A new membrane-associated 2,4,6-trichlorophenol reductive dehalogenase from Desulfitobacterium frappieri PCP-1 was isolated. Initial characterization of the crude preparation showed that the dechlorinating activity was sensitive to oxygen, and its optimum pH was 7.0. Its dechlorinating activity was not inhibited by sulphate, was completely inhibited by 1 mM sulphite, and partially inhibited by 5 mM sodium azide and by more than 5 mM nitrate. Several polychlorophenols were dechlorinated in the ortho position with respect to the hydroxy group. A dehalogenase was purified to apparent homogeneity. SDS gel electrophoresis revealed a single protein band with a molecular mass of 37 kDa. However, after two-dimensional gel electrophoresis, this band was composed of three isoforms. MS analyses showed that the three isoforms were from the same protein and the molecular mass of the most abundant isoform is 33800 Da. A mixture of iodopropane and titanium citrate caused a light-reversible inhibition of the dechlorinating activity, suggesting the involvement of a corrinoid cofactor. The apparent Km value for 2,4,6-trichlorophenol and pentachlorophenol were 18.3±2.8 μM and 26.8±2.9 μM respectively, at a methyl viologen concentration of 2 mM. The N-terminal amino acid sequence and an internal tryptic peptide sequence were determined. One open reading frame (ORF) was found in the Desulfitobacterium hafniense genome containing these peptides sequences. The corresponding ORF in D. frappieri PCP-1 was cloned and sequenced. This ORF, that we designated crdA, showed no homology with any known dehalogenase, suggesting a distinct reductive dehalogenase.
Abbreviations used: DTT, dithiothreitol; HIC, hydrophobic interaction chromatography; JGI, Joint Genome Institute; ORF, open reading frame; PCE, tetrachloroethene; PCP, pentachlorophenol; TCE, trichloroethene.
The nucleotide sequence data reported will appear in DDBJ, EMBL, GenBank® and GSDB Nucleotide Sequence Databases under the accession number AY043467.