E. coli display 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 (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.

synthesis, gene, Escherichia coli, Enzymes, WbwC

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
0,0,3	bDGalp	104.5	70.7	72.1	68.3	74.5	60.2
0,0,2	Ac
0,0	aDGalpN	94.7	48.2	77.1	68.4	71.6	60.0
0	P
	P


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
0,0,3	bDGalp	4.41	3.45	3.55	3.82	3.57	3.65
0,0,2	Ac	-	1.97
0,0	aDGalpN	5.45	4.32	4.00	4.19	4.13	3.67
0	P
	P


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
0,0,3	bDGalp	104.5/4.41	70.7/3.45	72.1/3.55	68.3/3.82	74.5/3.57	60.2/3.65
0,0,2	Ac	NMR TSV error 2: unequal length of 13C and 1H datasets
0,0	aDGalpN	94.7/5.45	48.2/4.32	77.1/4.00	68.4/4.19	71.6/4.13	60.0/3.67
0	P
	P

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

CC(=O)N[C@H]1[C@@H](OP(=O)(O)OP(=O)(O)O)O[C@H](CO)[C@H](O)[C@@H]1O[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O

543.308 g/mol (C₁₄H₂₇NO₁₇P₂)