Purification, crystallization and preliminary structural studies of dTDP-6-deoxy-d-xylo-4-hexulose 3,5-epimerase (RmlC), the third enzyme of the dTDP-l-rhamnose synthesis pathway, from Salmonella enterica serovar Typhimurium

• Published in: Acta crystallographica. Section D, Biological crystallography. Vol. 55; no. 3; pp. 706 - 708
• Main Authors: Giraud, Marie-France, Gordon, Fiona M., Whitfield, Chris, Messner, Paul, McMahon, Stephen A., Naismith, James H.
• Format: Journal Article
• Language: English
• Published: 5 Abbey Square, Chester, Cheshire CH1 2HU, England International Union of Crystallography 01.03.1999
• Subjects: 5-epimerase; Carbohydrate Epimerases - chemistry; Carbohydrate Epimerases - isolation & purification; Chromatography, High Pressure Liquid; Cloning, Molecular; Crystallization; drug design; dTDP-6-deoxy-d-xylo-4-hexulose 3; dTDP‐6‐deoxy‐d‐xylo‐4‐hexulose 3,5‐epimerase; epimerases; l-rhamnose; Protein Conformation; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Rhamnose - analogs & derivatives; Rhamnose - biosynthesis; Salmonella typhimurium - enzymology; tuberculosis
• ISSN: 1399-0047; 0907-4449; 1399-0047
• DOI: 10.1107/S0907444998015042

l‐Rhamnose is an essential component of the cell wall of many pathogenic bacteria. Its precusor, dTDP‐l‐rhamnose, is synthesized from α‐d‐glucose‐1‐phosphate and dTTP via a pathway requiring four distinct enzymes: RmlA, RmlB, RmlC and RmlD. RmlC was 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 were obtained using the sitting‐drop vapour‐diffusion method with ammonium sulfate as precipitant. Diffraction data were collected on a frozen crystal to a resolution of 2.17 Å. The crystal belongs to either space group P3121 or P3221, with unit‐cell parameters a = b = 71.56, c = 183.53 Å and α = β = 90, γ = 120°.