Maillard, Julien Schumacher, Wolfram Vazquez, Francisco Regeard, Christophe Hagen, Wilfred R. Holliger, Christof <jats:title>ABSTRACT</jats:title> <jats:p>The membrane-bound tetrachloroethene reductive dehalogenase (PCE-RDase) (PceA; EC 1.97.1.8), the terminal component of the respiratory chain of <jats:italic>Dehalobacter restrictus</jats:italic>, was purified 25-fold to apparent electrophoretic homogeneity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single band with an apparent molecular mass of 60 ± 1 kDa, whereas the native molecular mass was 71± 8 kDa according to size exclusion chromatography in the presence of the detergent octyl-β-<jats:sc>d</jats:sc>-glucopyranoside. The monomeric enzyme contained (per mol of the 60-kDa subunit) 1.0± 0.1 mol of cobalamin, 0.6 ± 0.02 mol of cobalt, 7.1± 0.6 mol of iron, and 5.8 ± 0.5 mol of acid-labile sulfur. Purified PceA catalyzed the reductive dechlorination of tetrachloroethene and trichloroethene to <jats:italic>cis</jats:italic>-1,2-dichloroethene with a specific activity of 250 ± 12 nkat/mg of protein. In addition, several chloroethanes and tetrachloromethane caused methyl viologen oxidation in the presence of PceA. The <jats:italic>K<jats:sub>m</jats:sub> </jats:italic> values for tetrachloroethene, trichloroethene, and methyl viologen were 20.4± 3.2, 23.7 ± 5.2, and 47 ± 10 μM, respectively. The PceA exhibited the highest activity at pH 8.1 and was oxygen sensitive, with a half-life of activity of 280 min upon exposure to air. Based on the almost identical N-terminal amino acid sequences of PceA of <jats:italic>Dehalobacter restrictus</jats:italic>, <jats:italic>Desulfitobacterium hafniense</jats:italic> strain TCE1 (formerly <jats:italic>Desulfitobacterium frappieri</jats:italic> strain TCE1), and <jats:italic>Desulfitobacterium hafniense</jats:italic> strain PCE-S (formerly <jats:italic>Desulfitobacterium frappieri</jats:italic> strain PCE-S), the <jats:italic>pceA</jats:italic> genes of the first two organisms were cloned and sequenced. Together with the <jats:italic>pceA</jats:italic> genes of <jats:italic>Desulfitobacterium hafniense</jats:italic> strains PCE-S and Y51, the <jats:italic>pceA</jats:italic> genes of <jats:italic>Desulfitobacterium hafniense</jats:italic> strain TCE1 and <jats:italic>Dehalobacter restrictus</jats:italic> form a coherent group of reductive dehalogenases with almost 100% sequence identity. Also, the <jats:italic>pceB</jats:italic> genes, which may code for a membrane anchor protein of PceA, and the intergenic regions of <jats:italic>Dehalobacter restrictus</jats:italic> and the three desulfitobacteria had identical sequences. Whereas the <jats:italic>cprB</jats:italic> (chlorophenol reductive dehalogenase) genes of chlorophenol-dehalorespiring bacteria are always located upstream of <jats:italic>cprA</jats:italic>, all <jats:italic>pceB</jats:italic> genes known so far are located downstream of <jats:italic>pceA.</jats:italic> The possible consequences of this feature for the annotation of putative reductive dehalogenase genes are discussed, as are the sequence around the iron-sulfur cluster binding motifs and the type of iron-sulfur clusters of the reductive dehalogenases of <jats:italic>Dehalobacter restrictus</jats:italic> and <jats:italic>Desulfitobacterium dehalogenans</jats:italic> identified by electron paramagnetic resonance spectroscopy.</jats:p> application/pdf https://sonar.ch/global/documents/96249 eng info:eu-repo/semantics/altIdentifier/doi/10.1128/aem.69.8.4628-4638.2003 info:eu-repo/semantics/altIdentifier/issn/0099-2240 info:eu-repo/semantics/openAccess Applied and Environmental Microbiology. - American Society for Microbiology. - 2003, vol. 69, no. 8, p. 4628-4638 Biotechnology Food Science Ecology Applied Microbiology and Biotechnology Characterization of the Corrinoid Iron-Sulfur Protein Tetrachloroethene Reductive Dehalogenase of Dehalobacter restrictus http://purl.org/coar/resource_type/c_6501