Purification of 1,3-propanediol dehydrogenase from Citrobacter freundii and cloning, sequencing, and overexpression of the corresponding gene in Escherichia coli.
1,3-Propanediol dehydrogenase (EC 1.1.1.202) was purified to homogeneity from Citrobacter freundii grown anaerobically on glycerol in continuous culture. The enzyme is an octamer of a polypeptide of 43,400 Da. When tested as a dehydrogenase, the enzyme was most active with substrates containing two primary alcohol groups separated by one or two carbon atoms. In the physiological direction, 3-hydroxypropionaldehyde was the preferred substrate. The apparent Km values of the enzyme for 3-hydroxypropionaldehyde and NADH were 140 and 33 microM, respectively. The enzyme was inhibited by chelators of divalent cations but could be reactivated by the addition of Fe2+. The dhaT gene, encoding the 1,3-propanediol dehydrogenase, was cloned, and its nucleotide sequence (1,164 bp) was determined. The deduced dhaT gene product (387 amino acids, 41,324 Da) showed a high level of similarity to a novel family (type III) of alcohol dehydrogenases. The dhaT gene was overexpressed in Escherichia coli 274-fold by using the T7 RNA polymerase/promoter system.
Daniel
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Institut für Mikrobiologie, Georg-August-Universität Göttingen, Germany.
Boenigk
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Gottschalk
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D000426:Alcohol Dehydrogenase; D000429:Alcohol Oxidoreductases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D002413:Cations, Divalent / Q000494:pharmacology; D016971:Citrobacter freundii / Q000201:enzymology* / Q000235:genetics*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D002413:Cations, Divalent; D004269:DNA, Bacterial; D000429:Alcohol Oxidoreductases; D000426:Alcohol Dehydrogenase; C052370:1,3-propanediol dehydrogenase