Purification and characterization of an oxygenase component in benzoate 1,2-dioxygenase system from Pseudomonas arvilla C-1.
The benzoate 1,2-dioxygenase system of Pseudomonas arvilla consists of two proteins, an NADH-cytochrome c reductase and an oxygenase. The oxygenase component was purified to apparent homogeneity by the criteria of polyacrylamide gel electrophoresis from benzoate-induced cells of P. arvilla. The molecular weight of the enzyme was determined to be 273,000 by sedimentation equilibrium analysis, 280,000 by electrophoresis on polyacrylamide gels of different concentrations, and 270,000 by Sepharose CL-6B gel filtration, respectively. The sedimentation coefficient, the Stokes radius, and the partial specific volume of the enzyme were calculated to be 10.0 S, 56 A, and 0.72 ml/g, respectively. The isoelectric point of the enzyme was estimated to be pH 4.5. The enzyme contained about 10 mol of iron and about 8 mol of labile sulfide/mol of enzyme. The iron-sulfur clusters of the enzyme were suggested to be (2Fe-2S*) from cluster-extrusion experiments (S*, sulfide, acid-labile sulfur). No significant amounts of heme or flavin were detected in the enzyme. The enzyme exhibited absorption spectrum with maxima at 279, 325, and 464 nm. The turnover number of the enzyme in the presence of saturating amounts of NADH-cytochrome c reductase, the other component of the benzoate 1,2-dioxygenase system, was calculated to be 22,000 at 24 degrees C. The apparent Km values for the reductase, benzoate, and molecular oxygen were 26 (0.97 mg of protein/ml), 3.9, and 4.3 microM, respectively.
D000596:Amino Acids / Q000032:analysis; D001565:Benzoates / Q000302:isolation & purification / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000302:isolation & purification; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D010105:Oxygenases / Q000302:isolation & purification / Q000378:metabolism*; D011549:Pseudomonas / Q000201:enzymology*; D013053:Spectrophotometry; D013379:Substrate Specificity
D000596:Amino Acids; D001565:Benzoates; D007506:Iron-Sulfur Proteins; D046911:Macromolecular Substances; D010105:Oxygenases; C019766:benzoate 1,2-dioxygenase