title: Purification and characterization of a novel 3-chlorobenzoate-reductive dehalogenase from the cytoplasmic membrane of Desulfomonile tiedjei DCB-1.
abstract: Although reductive dehalogenation by anaerobic microorganisms offers great potential for the degradation of halocarbons, little is known about the biochemical mechanisms involved. It has previously been demonstrated that the dehalogenase activity involved in 3-chlorobenzoate dehalogenation by Desulfomonile tiedjei DCB-1 is present in the membrane fraction of the cell extracts. We report herein the purification of a 3-chlorobenzoate-reductive dehalogenase from the cytoplasmic membrane of D. tiedjei DCB-1. The dehalogenase activity was monitored by the conversion of 3-chlorobenzoate to benzoate with reduced methyl viologen as a reducing agent. The membrane fraction of the cell extracts was obtained by ultracentrifugation, and the membrane proteins were solubilized with either the detergent CHAPS (3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate) or Triton X-100 in the presence of glycerol. The solubilized dehalogenase was purified by ammonium sulfate fractionation and a combination of anion exchange, hydroxyapatite, and hydrophobic interaction chromatographies. This procedure yielded about 7% of the total dehalogenase activity with a 120-fold increase in specific activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the purified dehalogenase consisted of two subunits with molecular weights of 64,000 and 37,000. The enzyme converted 3-chlorobenzoate to benzoate at its highest specific activity in 10 mM potassium phosphate buffer (pH 7.2) at 38 degrees C. The enzyme was yellow and probably a heme protein. The enzyme had an adsorbance peak at 408 nm. The dithionite-reduced enzyme displayed absorbance peaks at 416, 522, and 550 nm. The dithionite-reduced enzyme was able to complex with carbon monoxide. The nature of the heme chromophore is currently unknown.
authors.affiliation: Environmental Microbiology Group, Pacific Northwest Laboratory, Richland, Washington 99352, USA.
authors.forename: S
authors.initials: S
authors.lastname: Ni
authors.forename: J K
authors.initials: JK
authors.lastname: Fredrickson
authors.forename: L
authors.initials: L
authors.lastname: Xun
mesh_terms: D001565:Benzoates / Q000378:metabolism; D019817:Benzoic Acid; D001673:Biodegradation, Environmental; D002458:Cell Fractionation; D002462:Cell Membrane / Q000201:enzymology*; D002723:Chlorobenzoates / Q000378:metabolism; D006089:Gram-Negative Anaerobic Bacteria / Q000201:enzymology*; D006867:Hydrolases / Q000737:chemistry / Q000302:isolation & purification*; D010084:Oxidation-Reduction; D013053:Spectrophotometry; D013347:Subcellular Fractions / Q000378:metabolism
chemical_list: D001565:Benzoates; D002723:Chlorobenzoates; C036427:3-chlorobenzoic acid; D019817:Benzoic Acid; D006867:Hydrolases; C095469:3-chlorobenzoate dehalogenase