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Applied and Environmental Microbiology, August 2003, p. 4628-4638, Vol. 69, No. 8
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.8.4628-4638.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Characterization of the Corrinoid Iron-Sulfur Protein Tetrachloroethene Reductive Dehalogenase of Dehalobacter restrictus

Julien Maillard,1 Wolfram Schumacher,2,{dagger} Francisco Vazquez,2 Christophe Regeard,1 Wilfred R. Hagen,3,{ddagger} and Christof Holliger1*

ENAC-Laboratory for Environmental Biotechnology, Swiss Federal Institute of Technology (EPFL) CH-1015 Lausanne,1 Limnological Research Center, Swiss Federal Institute for Environmental Science and Technology (EAWAG), CH-6047 Kastanienbaum,Switzerland,2 Department of Biochemistry, Wageningen Agricultural University, NL-6703 HA Wageningen, The Netherlands3

Received 4 February 2003/ Accepted 7 May 2003

The membrane-bound tetrachloroethene reductive dehalogenase (PCE-RDase) (PceA; EC 1.97.1.8), the terminal component of the respiratory chain of Dehalobacter restrictus, 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-ß-D-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 cis-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 Km 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 Dehalobacter restrictus, Desulfitobacterium hafniense strain TCE1 (formerly Desulfitobacterium frappieri strain TCE1), and Desulfitobacterium hafniense strain PCE-S (formerly Desulfitobacterium frappieri strain PCE-S), the pceA genes of the first two organisms were cloned and sequenced. Together with the pceA genes of Desulfitobacterium hafniense strains PCE-S and Y51, the pceA genes of Desulfitobacterium hafniense strain TCE1 and Dehalobacter restrictus form a coherent group of reductive dehalogenases with almost 100% sequence identity. Also, the pceB genes, which may code for a membrane anchor protein of PceA, and the intergenic regions of Dehalobacter restrictus and the three desulfitobacteria had identical sequences. Whereas the cprB (chlorophenol reductive dehalogenase) genes of chlorophenol-dehalorespiring bacteria are always located upstream of cprA, all pceB genes known so far are located downstream of pceA. 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 Dehalobacter restrictus and Desulfitobacterium dehalogenans identified by electron paramagnetic resonance spectroscopy.

* Corresponding author. Mailing address: Swiss Federal Institute of Technology (EPFL), ENAC-Laboratory for Environmental Biotechnology, BÂtiment CH-B Ecublens, CH-1015 Lausanne, Switzerland. Phone: 41-21-6934724. Fax: 41-21-6934722. E-mail: christof.holliger{at}epfl.ch.

{dagger} Present address: Diagnostics GmbH, D-82377 Penzberg, Germany.

{ddagger} Present address: Kluyver Department of Biotechnology, Delft University of Technology, NL-2628 BC Delft, The Netherlands.

Applied and Environmental Microbiology, August 2003, p. 4628-4638, Vol. 69, No. 8
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.8.4628-4638.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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