Biochemical and molecular characterization of the oxidative branch of glycerol utilization by Citrobacter freundii.
Glycerol dehydrogenase (EC 1.1.1.6) and dihydroxyacetone kinase (EC 2.7.1.29) were purified from Citrobacter freundii. The dehydrogenase is a hexamer of a polypeptide of 43,000 Da. The enzyme exhibited a rather broad substrate specificity, but glycerol was the preferred substrate in the physiological direction. The apparent Kms of the enzyme for glycerol and NAD+ were 1.27 mM and 57 microM, respectively. The kinase is a dimer of a polypeptide of 57,000 Da. The enzyme was highly specific for the substrates dihydroxyacetone and ATP; the apparent Kms were 30 and 70 microM, respectively. The DNA region which contained the genes encoding glycerol dehydrogenase (dhaD) and dihydroxyacetone kinase (dhaK) was cloned and sequenced. Both genes were identified by N-terminal sequence comparison. The deduced dhaD gene product (365 amino acids) exhibited high degrees of homology to glycerol dehydrogenases from other organisms and less homology to type III alcohol dehydrogenases, whereas the dhaK gene product (552 amino acids) revealed no significant homology to any other protein in the databases. A large gene (dhaR) of 1,929 bp was found downstream from dhaD. The deduced gene product (641 amino acids) showed significant similarities to members of the sigma 54 bacterial enhancer-binding protein family.
Daniel
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Institut für Mikrobiologie, Georg-August-Universität Göttingen, Germany.
Stuertz
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Gottschalk
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D000595:Amino Acid Sequence; D001483:Base Sequence; D002413:Cations, Divalent / Q000378:metabolism; D016971:Citrobacter freundii / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D017124:Conserved Sequence; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics; D004789:Enzyme Activation; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D005990:Glycerol / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013401:Sugar Alcohol Dehydrogenases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D015534:Trans-Activators / Q000096:biosynthesis / Q000235:genetics
D002413:Cations, Divalent; D004268:DNA-Binding Proteins; C095090:DhaR protein, E coli; D029968:Escherichia coli Proteins; D015534:Trans-Activators; D013401:Sugar Alcohol Dehydrogenases; C020672:glycerol dehydrogenase; D017853:Phosphotransferases (Alcohol Group Acceptor); C023132:glycerone kinase; D005990:Glycerol