The sialyl-T antigen, sialylα2-3Galβ1-3GalNAc-, is a common O-glycan structure in human glycoproteins and is synthesized by sialyltransferase ST3Gal1. The enterohemorrhagic Escherichia coli serotype O104 has the rare ability to synthesize a sialyl-T antigen mimic. We showed here that the wbwA gene of the E. coli O104 antigen synthesis gene cluster encodes an α2,3-sialyltransferase WbwA that transfers sialic acid from CMP-sialic acid to Galβ1-3GalNAc-α-diphosphate-lipid acceptor. The NMR analysis of purified WbwA enzyme reaction product indicated that the sialyl-T antigen, sialylα2-3Galβ1-3GalNAc-α-diphosphate-lipid, was synthesized. We showed that the conserved HP motif, and Glu/Asp residues of two EDG motifs in WbwA are important for the activity. The characterization studies showed that WbwA from E. coli O104 is a monofunctional α2,3-sialyltransferase, and is distinct from human ST3Gal1 as well as all other known sialyltransferases due to its unique acceptor specificity. This work contributes to knowledge of the biosynthesis of bacterial virulence factors. IMPORTANCE: This is the first characterization of a sialyltransferase involved in the synthesis of an O antigen in E. coli. The enzyme contributes to the mimicry of human sialyl-T antigen and has a unique substrate specificity but very little sequence identity with other sialyltransferases. Thus the bacterial sialyltransferase is related to the human counterpart only by the similarity of biochemical activity.

O-antigen, sialyltransferase, Substrate Specificity, O antigen synthesis, E.coli O104, Enterohemorrhagic Escherichia coli, sialyl-T antigen, ST3Gal1, WbwA

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6	C7	C8	C9
0,0,3,3,5	Ac
0,0,3,3	aXNeup	?	?	?	66.1	51.4	?	?	?	?
0,0,3	bDGalp	104.2	68.7	75.4	66.8	?	?
0,0,2	Ac
0,0	aDGalpN	94.7	48.4	77.1	68.2	71.6	60.5
0	P
	P


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6	H7	H8	H9
0,0,3,3,5	Ac	-	1.9
0,0,3,3	aXNeup	-	-	1.70-2.67	3.58	3.75	3.54	?	?	?
0,0,3	bDGalp	4.47	3.44	3.98	3.86	?	?
0,0,2	Ac	-	1.9
0,0	aDGalpN	5.45	4.31	3.98	4.18	4.13	3.66
0	P
	P


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6	C7/H7	C8/H8	C9/H9
0,0,3,3,5	Ac	NMR TSV error 2: unequal length of 13C and 1H datasets
0,0,3,3	aXNeup			?/1.70-2.67	66.1/3.58	51.4/3.75	?/3.54	?/?	?/?	?/?
0,0,3	bDGalp	104.2/4.47	68.7/3.44	75.4/3.98	66.8/3.86	?/?	?/?
0,0,2	Ac	NMR TSV error 2: unequal length of 13C and 1H datasets
0,0	aDGalpN	94.7/5.45	48.4/4.31	77.1/3.98	68.2/4.18	71.6/4.13	60.5/3.66
0	P
	P

The spectrum also has 9 signals at unknown positions (not plotted).

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

CC(=O)N[C@@H]1[C@@H](O)C[C@](O[C@H]2[C@@H](O)[C@@H](CO)O[C@@H](O[C@H]3[C@@H](O)[C@@H](CO)O[C@H](OP(=O)(O)OP(=O)(O)O)[C@@H]3NC(C)=O)[C@@H]2O)(C(=O)O)O[C@H]1[C@H](O)[C@H](O)CO

834.564 g/mol (C₂₅H₄₄N₂O₂₅P₂)