The enzyme which cleaves the benzene ring of 6-chlorohydroxyquinol was purified to apparent homogeneity from an extract of 2,4,6-trichlorophenol-grown cells of Streptomyces rochei 303. Like the analogous enzyme from Azotobacter sp. strain GP1, it exhibited a highly restricted substrate specificity and was able to cleave only 6-chlorohydroxyquinol and hydroxyquinol and not catechol, chlorinated catechols, or pyrogallol. No extradiolcleaving activity was observed. In contrast to 6-chlorohydroxyquinol 1,2-dioxygenase from Azotobacter sp. strain GP1, the S. rochei enzyme had a distinct preference for 6-chlorohydroxyquinol over hydroxyquinol (k cat /K m ‫؍‬ 1.2 and 0.57 s ؊1 ⅐ M ؊1 , respectively). The enzyme from S. rochei appears to be a dimer of two identical 31-kDa subunits. It is a colored protein and was found to contain 1 mol of iron per mol of enzyme. The NH 2 -terminal amino acid sequences of 6-chlorohydroxyquinol 1,2-dioxygenase from S. rochei 303 and from Azotobacter sp. strain GP1 showed a high degree of similarity.Two pathways for the aerobic degradation of chlorophenols have been described so far: one via chlorocatechols and the other via chlorohydroquinones (2, 9, 10, 15). A predominant catabolic route for the compounds carrying one or two chlorine substituents was shown to be the modified ortho-cleavage pathway (14,18,24). In this pathway, chlorocatechols formed by introduction of a second hydroxy group are subjected to intradiol cleavage. In the case of chlorophenol degradation through the chlorohydroquinone pathway, the introduction of a third hydroxy group leads to the formation of hydroxyquinol or chlorohydroxyquinol (3, 13). Both unsubstituted and chlorinated 1,2,4-trihydroxybenzene were shown to be subject to ortho cleavage in Streptomyces rochei 303 (13). Extracts from cells grown in the presence of 2-chloro-or 2,4-dichlorophenol were found to prefer hydroxyquinol over 6-chlorohydroxyquinol as a ring fission substrate, whereas extracts from cells grown on 2,6-dichloro-or 2,4,6-trichlorophenol showed a preference for the chlorinated substrate (13). These results led us to suggest that two different dioxygenases play a role in the cleavage of the trihydroxylated aromatic ring: 6-chlorohydroxyquinol 1,2-dioxygenase in the ring fission of 6-chlorohydroxyquinol, the proposed intermediate of 2,4,6-trichloro-and 2,6-dichlorophenol degradation, and a second dioxygenase in the cleavage of hydroxyquinol, the proposed intermediate of 2,4-dichloro-and 2-chlorophenol degradation (Fig. 1) (13).In this paper, we describe the purification and characterization of 6-chlorohydroxyquinol 1,2-dioxygenase. Three other hydroxyquinol 1,2-dioxygenases have been purified and characterized so far (21,25,34). One of them takes part in the cleavage of hydroxyquinol, found as an intermediate of 4-hydroxybenzoate degradation in Trichosporon cutaneum (34).The second dioxygenase, from the basidiomycete Phanerochaete chrysosporium, catalyzes a key step in the degradation of vanillate, an intermediate in lignin degradation (25)....

Hydroxyquinol 1,2-dioxygenase was purified from cells of the soil bacterium Azotobacter sp. strain GP1 grown with 2,4,6-trichlorophenol as the sole source of carbon. The presumable function of this dioxygenase enzyme in the degradative pathway of 2,4,6-trichlorophenol is discussed. The enzyme was highly specific for 6-chlorohydroxyquinol (6-chloro-1,2,4-trihydroxybenzene) and hydroxyquinol (1,2,4-trihydroxybenzene) and was found to perform ortho cleavage of the hydroxyquinol compounds, yielding chloromaleylacetate and maleylacetate, respectively. With the conversion of 1 mol of 6-chlorohydroxyquinol, the consumption of 1 mol of O 2 and the formation of 1 mol of chloromaleylacetate were observed. Catechol was not accepted as a substrate. The enzyme has to be induced, and no activity was found in cells grown on succinate. The molecular weight of native hydroxyquinol 1,2-dioxygenase was estimated to 58,000, with a sedimentation coefficient of 4.32. The subunit molecular weight of 34,250 indicates a dimeric structure of the dioxygenase enzyme. The addition of Fe 2؉ ions significantly activated enzyme activity, and metal-chelating agents inhibited it. Electron paramagnetic resonance data are consistent with high-spin iron(III) in a rhombic environment. The NH 2-terminal amino acid sequence was determined for up to 40 amino acid residues and compared with sequences from literature data for other catechol and chlorocatechol dioxygenases. MATERIALS AND METHODS Chemicals. TCP was from Merck, Darmstadt, Germany; HQ was from Lancaster Synthesis, Morecombe, England. 6-Chlorohydroxyquinol was obtained in a three-step synthesis. In the first step, vanillin was chlorinated with sodium hypochlorite to 5-chlorovanillin (17). Ether cleavage of 5-chlorovanillin to 3-chloro-4,5-dihydroxybenzaldehyde was performed with aluminum chloride in pyridine (2). CHQ was synthesized by a modified Dakin oxidation (9): 50 mg of 3-chloro-4,5-dihydroxybenzaldehyde was dissolved in 300 l of sodium hydroxide (1 N), yielding a dark green solution. When the aldehyde was completely dissolved, 200 l of H 2 O 2 (30%) was added. A strong exothermic reaction followed. The color turned from green to dark red-brown. The reaction was allowed to continue for 2 min. The solution was acidified by the addition of 2 ml of H 2 SO 4 (1 N) and kept on ice. Upon cooling, unreacted aldehyde precipitated. The solution was filtered and extracted twice with ethylacetate. The extracts were combined, washed with acidified saturated sodium chloride solution (5 ml), and dried over anhydrous sodium sulfate. After decanting from sodium sulfate, ethylacetate was concentrated to dryness in a rotavapor, and the residue was dried in a desiccator over calcium chloride in an argon atmosphere. To avoid auto-oxidation and polymerization, synthesis and storage were performed under an argon atmosphere and in the dark. The synthesized CHQ showed a mass spectrum consistent with the proposed structure, and it was found to be identical in UV spectrum to a sample kindly provided by L. A. Golo...

We wish to acknowledge the invaluable and skilled assistance of Mr. D. Canwell in the preparation of the histological material.