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, fromSalmonella entericaserovar Typhimurium

Abstract

L-Rhamnose is an essential component of the cell wall of many pathogenic bacteria. Its precusor, 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. 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 A. The crystal belongs to either space group P3121 or P3221, with unit-cell parameters a = b = 71.56, c = 183.53 A and alpha = beta = 90, gamma = 120 degrees.

Genetic Analysis of the Cronobacter sakazakii O4 to O7 O-Antigen Gene Clusters and Development of a PCR Assay for Identification of All C. sakazakii O Serotypes

“…By amplifying gne using the primer pair targeting the gene (5-ACAGATTGGTGATGTTCG and 5-ATCAAAGCAATA TCCACC), we found the gne could be located elsewhere in the chromosomes of C. sakazakii O1 and O6 (data not shown). Similar cases have been also been identified in E. coli (12,13).…”

“…Genes involved in the biosynthesis of common sugar nucleotide precursors (GlcNAc, Glc, and Gal) were not located between the JUMPStart and gnd gene (43). Four proteins encoded by orf5-1, orf5-2, orf5-3, and orf5-4 share high-level identity (69 to 97%) with known RmlB, RmlD, RmlA, and RmlC proteins, which are responsible for L-Rha biosynthesis (12,13). Three proteins encoded by orf5-5, orf5-6, and orf5-7 share identity (63 to 76%) with QdtA, QdtC, and QdtB, which are involved in the synthesis of D-Qui3NAc (37).…”

The Aeromonas hydrophila wb * O34 Gene Cluster: Genetics and Temperature Regulation

“…This analysis revealed three sugar synthesis pathways, pathways for L-rhamnose, L-6dTal, and D-mannose. ORF1-, ORF2-, ORF3-, and ORF16-encoded proteins were homologous to RmlB (2), RmlA (57), RmlC, and RmlD (16,17), respectively ( Table 2). The products of rmlA through rmlD are responsible for the biosynthesis of dTDP-L-rhamnose from glucose-1-phosphate, and these four genes usually form a group in the wb* gene clusters (48).…”

Drug Targeting Mycobacterium tuberculosis Cell Wall Synthesis: Genetics of dTDP-Rhamnose Synthetic Enzymes and Development of a Microtiter Plate-Based Screen for Inhibitors of Conversion of dTDP-Glucose to dTDP-Rhamnose

“…The enzymes are soluble and can be readily prepared in large amounts. X-ray structural analysis is becoming available for the Salmonella homologs of these enzymes (1,(15)(16)(17), with efforts proceeding on the M. tuberculosis versions. Mechanisms of action and kinetic parameters are becoming known (18,19,34,42,45).…”

“…Although the rhamnosyl transferase is a good drug target in itself, there are many advantages in targeting the four enzymes required to make its required substrate, dTDP-Rha. These include the facts that the enzymes are soluble (22,28,46), that crystal structures of these enzymes from bacteria are forthcoming (1,(15)(16)(17), and that for one of these enzymes, RmlB, detailed mechanistic studies have been performed (18,34,42,45). Although the completion of the entire genome sequence of M. tuberculosis (8) greatly aids in the identification of the enzymes involved in dTDP-L-rhamnose synthesis, unambiguous identification from just the sequence data is problematical.…”