Characterization of the Corrinoid Iron-Sulfur Protein
Tetrachloroethene Reductive Dehalogenase of
Dehalobacter
restrictus
- Maillard, Julien ENAC-Laboratory for Environmental Biotechnology, Swiss Federal Institute of Technology (EPFL) CH-1015 Lausanne
- Schumacher, Wolfram Limnological Research Center, Swiss Federal Institute for Environmental Science and Technology (EAWAG), CH-6047 Kastanienbaum,Switzerland
- Vazquez, Francisco Limnological Research Center, Swiss Federal Institute for Environmental Science and Technology (EAWAG), CH-6047 Kastanienbaum,Switzerland
- Regeard, Christophe ENAC-Laboratory for Environmental Biotechnology, Swiss Federal Institute of Technology (EPFL) CH-1015 Lausanne
- Hagen, Wilfred R. Department of Biochemistry, Wageningen Agricultural University, NL-6703 HA Wageningen, The Netherlands
- Holliger, Christof ENAC-Laboratory for Environmental Biotechnology, Swiss Federal Institute of Technology (EPFL) CH-1015 Lausanne
Published in:
- Applied and Environmental Microbiology. - American Society for Microbiology. - 2003, vol. 69, no. 8, p. 4628-4638
English
ABSTRACT
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-β-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. TheKm
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 ofDehalobacter restrictus , Desulfitobacterium
hafniense strain TCE1 (formerly Desulfitobacterium
frappieri strain TCE1), and Desulfitobacterium hafniense
strain PCE-S (formerlyDesulfitobacterium frappieri strain
PCE-S), thepceA genes of the first two organisms were cloned
and sequenced. Together with thepceA genes of
Desulfitobacterium hafniense strains PCE-S and Y51, the
pceA genes of Desulfitobacterium hafniense
strain TCE1 andDehalobacter restrictus form a coherent group
of reductive dehalogenases with almost 100% sequence identity.
Also, thepceB genes, which may code for a membrane anchor
protein of PceA, and the intergenic regions ofDehalobacter
restrictus and the three desulfitobacteria had identical
sequences. Whereas thecprB (chlorophenol reductive
dehalogenase) genes of chlorophenol-dehalorespiring bacteria are always
located upstream ofcprA , all pceB genes known so far
are located downstream ofpceA. 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 ofDehalobacter restrictus and
Desulfitobacterium dehalogenans identified by electron
paramagnetic resonance
spectroscopy.
membrane-bound tetrachloroethene reductive dehalogenase (PCE-RDase)
(PceA; EC 1.97.1.8), the terminal component of the respiratory chain of
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-
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
250 ± 12 nkat/mg of protein. In addition, several
chloroethanes and tetrachloromethane caused methyl viologen oxidation
in the presence of PceA. The
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
hafniense
frappieri
strain PCE-S (formerly
PCE-S), the
and sequenced. Together with the
strain TCE1 and
of reductive dehalogenases with almost 100% sequence identity.
Also, the
protein of PceA, and the intergenic regions of
restrictus
sequences. Whereas the
dehalogenase) genes of chlorophenol-dehalorespiring bacteria are always
located upstream of
are located downstream 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
paramagnetic resonance
spectroscopy.
- Language
-
- English
- Open access status
- bronze
- Identifiers
-
- DOI 10.1128/aem.69.8.4628-4638.2003
- ISSN 0099-2240
- Persistent URL
- https://sonar.ch/global/documents/96249
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