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Purification, cloning and sequencing of an enzyme mediating the reductive dechlorination of 2,4,6-trichlorophenol from Desulfitobacterium frappieri PCP-1.

ABSTRACT: 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 K(m) value for 2,4,6-trichlorophenol and pentachlorophenol were 18.3+/-2.8 microM and 26.8+/-2.9 microM 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.

SUBMITTER: Boyer A

PROVIDER: S-EPMC1223485 | biostudies-other | 2003 Jul

REPOSITORIES: biostudies-other

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Project description:Dehalococcoides mccartyi strains are corrinoid-auxotrophic Bacteria and axenic cultures that require vitamin B12 (CN-Cbl) to conserve energy via organohalide respiration. Cultures of D. mccartyi strains BAV1, GT and FL2 grown with limiting amounts of 1 µg l(-1) CN-Cbl quickly depleted CN-Cbl, and reductive dechlorination of polychlorinated ethenes was incomplete leading to vinyl chloride (VC) accumulation. In contrast, the same cultures amended with 25 µg l(-1) CN-Cbl exhibited up to 2.3-fold higher dechlorination rates, 2.8-9.1-fold increased growth yields, and completely consumed growth-supporting chlorinated ethenes. To explore whether known cobamide-producing microbes supply Dehalococcoides with the required corrinoid cofactor, co-culture experiments were performed with the methanogen Methanosarcina barkeri strain Fusaro and two acetogens, Sporomusa ovata and Sporomusa sp. strain KB-1, as Dehalococcoides partner populations. During growth with H2/CO2, M. barkeri axenic cultures produced 4.2 ± 0.1 µg l(-1) extracellular cobamide (factor III), whereas the Sporomusa cultures produced phenolyl- and p-cresolyl-cobamides. Neither factor III nor the phenolic cobamides supported Dehalococcoides reductive dechlorination activity suggesting that M. barkeri and the Sporomusa sp. cannot fulfil Dehalococcoides' nutritional requirements. Dehalococcoides dechlorination activity and growth occurred in M. barkeri and Sporomusa sp. co-cultures amended with 10 µM 5',6'-dimethylbenzimidazole (DMB), indicating that a cobalamin is a preferred corrinoid cofactor of strains BAV1, GT and FL2 when grown with chlorinated ethenes as electron acceptors. Even though the methanogen and acetogen populations tested did not produce cobalamin, the addition of DMB enabled guided biosynthesis and generated a cobalamin that supported Dehalococcoides' activity and growth. Guided cobalamin biosynthesis may offer opportunities to sustain and enhance Dehalococcoides activity in contaminated subsurface environments.

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Purification, cloning and sequencing of an enzyme mediating the reductive dechlorination of 2,4,6-trichlorophenol from Desulfitobacterium frappieri PCP-1.

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