title: Purification and characterization of a tetrachloro-p-hydroquinone reductive dehalogenase from a Flavobacterium sp.
abstract: Tetrachloro-p-hydroquinone (TeCH) is the first intermediate in pentachlorophenol (PCP) degradation by Flavobacterium sp. strain ATCC 39723. We previously purified a PCP hydroxylase that oxidized PCP to TeCH. Subsequently, we identified the reductive dehalogenation of TeCH to 2,3,6-trichloro-p-hydroquinone and then to 2,6-dichloro-p-hydroquinone in a cell extract with the reduced form of glutathione as the reducing agent under anaerobic conditions. Here we report the purification of a TeCH reductive dehalogenase that reductively dehalogenated TeCH to trichlorohydroquinone and then to dichlorohydroquinone. The enzyme was purified by protamine sulfate treatment, ammonium sulfate fractionation, and phenyl-agarose, anion-exchange, and gel filtration column chromatographies. As determined by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses, the protein has a molecular weight of about 30,000; nondenaturing polyacrylamide gel electrophoresis analysis suggests that the native enzyme exists as a dimer. The enzyme used glutathione but not NADPH, NADH, dithiothreitol, or ascorbic acid as the reducing agent. The optimal pH was close to neutral.
authors.affiliation: Department of Bacteriology and Biochemistry, University of Idaho, Moscow 83843.
authors.forename: L
authors.initials: L
authors.lastname: Xun
authors.forename: E
authors.initials: E
authors.lastname: Topp
authors.forename: C S
authors.initials: CS
authors.lastname: Orser
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005417:Flavobacterium / Q000201:enzymology*; D006867:Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D006873:Hydroquinones / Q000378:metabolism*; D008969:Molecular Sequence Data
chemical_list: D001426:Bacterial Proteins; D006873:Hydroquinones; C044578:2,3,5,6-tetrachlorohydroquinone; D006867:Hydrolases; C078610:2,3,5,6-tetrachloro-4-hydroquinone reductive dehalogenase