Biochemical characterization of WbdN, a beta 1,3-glucosyltransferase involved in O-antigen synthesis in enterohemorrhagic Escherichia coli O157

The enterohemorrhagic O157 strain of Escherichia coli, which is one of the most well-known bacterial pathogens, has an O-antigen repeating unit structure with the sequence [-2-d-Rha4NAc alpha 1-3-l-Fuc alpha 1-4-d-Glc beta 1-3-d-GalNAc alpha 1-]. The O-antigen gene cluster of E. coli O157 contains the genes responsible for the assembly of this repeating unit and includes wbdN. In spite of cloning many O-antigen genes, biochemical characterization has been done on very few enzymes involved in O-antigen synthesis. In this work, we expressed the wbdN gene in E. coli BL21, and the His-tagged protein was purified. WbdN activity was characterized using the donor substrate UDP-[ super(14)C]Glc and the synthetic acceptor substrate GalNAc alpha -O-PO sub(3)-PO sub(3)-(CH sub(2)) sub(11)-O-Ph. The enzyme product was isolated by high pressure liquid chromatography, and mass spectrometry showed that one Glc residue was transferred to the acceptor by WbdN. Nuclear magnetic resonance analysis of the product structure indicated that Glc was beta 1-3 linked to GalNAc. WbdN contains a conserved DxD motif and requires divalent metal ions for full activity. WbdN activity has an optimal pH between 7 and 8 and is highly specific for UDP-Glc as the donor substrate. GalNAc alpha derivatives lacking the diphosphate group were inactive as substrates, and the enzyme did not transfer Glc to GlcNAc alpha -O-PO sub(3)-PO sub(3)-(CH sub(2)) sub(11)-O-Ph. Our results illustrate that WbdN is a specific UDP-Glc:GalNAc alpha -diphosphate-lipid beta 1,3-Glc-transferase. The enzyme is a target for the development of inhibitors to block O157-antigen synthesis.