Cloning, sequencing, and overexpression in Escherichia coli of the alpha-D-glucose-1-phosphate cytidylyltransferase gene isolated from Yersinia pseudotuberculosis.
A clone of Yersinia pseudotuberculosis DNA carrying the ascA gene was constructed, and the corresponding protein was successfully overexpressed in Escherichia coli. A protocol consisting of DEAE-cellulose and Sephadex G-100 column chromatography was developed and led to a nearly homogeneous purification of the ascA product. Initial characterization showed that the ascA-encoded protein is actually the alpha-D-glucose-1-phosphate cytidylyltransferase which catalyzes the first step of the biosynthesis of CDP-ascarylose (CDP-3,6-dideoxy-L-arabino-hexose), converting alpha-D-glucose-1-phosphate to CDP-D-glucose. In contrast to early studies suggesting that this enzyme was a monomeric protein of 111 kDa, the purified cytidylyltransferase from Y. pseudotuberculosis was found to consist of four identical subunits, each with a molecular mass of 29 kDa. This assignment is supported by the fact that the ascA gene, as a part of the ascarylose biosynthetic cluster, exhibits high sequence homology with other nucleotidylyltransferases, and its product shows high cytidylyltransferase activity. Subsequent amino acid comparison with other known nucleotidylyltransferases has allowed a definition of the important active-site residues within this essential catalyst. These comparisons have also afforded the inclusion of the cytidylyltransferase into the mechanistic convergence displayed by this fundamental class of enzyme.
Thorson
J S
JS
Department of Chemistry, University of Minnesota, Minneapolis 55455.
Kelly
T M
TM
Liu
H W
HW
D000595:Amino Acid Sequence; D001483:Base Sequence; D002412:Cations / Q000378:metabolism; D003001:Cloning, Molecular; D003570:Cytidine Triphosphate / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000032:analysis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009713:Nucleotidyltransferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D015011:Yersinia pseudotuberculosis / Q000235:genetics*
D002412:Cations; D008670:Metals; D011994:Recombinant Proteins; D003570:Cytidine Triphosphate; D009713:Nucleotidyltransferases; C020586:glucose-1-phosphate cytidylyltransferase