Structural determination and biosynthetic studies of the O-antigenic polysaccharide from the enterohemorrhagic Escherichia coli O91 using 13C-enrichment and NMR spectroscopy

The structure of the O-antigenic polysaccharide from the enterohemorrhagic Escherichia coli O91 has been determined using primarily NMR spectroscopy on the C-13-enriched polysaccharide. The O-antigen is composed of pentasaccharide repeating units with the following structure: -->4)-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->4)-beta-D-GlcpA-6-N-Gly-(1-->3)-beta-D-GlcpNAc-(1-->4)-alpha-D-Quip-3-N-[(R)-3-hydroxybutyramido]-(1-->. The bacterium was grown with D-[UL-C-13]glucose in the medium which resulted in an overall degree of labeling of similar to 65% in the sugar residues and similar to 50% in the N-acyl substituents, indicating some metabolic dilution in the latter. The C-13-enrichment of the polysaccharide proved valuable since NMR assignments could be made on the basis of C-13,C-13-connectivity inuniformly labeled residues. The biosynthesis of the (R)-3-hydroxybutyramido substituent via C-2 fragments was identified by NMR spectroscopy, The (R)-configuration at C3 is in accord with fatty acid biosynthesis. Additional cultures with specifically labeled D-[1-C-13]glucose or D-[6-C-13]glucose corroborated the direct incorporation of glucose as the building block for the hexose skeletons in the polysaccharide and the biosynthesis of acyl substituents occurring via the triose pool followed by decarboxylation to give acetyl building blocks labeled with C-13 at the methyl group.