PMID:12171937
| Citation |
Sivaraman, J, Sauvé, V, Matte, A and Cygler, M (2002) Crystal structure of Escherichia coli glucose-1-phosphate thymidylyltransferase (RffH) complexed with dTTP and Mg2+. J. Biol. Chem. 277:44214-9 |
|---|---|
| Abstract |
The enzyme glucose-1-phosphate thymidylyltransferase (RffH), the product of the rffh gene, catalyzes one of the steps in the synthesis of enterobacterial common antigen (ECA), a cell surface glycolipid found in Gram-negative enteric bacteria. In Escherichia coli two gene products, RffH and RmlA, catalyze the same enzymatic reaction and are homologous in sequence; however, they are part of different operons and function in different pathways. We report the crystal structure of RffH bound to deoxythymidine triphosphate (dTTP), the phosphate donor, and Mg(2+), refined at 2.6 A to an R-factor of 22.3% (R(free) = 28.4%). The crystal structure of RffH shows a tetrameric enzyme best described as a dimer of dimers. Each monomer has an overall alpha/beta fold and consists of two domains, a larger nucleotide binding domain (residues 1-115, 222-291) and a smaller sugar-binding domain (116-221), with the active site located at the domain interface. The Mg(2+) ion is coordinated by two conserved aspartates and the alpha-phosphate of deoxythymidine triphosphate. Its location corresponds well to that in a structurally similar domain of N-acetylglucosamine-1-phosphate uridylyltransferase (GlmU). Analysis of the RffH, RmlA, and GlmU complexes with substrates and products provides an explanation for their different affinities for Mg(2+) and leads to a proposal for the dynamics along the reaction pathway. |
| Links |
PubMed Online version:10.1074/jbc.M206932200 |
| Keywords |
Binding Sites; Catalysis; Cloning, Molecular; Crystallography, X-Ray; Dimerization; Escherichia coli/enzymology; Escherichia coli/metabolism; Magnesium/chemistry; Models, Molecular; Nucleotidyltransferases/chemistry; Nucleotidyltransferases/metabolism; Protein Conformation; Thymine Nucleotides/chemistry |
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