The Escherichia coli O9a O-polysaccharide (O-PS) is a prototype for bacterial glycan synthesis and export by an ATP-binding cassette (ABC) transporter-dependent pathway. The O9a O-PS possesses a tetrasaccharide repeat unit comprised of two α-(1→2)- and two α-(1→3)-linked mannose residues and is extended on a polyisoprenoid lipid carrier by the action of a polymerase (WbdA) containing two glycosyltransferase (GT) active sites. The N-terminal domain of WbdA possesses α-(1→2)-mannosyltransferase activity and we demonstrate in this study that the C-terminal domain is an α-(1→3)-mannosyltransferase. Previous studies established that the size of the O9a polysaccharide is determined by the chain-terminating dual kinase/methyltransferase (WbdD) that is tethered to the membrane and recruits WbdA into an active enzyme complex by protein-protein interactions. Here, we used bacterial two-hybrid analysis to identify a surface exposed alpha-helix in the C-terminal mannosyltransferase domain of WbdA as the site of interaction with WbdD. However, the C-terminal domain is unable to interact with WbdD in the absence of its N-terminal partner. Through deletion analysis, we demonstrate that the α-(1→2)-mannosyltransferase activity of the N-terminal domain is regulated by the activity of the C-terminal α-(1→3)-mannosyltransferase. In mutants where the C-terminal catalytic site is deleted, but the WbdD-interaction site remains, the N-terminal mannosyltransferase becomes an unrestricted polymerase creating a novel polymer comprised only of α-(1→2)-linked mannose residues. The WbdD protein therefore orchestrates critical localization and coordination of activities involved in chain extension and termination. Complex domain interactions are needed to position the polymerase components appropriately for assembly into a functional complex located at the cytoplasmic membrane.

Escherichia coli, O-polysaccharide, WbdA polymerase

13C NMR data:
missing...
1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
2,2,3	aDManp	5.08	?	?	?	?	?
2,2	aDManp	4.99	?	?	?	?	?
2	aDManp	5.25	?	?	?	?	?
	aDManp	5.33	?	?	?	?	?

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

*O[C@@H]1[C@H](O)[C@@H](O[C@@H]2[C@H](O)[C@@H](O[C@@H]3[C@@H](O[C@H]4[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]4*)O[C@H](CO)[C@@H](O)[C@@H]3O)O[C@H](CO)[C@H]2O)O[C@H](CO)[C@H]1O

648.564 g/mol (C₂₄H₄₀R₂O₂₀, R = next and previous repeats, not counted in mol weight)