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
Blankenfeldt W., Giraud MF., Leonard G., Rahim R., Creuzenet C., Lam JS., Naismith JH.
Glucose-1-phosphate thymidylyltransferase (RmlA; E.C. 184.108.40.206) 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 α-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 Å. Crystals belonged to space group P1, with unit-cell parameters a = 71.5, b = 73.1, c = 134.7 Å, α = 89.9, β = 80.9, γ= 81.1°. The asymmetric unit contains eight monomers in the form of two RmlA tetramers with a solvent content of 51%. Seleno-methionine-labelled protein has been obtained and crystallized.