title: Biochemical characterization of WbdN, a β1,3-glucosyltransferase involved in O-antigen synthesis in enterohemorrhagic Escherichia coli O157.
abstract: 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-Rha4NAcα1-3-l-Fucα1-4-d-Glcβ1-3-d-GalNAcα1-]. 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-[(14)C]Glc and the synthetic acceptor substrate GalNAcα-O-PO(3)-PO(3)-(CH(2))(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 β1-3 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. GalNAcα derivatives lacking the diphosphate group were inactive as substrates, and the enzyme did not transfer Glc to GlcNAcα-O-PO(3)-PO(3)-(CH(2))(11)-O-Ph. Our results illustrate that WbdN is a specific UDP-Glc:GalNAcα-diphosphate-lipid β1,3-Glc-transferase. The enzyme is a target for the development of inhibitors to block O157-antigen synthesis.
authors.affiliation: Department of Medicine, Division of Rheumatology,  Queen's University, Kingston, Ontario,  Canada.
authors.forename: Yin
authors.initials: Y
authors.lastname: Gao
authors.forename: Bin
authors.initials: B
authors.lastname: Liu
authors.forename: Scott
authors.initials: S
authors.lastname: Strum
authors.forename: John S
authors.initials: JS
authors.lastname: Schutzbach
authors.forename: Tatyana N
authors.initials: TN
authors.lastname: Druzhinina
authors.forename: Natalia S
authors.initials: NS
authors.lastname: Utkina
authors.forename: Vladimir I
authors.initials: VI
authors.lastname: Torgov
authors.forename: Leonid L
authors.initials: LL
authors.lastname: Danilov
authors.forename: Vladimir V
authors.initials: VV
authors.lastname: Veselovsky
authors.forename: Jason Z
authors.initials: JZ
authors.lastname: Vlahakis
authors.forename: Walter A
authors.initials: WA
authors.lastname: Szarek
authors.forename: Lei
authors.initials: L
authors.lastname: Wang
authors.forename: Inka
authors.initials: I
authors.lastname: Brockhausen
mesh_terms: D002851:Chromatography, High Pressure Liquid; D019453:Escherichia coli O157 / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D005964:Glucosyltransferases / Q000235:genetics / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D019081:O Antigens / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity; D014532:Uridine Diphosphate Glucose / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D019081:O Antigens; D005964:Glucosyltransferases; C585531:WbdN protein, E coli; D014532:Uridine Diphosphate Glucose