The two components of the inducible 2-halobenzoate 1,2-dioxygenase from Pseudomonas cepacia 2CBS were purified to homogeneity. Yellow component B is a monomer (Mr,37,500) with NADH-acceptor reductase activity. Ferricyanide, 2,6-dichlorophenol indophenol, and cytochrome c acted as electron acceptors. Component B was identified as an iron-sulfur flavoprotein containing 0.8 mol of flavin adenine dinucleotide, 1.7 mol of iron, and 1.7 mol of acid-labile sulfide per mol of enzyme. The isoelectric point was estimated to be pH 4.2. 1,2-dioxygenase, because it catalyzes the conversion of 2-fluoro-, 2-bromo-, 2-chloro-, and 2-iodobenzoate to catechol. 2-Halobenzoate 1,2-dioxygenase exhibited a very broad substrate specificity, but benzoate analogs with electron-withdrawing substituents at the ortho position were transformed preferentially.Chlorobenzoates are key intermediates in the degradative pathway of chlorobiphenyls (25). Due to their good water solubility and low toxicity, chlorobenzoates are favorable model compounds for studying the degradation of halogenated aromatic substances. The ortho-substituted halobenzoates are of special interest because they are more refractory than the other isomers to biodegradation. Steric hindrance and the effect of chlorine atoms on the electron density at the ortho position of the benzene ring were suggested to be responsible for the resistance of 2-halobenzoates (other than 2-fluorobenzoate) to hydroxylation by the benzoate dioxygenase system (50, 52).A number of reports describe the utilization of 2-chlorobenzoate by various Pseudomonas strains (20,33,62,77). Sylvestre et al. (62) suggested an initial attack of 2-chlorobenzoate by a 2-chlorobenzoate dioxygenase in Pseudomonas sp. strain B-300. Engesser and Schulte (20) postulated a 2-chlorobenzoate 1,2-dioxygenase system catalyzing the conversion of 2-chlorobenzoate to catechol in Pseudomonas putida CLB 250. However, these authors did not detect any 2-chlorobenzoate dioxygenase activity in cell extracts.Pseudomonas cepacia 2CBS utilizes 2-chlorobenzoate as a sole source of carbon and energy. In the first step of 2-chlorobenzoate degradation, 2-chlorobenzoate is converted to catechol, which is subject predominantly to metaring cleavage (23). The enzyme catalyzing the initial degradation step was shown to be a two-component dioxygenase system, previously termed 2-chlorobenzoate 1,2-dioxygenase (23).The benzoate dioxygenases of P. putida (arvilla) C-1 (74) and P. putida B13 (27,50,51) and the isofunctional TOL * Corresponding author.plasmid-encoded enzyme toluate 1,2-dioxygenase from P. putida (arvilla) mt-2 (27, 38, 70, 72) are unable to oxygenate 2-chlorobenzoate. Here we investigated the dehalogenating 2-halobenzoate 1,2-dioxygenase from P. cepacia 2CBS to compare these multicomponent enzyme systems.
MATERIALS AND METHODSMaterials. All chemicals were of the highest purity commercially available.Growth of P. cepacia 2CBS and preparation of crude extracts. P. cepacia 2CBS (23) was grown in chloride-free mineral salts medium (22)