Properties and sequence of the coenzyme B12-dependent glycerol dehydratase of Clostridium pasteurianum

Macis, Luciana

doi:10.1016/s0378-1097(98)00186-4

Cited by 32 publications

(64 citation statements)

References 18 publications

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“…To cover the different annealing temperatures of the degenerated primer pair, 10 PCRs per sample were performed in a gradient cycler. For calibration of the PCRs, the genes encoding the dehydratases of C. freundii (41) and Clostridium pasteurianum (28,29) were used as positive controls. The reactions were performed by using the isolated recombinant plasmids pMS2 and pFL1, respectively, as sources for the dehydratase genes of these organisms.…”

Section: Methodsmentioning

confidence: 99%

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Identification and Characterization of Coenzyme B ₁₂ -Dependent Glycerol Dehydratase- and Diol Dehydratase-Encoding Genes from Metagenomic DNA Libraries Derived from Enrichment Cultures

Knietsch¹,

Bowien²,

Whited³

et al. 2003

Appl Environ Microbiol

134

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To isolate genes encoding coenzyme B 12 -dependent glycerol and diol dehydratases, metagenomic libraries from three different environmental samples were constructed after allowing growth of the dehydratasecontaining microorganisms present for 48 h with glycerol under anaerobic conditions. The libraries were searched for the targeted genes by an activity screen, which was based on complementation of a constructed dehydratase-negative Escherichia coli strain. In this way, two positive E. coli clones out of 560,000 tested clones were obtained. In addition, screening was performed by colony hybridization with dehydratase-specific DNA fragments as probes. The screening of 158,000 E. coli clones by this method yielded five positive clones. Two of the plasmids (pAK6 and pAK8) recovered from the seven positive clones contained genes identical to those encoding the glycerol dehydratase of Citrobacter freundii and were not studied further. The remaining five plasmids (pAK2 to -5 and pAK7) contained two complete and three incomplete dehydratase-encoding gene regions, which were similar to the corresponding regions of enteric bacteria. Three (pAK2, -3, and -7) coded for glycerol dehydratases and two (pAK4 and -5) coded for diol dehydratases. We were able to perform high-level production and purification of three of these dehydratases. The glycerol dehydratases purified from E. coli Bl21/pAK2.1 and E. coli Bl21/pAK7.1 and the complemented hybrid diol dehydratase purified from E. coli Bl21/pAK5.1 were subject to suicide inactivation by glycerol and were cross-reactivated by the reactivation factor (DhaFG) for the glycerol dehydratase of C. freundii. The activities of the three environmentally derived dehydratases and that of glycerol dehydratase of C. freundii with glycerol or 1,2-propanediol as the substrate were inhibited in the presence of the glycerol fermentation product 1,3-propanediol. Taking the catalytic efficiency, stability against inactivation by glycerol, and inhibition by 1,3-propanediol into account, the hybrid diol dehydratase produced by E. coli Bl21/pAK5.1 exhibited the best properties of all tested enzymes for application in the biotechnological production of 1,3-propanediol.

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Section: Methodsmentioning

confidence: 99%

“…Conserved regions of coenzyme B 12 -dependent glycerol dehydratases (GDHs) and diol dehydratases (DDHs) selected for generation of degenerate oligonucleotides. Sequences from C. freundii (41); Clostridium pasteurianum (29); K. pneumoniae (44); K. oxytoca (45); and S. enterica serovar Typhimurium (6) are shown.…”

Section: Methodsmentioning

confidence: 99%

Identification and Characterization of Coenzyme B ₁₂ -Dependent Glycerol Dehydratase- and Diol Dehydratase-Encoding Genes from Metagenomic DNA Libraries Derived from Enrichment Cultures

Knietsch¹,

Bowien²,

Whited³

et al. 2003

Appl Environ Microbiol

134

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“…The reason is that the gene for the subunit B of the coenzyme B 12 -dependent glycerol dehydratase complex is missing. In this respect it is of interest that glycerol dehydratase activity was detected in C. pasteurianum and Clostridium butyricum (48). All genes for the synthesis of coenzyme B 12 , the cofactor of glycerol dehydratase, can be found in the genome of C. kluyveri.…”

Section: Ethanol Dehydrogenases and Acetaldehyde Dehydrogenases In Amentioning

confidence: 99%

The genome ofClostridium kluyveri, a strict anaerobe with unique metabolic features

