Characterization of Two UDP - Gal : GalNAc - diphosphate - lipid β 1 , 3 - 1 Galactosyltransferases WbwC from Escherichia coli 2 Serotypes O 104 and O 5 * S 3 4

25 E. coli display O antigens on the outer membrane that play an important role in bacterial interactions with 26 the environment. The O antigens of enterohemorrhagic E. coli O104 and O5 contain a Galβ1-3GalNAc27 disaccharide at the reducing end of the repeating unit. Several other O antigens contain this disaccharide 28 which is identical to the mammalian O-glycan core 1 or the cancer-associated Thomsen-Friedenreich (TF) 29 antigen. We identified the wbwC genes responsible for the synthesis of the disaccharide in E. coli 30 serotypes O104 and O5. To functionally characterize WbwC, an acceptor substrate analog, GalNAcα31 diphosphate-phenylundecyl, was synthesized. WbwC reaction products were isolated by high pressure 32 liquid chromatography and analyzed by mass spectrometry, nuclear magnetic resonance, galactosidase 33 and O-glycanase digestion and anti-TF antibody. The results clearly showed that the Galβ1-3GalNAcα34 linkage was synthesized, confirming WbwCECO104 and WbwCECO5 as UDP-Gal: GalNAcα-diphosphate35 lipid β1,3-Gal-transferases. Sequence analysis revealed a conserved DxDD motif, and mutagenesis 36 showed the importance of these Asp residues in catalysis. The purified enzymes require divalent cations 37 (Mn) for activity and are specific for UDP-Gal and GalNAc-diphosphate-lipid substrates. WbwC was 38 inhibited by bis-imidazolium salts having aliphatic chains of 18 to 22 carbons. This work will help to 39 elucidate mechanisms of polysaccharide synthesis in pathogenic bacteria and provide technology for 40 vaccine synthesis. 41 42 43 44 45 46 47 48 49 on A uust 5, 2017 by gest http/jb.asm .rg/ D ow nladed fom