title: The purification, crystallization and preliminary structural characterization of glucose-1-phosphate thymidylyltransferase (RmlA), the first enzyme of the dTDP-L-rhamnose synthesis pathway from Pseudomonas aeruginosa.
abstract: Glucose-1-phosphate thymidylyltransferase (RmlA; E.C. 2.7.7.24) is the first of four enzymes involved in the biosynthesis of dTDP-L-rhamnose, the precursor of L-rhamnose, a key component of the cell wall of many pathogenic bacteria. RmlA catalyses the condensation of thymidine triphosphate (dTTP) and alpha-D-glucose-1-phosphate (G1P), yielding dTDP-D-glucose. RmlA from Pseudomonas aeruginosa has been overexpressed and purified. Crystals of the enzyme have been grown using the sitting-drop vapour-diffusion technique with PEG 6000 and lithium sulfate as precipitant. Several diffraction data sets of single frozen crystals were collected to a resolution of 1.66 A. Crystals belonged to space group P1, with unit-cell parameters a = 71.5, b = 73.1, c = 134.7 A, alpha = 89.9, beta = 80.9, gamma = 81.1 degrees. The asymmetric unit contains eight monomers in the form of two RmlA tetramers with a solvent content of 51%. Selenomethionine-labelled protein has been obtained and crystallized.
authors.affiliation: Centre for Biomedical Sciences, North Haugh, The University, St Andrews, Fife KY16 9ST, Scotland.
authors.forename: W
authors.initials: W
authors.lastname: Blankenfeldt
authors.forename: M F
authors.initials: MF
authors.lastname: Giraud
authors.forename: G
authors.initials: G
authors.lastname: Leonard
authors.forename: R
authors.initials: R
authors.lastname: Rahim
authors.forename: C
authors.initials: C
authors.lastname: Creuzenet
authors.forename: J S
authors.initials: JS
authors.lastname: Lam
authors.forename: J H
authors.initials: JH
authors.lastname: Naismith
mesh_terms: D002851:Chromatography, High Pressure Liquid; D003460:Crystallization; D018360:Crystallography, X-Ray; D009702:Nucleoside Diphosphate Sugars / Q000096:biosynthesis*; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000302:isolation & purification*; D011487:Protein Conformation; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D013942:Thymine Nucleotides / Q000096:biosynthesis*
chemical_list: D009702:Nucleoside Diphosphate Sugars; D013942:Thymine Nucleotides; C073293:thymidine diphosphate rhamnose; D009713:Nucleotidyltransferases; C020587:glucose-1-phosphate thymidylyltransferase