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 bacteria. Its precursor, dTDP-L-rhamnose, is synthesized from alpha-D-glucose-1-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.
Giraud
M F
MF
Centre for Biomolecular Sciences, The University of St Andrews, St Andrews, Fife KY16 9ST, Scotland.
McMiken
H J
HJ
Leonard
G A
GA
Messner
P
P
Whitfield
C
C
Naismith
J H
JH
D002237:Carbohydrate Dehydrogenases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D003460:Crystallization; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D009702:Nucleoside Diphosphate Sugars / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012486:Salmonella typhimurium / Q000201:enzymology* / Q000235:genetics; D013942:Thymine Nucleotides / Q000096:biosynthesis
D009702:Nucleoside Diphosphate Sugars; D011994:Recombinant Proteins; D013942:Thymine Nucleotides; C073293:thymidine diphosphate rhamnose; D002237:Carbohydrate Dehydrogenases; C107144:dTDP-4-dehydrorhamnose reductase