The O-antigen is the outer part of the lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria and contains many repeats of an oligosaccharide unit. It contributes to antigenic variability and is essential to the full function and virulence of bacteria. Shigella is a Gram-negative human pathogen that causes diarrhea in humans. The O-antigen of Shigella boydii type 14 consists of repeating oligosaccharide units with the structure [→6 DGalp α1→4 DGlcpA β1→6 DGalp β1→4 DGalp β1→4 DGlcpNAc β1→]n. The wfeD gene in the O-antigen gene cluster of Shigella boydii type 14 was proposed to encode a galactosyltransferase (GalT) involved in O-antigen synthesis. We confirmed here that the wfeD gene product is a β4GalT that synthesizes the Galβ1-4GlcNAcα-R linkage. WfeD was expressed in E.coli and the activity characterized using UDP-[(3)H]Gal as the donor substrate and the synthetic acceptor substrate GlcNAcα-pyrophosphate-(CH2)11-O-phenyl. Enzyme product was analyzed by LC-MS, HPLC, NMR and galactosidase digestion. The enzyme was shown to be specific for the UDP-Gal donor substrate and required pyrophosphate in the acceptor substrate. Divalent metal ions such as Mn(2+), Ni(2+), and surprisingly also Pb(2+), enhanced the enzyme activity. Mutational analysis showed that the Glu101 residue within a DxD motif is essential for activity, possibly by forming the catalytic nucleophile. The Lys211 residue was also shown to be required for activity and may be involved in binding the negatively charged acceptor substrate. Our study revealed that the β4-GalT WfeD is a novel enzyme that has virtually no sequence similarity to mammalian β4GalT although it catalyzes a similar reaction.

structure, virulence, O-antigen, gene cluster, specific, galactosyltransferase, Shigella boydii

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
0,0,4	bDGalp	103.4	70.8	73.1	68.8	?	?
0,0,2	Ac
0,0	?DGlcpN	94.7	53.1	69.4	79.1	?	?
0	P
	P


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
0,0,4	bDGalp	4.345	3.538	3.465	3.792	?	?
0,0,2	Ac
0,0	?DGlcpN	5.561	4.027	3.846	3.626	?	?
0	P
	P


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
0,0,4	bDGalp	103.4/4.345	70.8/3.538	73.1/3.465	68.8/3.792	?/?	?/?
0,0,2	Ac
0,0	?DGlcpN	94.7/5.561	53.1/4.027	69.4/3.846	79.1/3.626	?/?	?/?
0	P
	P

The spectrum also has 4 signals at unknown positions (not plotted).

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

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

543.308 g/mol (C₁₄H₂₇NO₁₇P₂)