Crystal Structure of Escherichia coli Glucose-1-Phosphate Thymidylyltransferase (RffH) Complexed with dTTP and Mg2

• Published in: Journal of molecular biology 2002-11, Vol.277 (46), p.44214-44219
• Main Authors: Sivaraman, J, Sauvé, Véronique, Matte, Allan, Cygler, Miroslaw
• Format: Article
• Language: English
• Subjects: Binding Sites; Catalysis; Cloning, Molecular; CRYSTAL STRUCTURE; Crystallography, X-Ray; Dimerization; ESCHERICHIA COLI; Escherichia coli - enzymology; Escherichia coli - metabolism; Magnesium - chemistry; MATERIALS SCIENCE; Models, Molecular; MOLECULAR BIOLOGY; national synchrotron light source; Nucleotidyltransferases - chemistry; Nucleotidyltransferases - metabolism; Protein Conformation; Thymine Nucleotides - chemistry
• ISSN: 0021-9258; 0022-2836; 1083-351X; 1089-8638
• DOI: 10.1074/jbc.M206932200

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 Å 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 α/β 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 α-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.