title: Purification and characterization of the tetrachloroethene reductive dehalogenase of strain PCE-S.
abstract: The membrane-associated tetrachloroethene reductive dehalogenase from the tetrachloroethene-reducing anaerobe, strain PCE-S, was purified 165-fold to apparent homogeneity in the presence of the detergent Triton X-100. The purified dehalogenase catalyzed the reductive dechlorination of tetrachloroethene to trichloroethene and of trichloroethene to cis-1,2-dichloroethene with reduced methyl viologen as the electron donor, showing a specific activity of 650 nkat/mg protein. The apparent Km values of the enzyme for tetrachloroethene, trichloroethene, and methyl viologen were 10 microM, 4 microM, and 0.3 mM, respectively. SDS-PAGE revealed a single protein band with an apparent molecular mass of 65 kDa. The apparent molecular mass of the native enzyme was 200 kDa as determined by gel filtration. Tetrachloroethene dehalogenase contained 0.7 +/- 0.3 mol corrinoid, 1.0 +/- 0.3 mol cobalt, 7.8 +/- 0.5 mol iron, and 10.3 +/- 2.0 mol acid-labile sulfur per mol subunit. The pH optimum was approximately 7.2, and the temperature optimum was approximately 50 degrees C. The dehalogenase was oxygen-sensitive with a half-life of approximately 50 min. The N-terminal amino acid sequence of the enzyme was determined, and no significant similarity was found to any part of the amino acid sequence of the tetrachloroethene (PCE) reductive dehalogenase from Dehalospirillum multivorans.
authors.affiliation: Institut für Mikrobiologie, Universität Stuttgart, Allmandring 31, D-70569 Stuttgart, Germany.
authors.forename: E
authors.initials: E
authors.lastname: Miller
authors.forename: G
authors.initials: G
authors.lastname: Wohlfarth
authors.forename: G
authors.initials: G
authors.lastname: Diekert
mesh_terms: D001421:Bacteria, Anaerobic / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D006094:Gram-Positive Bacteria / Q000201:enzymology*; D010088:Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification*
chemical_list: D010088:Oxidoreductases; C112990:tetrachloroethene dehalogenase