Purification, cloning, and sequencing of a 3,5-dichlorophenol reductive dehalogenase from Desulfitobacterium frappieri PCP-1.
A membrane-associated 3,5-dichlorophenol reductive dehalogenase was isolated from Desulfitobacterium frappieri PCP-1. The highest dehalogenase activity was observed with the biomass cultured at 22 degrees C, compared to 30 and 37 degrees C, where the cell suspensions were 2.2 and 9.6 times less active, respectively. The reductive dehalogenase was purified 12.7-fold to apparent homogeneity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single band with an apparent molecular mass of 57 kDa. Its dechlorinating activity was not inhibited by sulfate and nitrate but was completely inhibited by 2.5 mM sulfite and 10 mM KCN. A mixture of iodopropane and titanium citrate caused a light-reversible inhibition of the dechlorinating activities, suggesting the involvement of a corrinoid cofactor. Several polychlorophenols were dechlorinated at the meta and para positions. The apparent K(m) for 3,5-dicholorophenol was 49.3 +/- 3.1 microM at a methyl viologen concentration of 2 mM. Six internal tryptic peptides were sequenced by mass spectrometry. One open reading frame (ORF) was found in the Desulfitobacterium hafniense genome containing these peptide sequences. This ORF corresponds to a gene coding for a CprA-type reductive dehalogenase. The corresponding ORF (named cprA5) in D. frappieri PCP-1 was cloned and sequenced. The cprA5 gene codes for a 548-amino-acid protein that contains a twin-arginine-type signal for secretion. The gene product has a cobalamin binding site motif and two iron-sulfur binding motifs and shows 66% identity (76 to 77% similarity) with some tetrachloroethene reductive dehalogenases. This is the first CprA-type reductive dehalogenase that can dechlorinate chlorophenols at the meta and para positions.
Thibodeau
Jacinthe
J
Institut National de la Recherche Scientifique, INRS-Institut Armand-Frappier, Université du Québec, 531 boul. des Prairies, Laval, QC H7V 1B7, Canada.
Gauthier
Annie
A
Duguay
Marie
M
Villemur
Richard
R
Lépine
François
F
Juteau
Pierre
P
Beaudet
Réjean
R
D000595:Amino Acid Sequence; D002733:Chlorophenols / Q000378:metabolism*; D003001:Cloning, Molecular; D045858:Desulfitobacterium / Q000201:enzymology* / Q000254:growth & development; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
D002733:Chlorophenols; D010088:Oxidoreductases; C116190:3,5-dichlorophenol