title: Characterization of two UDP-Gal:GalNAc-diphosphate-lipid β1,3-galactosyltransferases WbwC from Escherichia coli serotypes O104 and O5.
abstract: Escherichia coli displays O antigens on the outer membrane that play an important role in bacterial interactions with the environment. The O antigens of enterohemorrhagic E. coli O104 and O5 contain a Galβ1-3GalNAc disaccharide at the reducing end of the repeating unit. Several other O antigens contain this disaccharide, which is identical to the mammalian O-glycan core 1 or the cancer-associated Thomsen-Friedenreich (TF) antigen. We identified the wbwC genes responsible for the synthesis of the disaccharide in E. coli serotypes O104 and O5. To functionally characterize WbwC, an acceptor substrate analog, GalNAcα-diphosphate-phenylundecyl, was synthesized. WbwC reaction products were isolated by high-pressure liquid chromatography and analyzed by mass spectrometry, nuclear magnetic resonance, galactosidase and O-glycanase digestion, and anti-TF antibody. The results clearly showed that the Galβ1-3GalNAcα linkage was synthesized, confirming WbwCECO104 and WbwCECO5 as UDP-Gal:GalNAcα-diphosphate-lipid β1,3-Gal-transferases. Sequence analysis revealed a conserved DxDD motif, and mutagenesis showed the importance of these Asp residues in catalysis. The purified enzymes require divalent cations (Mn(2+)) for activity and are specific for UDP-Gal and GalNAc-diphosphate lipid substrates. WbwC was inhibited by bis-imidazolium salts having aliphatic chains of 18 to 22 carbons. This work will help to elucidate mechanisms of polysaccharide synthesis in pathogenic bacteria and provide technology for vaccine synthesis.
authors: Department of Medicine and Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, Canada.
authors: Shuo
authors: S
authors: Wang
authors: Department of Medicine and Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, Canada.
authors: Diana
authors: D
authors: Czuchry
authors: TEDA School of Biological Sciences and Biotechnology, Nankai University, Tianjin, China.
authors: Bin
authors: B
authors: Liu
authors: Department of Medicine and Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, Canada.
authors: Anna N
authors: AN
authors: Vinnikova
authors: Department of Medicine and Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, Canada.
authors: Yin
authors: Y
authors: Gao
authors: Department of Chemistry, Queen's University, Kingston, Ontario, Canada.
authors: Jason Z
authors: JZ
authors: Vlahakis
authors: Department of Chemistry, Queen's University, Kingston, Ontario, Canada.
authors: Walter A
authors: WA
authors: Szarek
authors: TEDA School of Biological Sciences and Biotechnology, Nankai University, Tianjin, China.
authors: Lei
authors: L
authors: Wang
authors: TEDA School of Biological Sciences and Biotechnology, Nankai University, Tianjin, China fenglu63@nankai.edu.cn brockhau@queensu.ca.
authors: Lu
authors: L
authors: Feng
authors: Department of Medicine and Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, Canada fenglu63@nankai.edu.cn brockhau@queensu.ca.
authors: Inka
authors: I
authors: Brockhausen
mesh_terms: D000595:Amino Acid Sequence; D054324:Enterohemorrhagic Escherichia coli / Q000145:classification / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D005700:Galactosyltransferases / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic; D012703:Serotyping
chemical_list: D029968:Escherichia coli Proteins; D005700:Galactosyltransferases; C000592114:WbwC protein, E coli