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.
Department of Biotechnology, Technical University of Denmark, Lyngby.
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N
Christiansen
B K
BK
Ahring
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G
Wohlfarth
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G
Diekert
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