Overproduction and characterization of recombinant UDP-glucose pyrophosphorylase from Escherichia coli K-12.
Using oligonucleotide probes synthesized on the basis of partial amino acid sequences, we have cloned and sequenced the gene of Escherichia coli K-12 encoding UDP-glucose pyrophosphorylase. The gene consists of 906 base pairs and encodes a polypeptide of 302 amino acid residues with a calculated molecular weight of 32,941. Its nucleotide sequence was found to be identical with that recently registered (EMBL, X59940) for a gene coding for an unknown 33-kDa protein, which was later annotated as UDP-glucose pyrophosphorylase on the basis of genetic studies. The UDP-glucose pyrophosphorylase gene, mapped at 27.3 min in the E. coli chromosome, complemented the galU mutation, which renders the bacterium unable to ferment galactose. The recombinant enzyme overproduced in E. coli cells and purified to homogeneity catalyzed the synthesis and pyrophosphorolysis of UDP-glucose by a sequential mechanism. The enzyme required Mg2+ for maximal activity and was inhibited by free UTP and pyrophosphate. The E. coli enzyme shows significant sequence similarities with the enzymes from Acetobacter xylinum and Salmonella typhimurium. However, little or no similarity was found with the eukaryotic enzymes that are involved in the biosynthesis of storage carbohydrates, or with other enzymes acting on similar sugar nucleotides. Thus, UDP-glucose pyrophosphorylases participating in diverse metabolic pathways can be classified structurally into the prokaryotic and eukaryotic groups, even though they have almost identical catalytic properties.
Institute of Scientific and Industrial Research, Osaka University.
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SA
Hossain
K
K
Tanizawa
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Y
Kazuta
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T
Fukui
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology*; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D009154:Mutation; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis; D017386:Sequence Homology, Amino Acid; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase / Q000032:analysis / Q000096:biosynthesis*
D011994:Recombinant Proteins; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase