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Pyrazines are a class of compounds that occur almost ubiquitously in nature. Pyrazines can be synthesised chemically or biologically, and are used as flavouring additives. The major formation of pyrazines occurs during heating of food. There is very little information available on the degradation of these compounds. In humans and animals, pyrazines are excreted as glucuronates or bound to glutathione via the kidney after hydroxylation, but the pyrazine ring is not cleaved. Bacteria have been isolated, which are able to use various substituted pyrazines as a sole carbon and energy source. In a few cases, the initial metabolites have been characterised; however, the mechanism of ring cleavage and the further degradation pathways are still unknown and await further investigation.

A novel phenol hydroxylase (PheA) that catalyzes the first step in the degradation of phenol in Bacillus thermoglucosidasius A7 is described. The two-protein system, encoded by the pheA1 and pheA2 genes, consists of an oxygenase (PheA1) and a flavin reductase (PheA2) and is optimally active at 55°C. PheA1 and PheA2 were separately expressed in recombinant Escherichia coli BL21(DE3) pLysS cells and purified to apparent homogeneity. The pheA1 gene codes for a protein of 504 amino acids with a predicted mass of 57.2 kDa. PheA1 exists as a homodimer in solution and has no enzyme activity on its own. PheA1 catalyzes the efficient ortho-hydroxylation of phenol to catechol when supplemented with PheA2 and FAD/NADH. The hydroxylase activity is strictly FAD-dependent, and neither FMN nor riboflavin can replace FAD in this reaction. The pheA2 gene codes for a protein of 161 amino acids with a predicted mass of 17.7 kDa. PheA2 is also a homodimer, with each subunit containing a highly fluorescent FAD prosthetic group. PheA2 catalyzes the NADH-dependent reduction of free flavins according to a Ping Pong Bi Bi mechanism. PheA2 is structurally related to ferric reductase, an NAD(P)H-dependent reductase from the hyperthermophilic Archaea Archaeoglobus fulgidus that catalyzes the flavin-mediated reduction of iron complexes. However, PheA2 displays no ferric reductase activity and is the first member of a newly recognized family of shortchain flavin reductases that use FAD both as a substrate and as a prosthetic group.

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)

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