Expression and characterization of MMP1090 UDP-Gal/UDP-GalNAc Epimerase and investigation into UDP-sugar assays
A gene found in Methanococcus maripaludis, MMP1090, is a homolog of known UDP-glucose (UDP-Glc)/UDP-galactose (UDP-Gal) and UDP-N-acetylglucosamine (UDP-GlcNAc)/UDP-Nacetylgalactosamine (UDP-GalNAc) 4-epimerases. These UDP-sugars are integral in the biosynthetic pathways of many organisms, from sugar metabolism in humans to cell wall formation in Escherichia coli. The MMP1090 epimerase is expected to function through abstracting a hydrogen on the C4 carbon of the UDP-sugar and inducing a rotation about the phosphorus-oxygen bond connecting the UDP to the sugar. This enzyme was successfully expressed and purified; subsequently, the protein successfully epimerized UDP-Glc and UDPGlcNAc to UDP-Gal and UDP-GalNAc, and kinetic parameters were determined. Utilizing this epimerase, the possibility of a coupled-concentration assay for UDP-Gal using E. coli as the test organism was explored, where the assay reaction involves epimerization of UDP-Gal to UDPGlc followed by oxidation by a dehydrogenase and reduction of NAD+ which can be detected by fluorescence. By using an optimized UDP-Glc concentration assay, growth conditions that could affect UDP-Glc levels in E. coli were investigated. Glucose supplemented LB-medium was shown to cause significantly elevated UDP-Glc in E. coli—signifying increased sugar metabolism—compared to E. coli grown in normal LB-medium. UDP-Glc levels in E. coli grown in aerobic conditions appear to show no significant difference when compared with E. coli grown in semi-anaerobic conditions.