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

L-Rhamnose is an essential component of the cell wall of many pathogenic ba cteria. Its precursor, dTDP-L-rhamnose, is synthesized from cr-D-glucose-l- phosphate and dTTP via a pathway requiring four distinct enzymes: RmlA, Rml B, RmlC and RmlD. RmlD catalyses the terminal step of this pathway by conve rting dTDP-6-deoxy-L-lyxo-4-hexulose to dTDP-L-rhamnose. RmlD from Salmonel la enterica serovar Typhimurium has been overexpressed in Escherichia coli. The recombinant protein was purified by a two-step protocol involving anio n-exchange and hydrophobic chromatography. Dynamic light-scattering experim ents indicated that the recombinant protein is monodisperse. Crystals of na tive and selenomethionine-enriched RmlD have been obtained using the sittin g-drop vapour-diffusion method with polyethylene glycol as precipitant. Dif fraction data have been collected from orthorhombic crystals of both native and selenomethionyl-derivatized protein, allowing tracing of the protein s tructure.