Cloning, sequencing, and expression of the Zymomonas mobilis phosphoglycerate mutase gene (pgm) in Escherichia coli.
Phosphoglycerate mutase is an essential glycolytic enzyme for Zymomonas mobilis, catalyzing the reversible interconversion of 3-phosphoglycerate and 2-phosphoglycerate. The pgm gene encoding this enzyme was cloned on a 5.2-kbp DNA fragment and expressed in Escherichia coli. Recombinants were identified by using antibodies directed against purified Z. mobilis phosphoglycerate mutase. The pgm gene contains a canonical ribosome-binding site, a biased pattern of codon usage, a long upstream untranslated region, and four promoters which share sequence homology. Interestingly, adhA and a D-specific 2-hydroxyacid dehydrogenase were found on the same DNA fragment and appear to form a cluster of genes which function in central metabolism. The translated sequence for Z. mobilis pgm was in full agreement with the 40 N-terminal amino acid residues determined by protein sequencing. The primary structure of the translated sequence is highly conserved (52 to 60% identity with other phosphoglycerate mutases) and also shares extensive homology with bisphosphoglycerate mutases (51 to 59% identity). Since Southern blots indicated the presence of only a single copy of pgm in the Z. mobilis chromosome, it is likely that the cloned pgm gene functions to provide both activities. Z. mobilis phosphoglycerate mutase is unusual in that it lacks the flexible tail and lysines at the carboxy terminus which are present in the enzyme isolated from all other organisms examined.
Yomano
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LP
Department of Microbiology and Cell Science, University of Florida, Gainesville 32611.
Scopes
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RK
Ingram
L O
LO
D019794:2,3-Diphosphoglycerate; D000429:Alcohol Oxidoreductases / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D001731:Bisphosphoglycerate Mutase / Q000096:biosynthesis / Q000235:genetics*; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004163:Diphosphoglyceric Acids / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics*; D006019:Glycolysis; D006880:Hydroxy Acids / Q000378:metabolism; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic; D017355:Zymomonas / Q000201:enzymology / Q000235:genetics*
D004163:Diphosphoglyceric Acids; D006880:Hydroxy Acids; D012333:RNA, Messenger; D011994:Recombinant Proteins; D019794:2,3-Diphosphoglycerate; D000429:Alcohol Oxidoreductases; D001731:Bisphosphoglycerate Mutase