The biochemistry of pentachlorophenol (PCP) degradation by Flavobacterium sp. strain ATCC 39723 has been studied, and two enzymes responsible for the conversion of PCP to 2,6-dichloro-p-hydroquinone (2,6-DiCH) have previously been purified and characterized. In this study, enzymatic activities consuming 2,6-DiCH were identified from the cell extracts of strain ATCC 39723. The enzyme was purified to apparent homogeneity by a purification scheme consisting of seven steps. Gel filtration chromatography showed a native molecular weight of about 40,000, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein of 42,500 Da. The purified enzyme converted 2,6-DiCH to 6-chlorohydroxyquinol (6-chloro-1,2,4-trihydroxybenzene), which was easily oxidized by molecular oxygen and hard to detect. The end product, 6-chlorohydroxyquinol, was detected only in the presence of a reductase and NADH in the reaction mixture. The enzyme dechlorinated 2,6-DiCH but not 2,5-DiCH. The enzyme required Fe 2؉ for activity and was severely inhibited by metal chelating agents. The optimal conditions for activity were pH 7.0 and 40؇C. The K m for 2,6-DiCH was 35 M, and the k cat was 0.011 s ؊1 .Chlorophenols have been widely used as disinfectants and preservatives and have entered the environment in considerable amounts (7). They are a major group of environmental pollutants (7, 10). Several microorganisms that can mineralize chlorophenols have been isolated, and the metabolisms of chlorophenols have been extensively studied (1,8,12,16,18). There are two major classes of metabolic pathways for the degradation of chlorophenols. The breakdown of mono-or dichlorophenols is usually through a catechol intermediate before ring cleavage (6). On the other hand, most polychlorinated phenols are degraded through a chlorohydroxyquinol (CHQ) intermediate before ring cleavage (1,3,24).The reaction mechanisms and enzymes responsible for the degradation of polychlorinated phenols have been studied in several microorganisms (11,20,(22)(23)(24). Flavobacterium sp. strain ATCC 39723 oxidizes pentachlorophenol (PCP) to tetrachloro-p-hydroquinone (TeCH) by PCP 4-monooxygenase (20, 21) and then converts TeCH to 2,6-dichloro-p-hydroquinone (2,6-DiCH) by TeCH reductive dehalogenase (22). Ring cleavage dioxygenases using 6-CHQ (6-chloro-1,2,4-trihydroxybenzene) and hydroxyquinol (1,2,4-trihydroxybenzene) as substrates have been purified and characterized from Azotobacter sp. strain GP1 (11) and Streptomyces rochei 303 (24). The missing information is how 2,6-DiCH is converted to 6-CHQ. Since 2,6-DiCH is a common metabolite in the degradation of polychlorinated phenols, such as PCP, 2,3,5,6-tetrachlorophenol, and 2,4,6-trichlorophenol (2,4,6-TCP), by various microorganisms (8,12,22), it is important to understand its metabolism.In this report, we present the identification, purification, and characterization of 2,6-DiCH chlorohydrolase that converted 2,6-DiCH to 6-CHQ from strain ATCC 39723. MATERIALS AND METHODSChemicals. 2,4,6-TCP, 2,6-di...