The majority of hetero-polysaccharide biosynthesis in Gram-negative bacteria utilizes the wzy-dependent pathway, in which repeating O-units are first synthesized in the cytosol and then subsequently translocated to the periplasmic face of the inner membrane where polymerization is initiated by the Wzy polymerase. Wzy proteins share little primary sequence homology and are specific for their cognate O-unit structures. Our previous studies on O-polysaccharide biosynthesis in Escherichia coli O86 identified the wbnI gene, which encodes a galactosyltransferase responsible for the introduction of α-(1→3)-Galp residues as side chains of the polysaccharide. In this work, we functionally inactivated the wbnI gene and showed that the mutant strain produced a different polysaccharide without the side chain Galp residue. The yield of the polysaccharide was substantially lower than the one produced by the wild-type strain. This study indicated that the complete O-unit structure is the preferred substrate for the polymerization, thus further confirming the specificity of Wzy. On the other hand, these studies also suggest that the Wzy polymerase might have moderate tolerance of side-chain truncated O-unit substrates.

Lipopolysaccharide, glycosyltransferase, polymerization, O-Repeating unit, wzy-Dependent pathway

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
3,3,2	aLFucp	98.9	69.9	70.3	83.3	67.6	15.3
3,3	bDGalp	102.2	77.4	74.4	69.8	74.8	61.8
3,2	Ac	174.4	22.1
3	aDGalpN	92.9	49.7	75.4	69.5	71.9	61.6
2	Ac	173.8	22.4
	bDGalpN	92.9	51.8	75.8	64.6	76.2	61.7


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
3,3,2	aLFucp	5.23	3.81	3.64	3.83	4.28	1.11
3,3	bDGalp	4.64	3.64	3.84	3.93	3.67	3.84
3,2	Ac	-	1.97
3	aDGalpN	5.23	4.14	3.80	4.07	3.67	3.78
2	Ac	-	1.99
	bDGalpN	5.08	4.25	3.96	4.20	3.85	3.79


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
3,3,2	aLFucp	98.9/5.23	69.9/3.81	70.3/3.64	83.3/3.83	67.6/4.28	15.3/1.11
3,3	bDGalp	102.2/4.64	77.4/3.64	74.4/3.84	69.8/3.93	74.8/3.67	61.8/3.84
3,2	Ac		22.1/1.97
3	aDGalpN	92.9/5.23	49.7/4.14	75.4/3.80	69.5/4.07	71.9/3.67	61.6/3.78
2	Ac		22.4/1.99
	bDGalpN	92.9/5.08	51.8/4.25	75.8/3.96	64.6/4.20	76.2/3.85	61.7/3.79

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

*O[C@@H]1[C@H](C)O[C@@H](O[C@@H]2[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]2O[C@@H]2[C@@H](NC(C)=O)[C@@H](O[C@@H]3[C@@H](NC(C)=O)[C@H](*)O[C@H](CO)[C@@H]3O)O[C@H](CO)[C@@H]2O)[C@@H](O)[C@@H]1O

714.671 g/mol (C₂₈H₄₆R₂N₂O₁₉, R = next and previous repeats, not counted in mol weight)