Abstract
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.