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Purification and characterization of an oxygenase component in benzoate 1,2-dioxygenase system from Pseudomonas arvilla C-1.

1980; Elsevier BV; Volume: 255; Issue: 11 Linguagem: Inglês

10.1016/s0021-9258(19)70748-0

1083-351X

Mutsuo Yamaguchi, Hiroshi Fujisawa,

Chemical Reactions and Isotopes

The benzoate 1,bdioxygenase 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. untilla.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-GB 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°C.The apparent K,,, values for the reductase, benzoate, and molecular oxygen were 26 (0.97 mg of protein/ml), 3.9, and 4.3 PM, respectively. A double hydroxylation reaction was first demonstrated byHayaishi and his coworkers (1,2) with anthranilate dioxygenase which catalyzes the formation of catechol from anthranilate.They demonstrated by experiments with "0 that both atoms of oxygen incorporated in two hydroxyl groups of catechol were exclusively derived from the same oxygen molecule.Similar dioxygenases, catalyzing double hydroxylation of aromatic compounds including benzene (3, 4), toluene ( 5 ) , phthalate (6), pyrazon (7), and naphthalene (8), have been isolated from soil bacteria and they have been demonstrated to be multienzyme systems consisting of two or three protein components.However, detailed studies of the reaction mechanism of double hydroxylation by dioxygenases have not been reported, since the enzyme systems are unstable and pure enzymes have not been available for use in studying the reaction mechanism.The benzoate 1,2-dioxygenase system catalyzes the double hydroxylation of benzoate with the incorporation of 2 atoms