Purification and characterization of the 3-chloro-4-hydroxy-phenylacetate reductive dehalogenase of Desulfitobacterium hafniense.
The membrane-bound 3-chloro-4-hydroxyphenylacetate (Cl-OHPA) reductive dehalogenase from the chlorophenol-reducing anaerobe Desulfitobacterium hafniense was purified 11.3-fold to apparent homogeneity in the presence of the detergent CHAPS. The purified dehalogenase catalyzed the reductive dechlorination of Cl-OHPA to 4-hydroxyphenylacetate with reduced methyl viologen as the electron donor at a specific activity of 103.2 nkat/mg protein. SDS-PAGE revealed a single protein band with an apparent molecular mass of 46.5 kDa. The enzyme contained 0.68 +/- 0.2 mol corrinoid, 12.0 +/- 0.7 mol iron, and 13.0 +/- 0.7 mol acid-labile sulfur per mol subunit. The N-terminal amino acid sequence of the enzyme was determined and no significant similarity was found to any protein present in the gene bank.
Christiansen
N
N
Department of Biotechnology, Technical University of Denmark, Lyngby.
Ahring
B K
BK
Wohlfarth
G
G
Diekert
G
G
D000085:Acetates / Q000378:metabolism; D002793:Cholic Acids; D002852:Chromatography, Ion Exchange; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D006089:Gram-Negative Anaerobic Bacteria / Q000201:enzymology*; D008970:Molecular Weight; D010088:Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism*; D010636:Phenols / Q000378:metabolism; D010642:Phenoxyacetates; D013053:Spectrophotometry; D013379:Substrate Specificity
C104563:3-chloro-4-hydroxyphenoxyacetic acid; D000085:Acetates; D002793:Cholic Acids; D003902:Detergents; D010636:Phenols; D010642:Phenoxyacetates; D010088:Oxidoreductases; C115216:3-chloro-4-hydroxyphenylacetate reductive dehalogenase; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate