A limited range of different structures is observed in O-antigenic polysaccharides (OPSs) from Klebsiella pneumoniae lipopolysaccharides. Among these, several are based on modifications of a conserved core element of serotype O2a OPS, which has a disaccharide repeat structure [→3)-α-Galp-(1→3)-β-Galf-(1→]. Here, we describe the enzymatic pathways for a highly unusual modification strategy involving the attachment of a second glycan repeat unit structure to the non-reducing terminus of O2a. This occurs by the addition of the O1 [→3)-α-Galp-(1→3)-β-Galp-(1→] or O2c [→3)-β-GlcpNAc-(1→5)-β-Galf-(1→] antigens. The organization of the enzyme activities performing these modifications differs, with the enzyme WbbY possessing two glycosyltransferase catalytic sites solely responsible for O1-antigen polymerization and forming a complex with the O2a glycosyltransferase, WbbM. In contrast, O2c polymerization requires glycosyltransferases WbmV and WbmW, which interact with one another, but apparently not with WbbM. Using defined synthetic acceptors and site-directed mutants to assign the activities of the WbbY catalytic sites, we found that the C-terminal WbbY domain is a UDP-Galp-dependent GT-A galactosyltransferase adding β-(1→3)-linked D-Galp, whereas the WbbY N terminus includes a GT-B enzyme adding α-(1→3)-linked D-Galp These activities build the O1 antigen on a terminal Galp in the O2a domain. Using similar approaches, we identified WbmV as the UDP-GlcNAc-dependent transferase and noted that WbmW represents a UDP-Galf-dependent enzyme and that both are GT-A members. WbmVW polymerizes the O2c antigen on a terminal Galf Our results provide mechanistic and conceptual insights into an important strategy for polysacharide antigen diversification in bacteria.

polysaccharide, O antigen, Gram-negative bacteria, glycosyltransferase, Klebsiella pneumoniae, serotyping, lipopolysaccharide (LPS), antigenic diversity, cell surface, enzyme complex

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
3,3,3	aDGalp	96.7	68.7	80.4	70.5	71.9	62.3
3,3	bDGalp	105.5	71.0	78.6	66.2	76.1	62.3
3	aDGalp	100.9	68.6	80.5	?	?	?
	bDGalf	108.9	80.5	86.2	83.4	72.3	64.2


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
3,3,3	aDGalp	5.19	4.07	4.16	4.29	4.24	3.75
3,3	bDGalp	4.71	3.78	3.80	4.20	3.70	3.78-3.82
3	aDGalp	5.07	4.00	4.00	4.25	?	?
	bDGalf	4.97	4.22	4.04	4.14	3.86	3.67


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
3,3,3	aDGalp	96.7/5.19	68.7/4.07	80.4/4.16	70.5/4.29	71.9/4.24	62.3/3.75
3,3	bDGalp	105.5/4.71	71.0/3.78	78.6/3.80	66.2/4.20	76.1/3.70	62.3/3.78-3.82
3	aDGalp	100.9/5.07	68.6/4.00	80.5/4.00	?/4.25	?/?	?/?
	bDGalf	108.9/4.97	80.5/4.22	86.2/4.04	83.4/4.14	72.3/3.86	64.2/3.67

The spectrum also has 3 signals at unknown positions (not plotted).

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

Warning = sub-repeat ending at bDGalp: unknown unit count was assumed to be 3
OC[C@@H](O)[C@@H]1O[C@@H](O)[C@H](O)[C@H]1O[C@H]1O[C@H](CO)[C@H](O)[C@H](O[C@@H]2O[C@H](CO)[C@H](O)[C@H](O[C@H]3O[C@H](CO)[C@H](O)[C@H](O[C@@H]4O[C@H](CO)[C@H](O)[C@H](O[C@H]5O[C@H](CO)[C@H](O)[C@H](O[C@@H]6O[C@H](CO)[C@H](O)[C@H](O[C@H]7O[C@H](CO)[C@H](O)[C@H](O)[C@H]7O)[C@H]6O)[C@H]5O)[C@H]4O)[C@H]3O)[C@H]2O)[C@H]1O

1315.143 g/mol (C₄₈H₈₂O₄₁)