Tetrachloroethene reductive dehalogenase of Dehalospirillum multivorans : substrate specificity of the native enzyme and its corrinoid cofactor

Neumann, Anke; Siebert, Anke; Trescher, Tina; Reinhardt, Simone; Wohlfarth, Gert; Diekert, Gabriele

doi:10.1007/s00203-002-0409-3

Cited by 98 publications

(78 citation statements)

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“…Cultivation of Dehalobacter restrictus depends on the presence of cobalamin in the medium (14), and growth on cobinamide-containing medium ceased after a few transfers (data not shown). All these results indicated that the corrinoid present in PCE-RDase is a cobalamin, which is in contrast to the recent findings obtained with Dehalospirillum multivorans; for the latter organism it has been shown that the corrinoid isolated from the PCE-RDase had different catalytic dechlorination properties than commercially available cobalamin (24). The cobalamin content determined spectrophotometrically by measuring the difference between A 580 and A 640 was 0.97 Ϯ 0.07 mol of cobalamin/mol of enzyme (Fig.…”

Section: Resultsmentioning

confidence: 56%

“…Similar experiments with PCE-RDase of Dehalobacter restrictus and increasing amounts of the D˙donor d 7 -isopropyl alcohol providing evidence for a radical mechanism with free cobalamin did not indicate that a dissociative one-electron transfer is involved in the cobalamin enzyme-catalyzed reaction (data not shown). The dechlorination of trans-1,3-dichloropropene to a mixture of trans-1-chloropropene, cis-1-chloropropene, and 3-chloropropene by the PCE-RDases of Dehalospirillum multivorans and Desulfitobacterium hafniense strain PCE-S, on the other hand, is in accordance with a dechlorination mechanism involving a radical intermediate (24). Rapid freezing experiments combined with EPR analysis might provide additional indications of the reaction mechanism involved.…”

Section: Resultsmentioning

confidence: 86%

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Characterization of the Corrinoid Iron-Sulfur ProteinTetrachloroethene Reductive Dehalogenase of Dehalobacterrestrictus

Maillard

Schumacher

Vazquez

et al. 2003

Appl Environ Microbiol

149

155

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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-␤-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 K m values for tetrachloroethene, trichloroethene, and methyl viologen were 20.4 ؎ 3.2, 23.7 ؎ 5.2, and 47 ؎ 10 M, 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.Tetrachloroethene (PCE), a frequently detected groundwater contaminant, is used by different bacteria as a terminal electron acceptor in a process called dehalorespiration (10,14,16,20,21,32,35,40,46). Six such isolates belong phylogenetically to the subphylum Firmicutes of the gram-positive bacteria, whereas other isolates are affiliated with phylogenetic groups such as the ␦ and ε subclasses of the Proteobacteria and the green no...

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Section: Resultsmentioning

confidence: 56%

Section: Resultsmentioning

confidence: 86%

Characterization of the Corrinoid Iron-Sulfur ProteinTetrachloroethene Reductive Dehalogenase of Dehalobacterrestrictus

Maillard

Schumacher

Vazquez

et al. 2003

Appl Environ Microbiol

149

155

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“…Propyl iodide (50 M) completely inhibited dechlorination in the dark; the inhibition could be reversed by illumination. These findings provide strong evidence for the involvement of a corrinoid in the dichloroelimination reaction (19,20). In vivo studies with three different 2,3-DCB stereoisomers as electron acceptors were performed (Fig.…”

Section: Resultsmentioning

confidence: 67%

Stereoselective Microbial Dehalorespiration with Vicinal Dichlorinated Alkanes

Wildeman

Diekert

Langenhove

et al. 2003

Appl Environ Microbiol

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The suspected carcinogen 1,2-dichloroethane (1,2-DCA) is the most abundant chlorinated C 2 groundwater pollutant on earth. However, a reductive in situ detoxification technology for this compound does not exist. Although anaerobic dehalorespiring bacteria are known to catalyze several dechlorination steps in the reductive-degradation pathway of chlorinated ethenes and ethanes, no appropriate isolates that selectively and metabolically convert them into completely dechlorinated end products in defined growth media have been reported. Here we report on the isolation of Desulfitobacterium dichloroeliminans strain DCA1, a nutritionally defined anaerobic dehalorespiring bacterium that selectively converts 1,2-dichloroethane and all possible vicinal dichloropropanes and -butanes into completely dechlorinated end products. Menaquinone was identified as an essential cofactor for growth of strain DCA1 in pure culture. Strain DCA1 converts chiral chlorosubstrates, revealing the presence of a stereoselective dehalogenase that exclusively catalyzes an energyconserving anti mechanistic dichloroelimination. Unlike any known dehalorespiring isolate, strain DCA1 does not carry out reductive hydrogenolysis reactions but rather exclusively dichloroeliminates its substrates. This unique dehalorespiratory biochemistry has shown promising application possibilities for bioremediation purposes and fine-chemical synthesis.

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“…The positions of the respective excised protein bands were determined by the method as described by Adrian et al (2007). Gel fragments were used for in-gel activity assays and the dechlorination activity of PCE was conducted as previously described (Neumann et al, 2007). The protein band identified to show dechlorination activity was eluted and concentrated for a second gel electrophoresis step (Adrian et al, 2007).…”

Section: Genome Walking Of the Rdase Genesmentioning

confidence: 99%

Identification and transcriptional analysis of trans-DCE-producing reductive dehalogenases in Dehalococcoides species

et al. 2010

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During microbial reductive dechlorination of tetrachloroethene (PCE) and trichloroethene (TCE), trans-1, 2-dichloroethene (trans-DCE) has been observed to be produced predominantly by certain mixed and pure cultures. However, the reductive dehalogenase (RDase) genes involved in trans-DCE generation remain elusive. In this study, identification and transcriptional analysis of RDases were conducted on trans-DCE-producing Dehalococcoides sp. strain MB. Two pairs of degenerate primers targeting the conserved regions of RDases in known Dehalococcoides species were applied to amplify the putative RDase genes of strain MB. Cloning and restriction analysis revealed the presence of seven unique RDase gene fragments (dceA1 to dceA7) that possess sequence identity to known RDase genes. Gene expression analysis of the PCE-grown culture MB exhibited 10-fold regulation of the RDase gene dceA6 (designated mbrA gene), suggesting that it is involved in the production of trans-DCE. This is in agreement with the molecular size of the most abundant protein that is resolved on the denaturing protein gel. Complete sequence of the mbrA gene was obtained by chromosome walking, and the upstream of it is a regulator of transcription, indicating that the expression of this functional gene is tightly controlled in the microbe. The mbrA gene was subsequently found to be present in other trans-DCE-producing cultures containing Dehalococcoides sp. The new mbrA gene identified in this study may serve as an important biomarker for evaluating, predicting and elucidating the biological production of trans-DCE in the chloroethene-contaminated sites.

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Tetrachloroethene reductive dehalogenase of Dehalospirillum multivorans : substrate specificity of the native enzyme and its corrinoid cofactor

Cited by 98 publications

References 0 publications

Characterization of the Corrinoid Iron-Sulfur ProteinTetrachloroethene Reductive Dehalogenase of Dehalobacterrestrictus

Characterization of the Corrinoid Iron-Sulfur ProteinTetrachloroethene Reductive Dehalogenase of Dehalobacterrestrictus

Stereoselective Microbial Dehalorespiration with Vicinal Dichlorinated Alkanes

Identification and transcriptional analysis of trans-DCE-producing reductive dehalogenases in Dehalococcoides species

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