title: Purification and characterization of 2,6-dichloro-p-hydroquinone chlorohydrolase from Flavobacterium sp. strain ATCC 39723.
abstract: 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 Fe2+ for activity and was severely inhibited by metal chelating agents. The optimal conditions for activity were pH 7.0 and 40 degrees C. The Kcat for 2,6-DiCH was 35 microM, and the kcat was 0.011 s-1.
authors.affiliation: Environmental Microbiology Group, Battelle Pacific Northwest National Laboratory, Washington State University, Tri-Cities, Richland 99352, USA.
authors.forename: J Y
authors.initials: JY
authors.lastname: Lee
authors.forename: L
authors.initials: L
authors.lastname: Xun
mesh_terms: D001673:Biodegradation, Environmental; D004790:Enzyme Induction; D005417:Flavobacterium / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006873:Hydroquinones / Q000378:metabolism*; D008970:Molecular Weight; D010084:Oxidation-Reduction; D013696:Temperature
chemical_list: C091167:6-chlorohydroxyquinol; D006873:Hydroquinones; C104629:2,6-dichloro-4-hydroquinone; D006867:Hydrolases; C104630:2,6-dichloro-4-hydroquinone chlorohydrolase