Cloning, sequencing, and overexpression in Escherichia coli of the alpha-D-glucose-1-phosphate cytidylyltransferase gene isolated from Yersinia pseudotuberculosis.
AUTOR(ES)
Thorson, J S
RESUMO
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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=205285Documentos Relacionados
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