Overexpression, purification, crystallization and preliminary structural study of dTDP-6-deoxy-L-lyxo-4-hexulose reductase (RmlD), the fourth enzyme of the dTDP-L-rhamnose synthesis pathway, from Salmonella enterica serovar Typhimurium
Giraud M-F., McMiken HJ., Leonard GA., Messner P., Whitfield C., Naismith JH.
<jats:p>L-Rhamnose is an essential component of the cell wall of many pathogenic bacteria. Its precursor, dTDP-L-rhamnose, is synthesized from α-D-glucose-1-phosphate and dTTP <jats:italic>via</jats:italic> 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-<jats:italic>lyxo</jats:italic>-4-hexulose to dTDP-L-rhamnose. RmlD from <jats:italic>Salmonella enterica</jats:italic> serovar Typhimurium has been overexpressed in <jats:italic>Escherichia coli</jats:italic>. 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.</jats:p>