Overexpression, purification, crystallisation and preliminary structural study of dTDP-6-deoxy-lyxo-4-reductase (RmlD) the fourth enzyme of the dTDP-rhanose synthesis pathway, from Salmonella entrica serovar Typhimurim

MF Giraud, HJ McMiken, GA Leonard, P Messner, C Whitfield, James Henderson Naismith

Research output: Contribution to journalArticlepeer-review

44 Citations (Scopus)

Abstract

L-Rhamnose is an essential component of the cell wall of many pathogenic bacteria. Its precursor, dTDP-L-rhamnose, is synthesized from cr-D-glucose-l-phosphate and dTTP via a pathway requiring four distinct enzymes: RmlA, RmlB, RmlC and RmlD. RmlD catalyses the terminal step of this pathway by converting dTDP-6-deoxy-L-lyxo-4-hexulose to dTDP-L-rhamnose. RmlD from Salmonella enterica serovar Typhimurium has been overexpressed in Escherichia coli. The recombinant protein was purified by a two-step protocol involving anion-exchange and hydrophobic chromatography. Dynamic light-scattering experiments indicated that the recombinant protein is monodisperse. Crystals of native and selenomethionine-enriched RmlD have been obtained using the sitting-drop vapour-diffusion method with polyethylene glycol as precipitant. Diffraction data have been collected from orthorhombic crystals of both native and selenomethionyl-derivatized protein, allowing tracing of the protein structure.

Original languageEnglish
Pages (from-to)2043-2046
Number of pages4
JournalActa Crystallographica. Section D, Biological crystallography
VolumeD55
Publication statusPublished - Dec 1999

Keywords

  • ESCHERICHIA-COLI
  • GENE-CLUSTER
  • O-ANTIGEN
  • BIOSYNTHESIS
  • LIPOPOLYSACCHARIDE
  • SEQUENCE
  • BINDING
  • REGION

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