; D050548:Catechol 1,2-Dioxygenase; D011520:Protocatechuate-3,4-Dioxygenase