Seedorf

Fricke

Veith

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

419

386

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Clostridium kluyveri is unique among the clostridia; it grows anaerobically on ethanol and acetate as sole energy sources. Fermentation products are butyrate, caproate, and H2. We report here the genome sequence of C. kluyveri, which revealed new insights into the metabolic capabilities of this well studied organism. A membrane-bound energy-converting NADH:ferredoxin oxidoreductase (RnfCDGEAB) and a cytoplasmic butyryl-CoA dehydrogenase complex (Bcd/EtfAB) coupling the reduction of crotonyl-CoA to butyryl-CoA with the reduction of ferredoxin represent a new energy-conserving module in anaerobes. The genes for NAD-dependent ethanol dehydrogenase and NAD(P)-dependent acetaldehyde dehydrogenase are located next to genes for microcompartment proteins, suggesting that the two enzymes, which are isolated together in a macromolecular complex, form a carboxysome-like structure. Unique for a strict anaerobe, C. kluyveri harbors three sets of genes predicted to encode for polyketide/nonribosomal peptide synthetase hybrides and one set for a nonribosomal peptide synthetase. The latter is predicted to catalyze the synthesis of a new siderophore, which is formed under iron-deficient growth conditions. butyryl-CoA dehydrogenase ͉ electron transfer flavoproteins ͉ genome sequence ͉ Rnf-dependent energy conservation

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“…14,15,23) A comparison of the amino acid sequences of the corresponding subunits between diol and glycerol dehydratases 20,[23][24][25][26][27] showed high identities between them, except that the N-terminal 32 and 37 amino acid residues of the and subunits, respectively, of diol dehydratase are lacking in the corresponding subunits of glycerol dehydratase. Hence it was suggested that these regions might determine the solubility of these enzymes, although the hydropathy plots of diol dehydratase do not show any prominent hydrophobic peaks in these missing regions.…”

mentioning

confidence: 99%

The N-Terminal Regions of β and γ Subunits Lower the Solubility of Adenosylcobalamin-Dependent Diol Dehydratase

Tobimatsu

Kawata

Toraya

2005

Bioscience, Biotechnology, and Biochemistry

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Adenosylcobalamin-dependent diol dehydratase is one of essential components of carboxysome-like polyhedral bodies. It exists as a heterohexamer ðÞ 2 , and its activity is recovered in a precipitant fraction of Klebsiella oxytoca and overexpressing Escherichia coli cells. Limited proteolysis of the enzyme with trypsin converted the enzyme into a highly soluble form without loss of enzyme activity. The N-terminal amino acid sequencing of the enzyme thus solubilized indicated that the N-terminal 20 and 16 amino acid residues had been removed from the and subunits, respectively. Mutant enzymes with the same N-terminal truncations of either or both of the and subunits were expressed on a high level in E. coli cells. All the mutant enzymes obtained were expressed in a soluble, active form. These results indicate that the N-terminal regions of the and subunits lower the solubility of diol dehydratase. The mutant enzyme with the N-terminal truncations of both and subunits was essentially indistinguishable in catalytic properties from recombinant wild-type enzyme or the enzyme purified from K. oxytoca in a soluble form.

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Properties and sequence of the coenzyme B12-dependent glycerol dehydratase of Clostridium pasteurianum

Cited by 32 publications

References 18 publications

Identification and Characterization of Coenzyme B ₁₂ -Dependent Glycerol Dehydratase- and Diol Dehydratase-Encoding Genes from Metagenomic DNA Libraries Derived from Enrichment Cultures

Identification and Characterization of Coenzyme B ₁₂ -Dependent Glycerol Dehydratase- and Diol Dehydratase-Encoding Genes from Metagenomic DNA Libraries Derived from Enrichment Cultures

The genome ofClostridium kluyveri, a strict anaerobe with unique metabolic features

The N-Terminal Regions of β and γ Subunits Lower the Solubility of Adenosylcobalamin-Dependent Diol Dehydratase

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Properties and sequence of the coenzyme B12-dependent glycerol dehydratase of Clostridium pasteurianum

Cited by 32 publications

References 18 publications

Identification and Characterization of Coenzyme B 12 -Dependent Glycerol Dehydratase- and Diol Dehydratase-Encoding Genes from Metagenomic DNA Libraries Derived from Enrichment Cultures

Identification and Characterization of Coenzyme B 12 -Dependent Glycerol Dehydratase- and Diol Dehydratase-Encoding Genes from Metagenomic DNA Libraries Derived from Enrichment Cultures

The genome ofClostridium kluyveri, a strict anaerobe with unique metabolic features

The N-Terminal Regions of β and γ Subunits Lower the Solubility of Adenosylcobalamin-Dependent Diol Dehydratase

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Product

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Identification and Characterization of Coenzyme B ₁₂ -Dependent Glycerol Dehydratase- and Diol Dehydratase-Encoding Genes from Metagenomic DNA Libraries Derived from Enrichment Cultures

Identification and Characterization of Coenzyme B ₁₂ -Dependent Glycerol Dehydratase- and Diol Dehydratase-Encoding Genes from Metagenomic DNA Libraries Derived from Enrichment Cultures