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Characterization of Two UDP-Gal:GalNAc-Diphosphate-Lipid β1,3-Galactosyltransferases WbwC from Escherichia coli Serotypes O104 and O5.
- Source :
-
Journal of Bacteriology . Sep2014, Vol. 196 Issue 17, p3122-3133. 12p. - Publication Year :
- 2014
-
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 aGa1β1-3GalNAc 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 anti-body. The results clearly showed that the Gaipi-3GalNAca 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 (Mn2+) 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. [ABSTRACT FROM AUTHOR]
Details
- Language :
- English
- ISSN :
- 00219193
- Volume :
- 196
- Issue :
- 17
- Database :
- Academic Search Index
- Journal :
- Journal of Bacteriology
- Publication Type :
- Academic Journal
- Accession number :
- 97478151
- Full Text :
- https://doi.org/10.1128/JB.01698-14