The purification, crystallization and preliminary structural characterization of glucose-1-phosphate thymidylyltransferase (Rm1A), the first enzyme of the dTDP-L-rhamnose synthesis pathway from Pseudomonas aeruginosa

Glucose-1-phosphate thymidylyltransferase (RmlA; E.C. 2.7.7.24) is the firs t of four enzymes involved in the biosynthesis of dTDP-L-rhamnose, the prec ursor of L-rhamnose, a key component of the cell wall of many pathogenic ba cteria. RmlA catalyses the condensation of thymidine triphosphate (dTTP) an d alpha -D-glucose-1-phosphate (G1P), yielding dTDP-D-glucose. RmlA from Ps eudomonas aeruginosa has been overexpressed and purified. Crystals of the e nzyme have been grown using the sitting-drop vapour-diffusion technique wit h PEG 6000 and lithium sulfate as precipitant. Several diffraction data set s of single frozen crystals were collected to a resolution of 1.66 Angstrom . Crystals belonged to space group P1, with unit-cell parameters a = 71.5, b = 73.1, c = 134.7 Angstrom, alpha = 89.9, beta = 80.9, gamma = 81.1 degre es. The asymmetric unit contains eight monomers in the form of two RmlA tet ramers with a solvent content of 51%. Selenomethionine-labelled protein has been obtained and crystallized.