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

W Blankenfeldt, MF Giraud, G Leonard, R Rahim, C Creuzenet, J Lam, James Henderson Naismith

Research output: Contribution to journalArticlepeer-review

28 Citations (Scopus)

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 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 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.

Original languageEnglish
Pages (from-to)1501-1504
Number of pages4
JournalActa Crystallographica. Section D, Biological crystallography
VolumeD56
Publication statusPublished - Nov 2000

Keywords

  • ENTERICA SEROVAR TYPHIMURIUM
  • 3,5-EPIMERASE
  • BIOSYNTHESIS
  • REDUCTASE
  • PROTEINS
  • BINDING

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