The purification, crystallization and structural elucidation of dTDP-D-glucose 4,6-dehydratase (RmlB), the second enzyme of the dTDP-L-rhamnose synthesis pathway from Salmonella enterica serovar typhimurium.
dTDP-D-glucose 4,6-dehydratase (RmlB) is the second of four enzymes involved in the dTDP-L-rhamnose pathway and catalyzes the dehydration of dTDP-D-glucose to dTDP-4-keto-6-deoxy-D-glucose. The ultimate product of the pathway, dTDP-L-rhamnose, is the precursor of L-rhamnose, which is a key component of the cell wall of many pathogenic bacteria. RmlB from Salmonella enterica serovar Typhimurium has been overexpressed and purified, and crystals of the enzyme have been grown using the sitting-drop vapour-diffusion technique with lithium sulfate as precipitant. Diffraction data have been obtained to a resolution of 2.8 A on a single frozen RmlB crystal which belongs to space group P2(1), with unit-cell parameters a = 111.85, b = 87.77, c = 145.66 A, beta = 131.53 degrees. The asymmetric unit contains four monomers in the form of two RmlB dimers with a solvent content of 62%. A molecular-replacement solution has been obtained and the model is currently being refined.
Allard
S T
ST
Centre for Biomedical Sciences, North Haugh, The University, St Andrews, Fife KY16 9ST, Scotland.
Giraud
M F
MF
Whitfield
C
C
Messner
P
P
Naismith
J H
JH
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002384:Catalysis; D003460:Crystallization; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006836:Hydro-Lyases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D009702:Nucleoside Diphosphate Sugars / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012486:Salmonella typhimurium / Q000145:classification / Q000201:enzymology*; D013942:Thymine Nucleotides / Q000096:biosynthesis*; D014961:X-Ray Diffraction
D001426:Bacterial Proteins; D009702:Nucleoside Diphosphate Sugars; D011994:Recombinant Proteins; D013942:Thymine Nucleotides; C073293:thymidine diphosphate rhamnose; D006836:Hydro-Lyases; C022771:dTDPglucose 4,6-dehydratase