Publications

• Kinetic and structural analysis of alpha-D-Glucose-1-phosphate cytidylyltransferase from Salmonella typhi.

  Koropatkin N.M., Cleland W.W., Holden H.M.

  Tyvelose is a 3,6-dideoxyhexose found in the O-antigen of the surface lipopolysaccharides of some pathogenic bacteria. It is synthesized via a complex biochemical pathway that is initiated by the formation of CDP-D-glucose. The production of this ligand is catalyzed by the enzyme glucose-1-phospha ... >> More

  Tyvelose is a 3,6-dideoxyhexose found in the O-antigen of the surface lipopolysaccharides of some pathogenic bacteria. It is synthesized via a complex biochemical pathway that is initiated by the formation of CDP-D-glucose. The production of this ligand is catalyzed by the enzyme glucose-1-phosphate cytidylyltransferase, which utilizes alpha-D-glucose 1-phosphate and MgCTP as substrates. Previous x-ray crystallographic investigations have demonstrated that the Salmonella typhi enzyme complexed with the product CDP-glucose is a fully integrated hexamer displaying 32 point group symmetry. The binding pocket for CDP-glucose is shared between two subunits. Here we describe both a detailed kinetic analysis of the cytidylyltransferase and a structural investigation of the enzyme complexed with MgCTP. These data demonstrate that the reaction catalyzed by the cytidylyltransferase proceeds via a sequential rather than a Bi Bi ping-pong mechanism as was previously reported. Additionally, the enzyme utilizes both CTP and UTP equally well as substrates. The structure of the enzyme with bound MgCTP reveals that the binding pocket for the nucleotide is contained within one subunit rather than shared between two. Key side chains involved in nucleotide binding include Thr(14), Arg(15), Lys(25), and Arg(111). In the previous structure of the enzyme complexed with CDP-glucose, those residues defined by Thr(14) to Ile(21) were disordered. The kinetic and x-ray crystallographic data presented here support a mechanism for this enzyme that is similar to that reported for the glucose-1-phosphate thymidylyltransferases. << Less

  J. Biol. Chem. 280:10774-10780(2005) [PubMed] [EuropePMC]

• Molecular structure of alpha-D-glucose-1-phosphate cytidylyltransferase from Salmonella typhi.

  Koropatkin N.M., Holden H.M.

  Dideoxysugars, which display biological activities ranging from mediating cell-cell interactions to serving as components in some antibiotics, are synthesized in various organisms via complex biochemical pathways that begin with the attachment of alpha-D-glucose 1-phosphate to either CTP or dTTP. ... >> More

  Dideoxysugars, which display biological activities ranging from mediating cell-cell interactions to serving as components in some antibiotics, are synthesized in various organisms via complex biochemical pathways that begin with the attachment of alpha-D-glucose 1-phosphate to either CTP or dTTP. Here we describe the three-dimensional structure of the alpha-D-glucose-1-phosphate cytidylyltransferase from Salmonella typhi, which catalyzes the first step in the production of CDP-tyvelose. For this investigation, the enzyme was crystallized in the presence of its product, CDP-glucose. In contrast to previous reports, the enzyme exists as a fully integrated hexamer with 32-point group symmetry. Each subunit displays a "bird-like" appearance with the "body" composed predominantly of a seven-stranded mixed beta-sheet and the two "wings" formed by beta-hairpin motifs. These two wings mediate subunit-subunit interactions along the 3-fold and 2-fold rotational axes, respectively. The six active sites of the hexamer are situated between the subunits related by the 2-fold rotational axes. CDP-glucose is anchored to the protein primarily by hydrogen bonds with backbone carbonyl oxygens and peptidic NH groups. The side chains of Arg111 and Asn188 from one subunit and Glu178 and Lys179 from the second subunit are also involved in hydrogen bonding with the ligand. The topology of the main core domain bears striking similarity to that observed for glucose-1-phosphate thymidylyltransferase and 4-diphosphocytidyl-2-C-methylerythritol synthetase. << Less

  J. Biol. Chem. 279:44023-44029(2004) [PubMed] [EuropePMC]