Crystal structure of Escherichia coli glucose-1-phosphate thymidylyltransferase (RffH) complexed with dTTP and Mg2+.
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.
Sivaraman
J
J
Department of Biochemistry, McGill University, Montréal, Québec H3G 1Y6, Canada.
Sauvé
Véronique
V
Matte
Allan
A
Cygler
Miroslaw
M
D001665:Binding Sites; D002384:Catalysis; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D008274:Magnesium / Q000737:chemistry*; D008958:Models, Molecular; D009713:Nucleotidyltransferases / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D013942:Thymine Nucleotides / Q000737:chemistry*
D013942:Thymine Nucleotides; D009713:Nucleotidyltransferases; C023226:UDPacetylglucosamine pyrophosphorylase; C020587:glucose-1-phosphate thymidylyltransferase; D008274:Magnesium; C024157:thymidine 5'-triphosphate