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Taxonomic group: bacteria / Proteobacteria (Phylum: Proteobacteria)
Associated disease: infection due to Escherichia coli [ICD11: XN6P4

]

NCBI PubMed ID: 31416837
Publication DOI: 10.1074/jbc.RA119.010323
Journal NLM ID: 2985121R
Publisher: Baltimore, MD: American Society for Biochemistry and Molecular Biology
Correspondence: cwhitfie

uoguelph.ca
Institutions: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada, Advanced Analysis Centre, University of Guelph, Guelph, ON, Canada, TEDA Institute of Biological Sciences and Biotechnology, Nankai University, 3 Hongda St. TEDA, Tianjin, China

Escherichia coli serotype O9a provides a model for export of lipopolysaccharide (LPS) O-antigen polysaccharide (O-PS) via ABC transporters. In O9a biosynthesis, a chain-terminator enzyme, WbdD, caps the nonreducing end of the glycan with a methylphosphate moiety and thereby establishes chain-length distribution. A carbohydrate-binding module (CBM) in the ABC transporter recognizes terminated glycans, ensuring that only mature O-PS is exported and incorporated into LPS. Here, we addressed two questions arising from this model. Are both residues in the binary terminator necessary for termination and export? And is a terminal methylphosphate moiety sufficient for export of heterologous glycans? To answer the first question, we uncoupled WbdD kinase and methyltransferase activities. WbdD mutants revealed that although the kinase activity is solely responsible for chain-length regulation, both activities are essential for CBM recognition and export. Consistent with this observation, a saturation transfer difference NMR experiment revealed a direct interaction between the CBM and the terminal methyl group. To determine whether methylphosphate is the sole determinant of substrate recognition by the CBM, we exploited Klebsiella pneumoniae O7, whose O-PS repeat-unit structure differs from O9a, but, as shown here, offers the second confirmed example of a terminal methylphosphate serving in substrate recognition. In vitro and in vivo experiments indicated that each CBM can bind the O-PS only with the native repeat unit, revealing that methylphosphate is essential but not sufficient for substrate recognition and export. Our findings provide important new insight into the structural determinants in a prototypical quality control system for glycan assembly and export.

glycolipid, ABC transporter, lipopolysaccharide (LPS), glycobiology, membrane transport, bacterial outer membrane, carbohydrate-binding module (CBM), glycan export

Structure type: oligomer
Location inside paper: fig.1B, E. coli O9a
Compound class: O-polysaccharide, O-antigen
Contained glycoepitopes: IEDB_115576,IEDB_130701,IEDB_135813,IEDB_136104,IEDB_137340,IEDB_140116,IEDB_141111,IEDB_141807,IEDB_141830,IEDB_143632,IEDB_144983,IEDB_151531,IEDB_152206,IEDB_164174,IEDB_164175,IEDB_164176,IEDB_174840,IEDB_241100,IEDB_76933,IEDB_983930,SB_136,SB_196,SB_197,SB_44,SB_67,SB_72

Methods: 13C NMR, 1H NMR, methylation, NMR-2D, GC-MS, SDS-PAGE, sugar analysis, DNA techniques, 31P NMR, acid hydrolysis, GLC, GC, Western blotting, STD NMR, binding assays, immunofluorescence microscopy
Enzymes that release or process the structure: WbdD, WbdA, WbdB, WbdC, WecA

Related record ID(s): 1073
NCBI Taxonomy refs (TaxIDs): 1010797
Show glycosyltransferases

There is only one chemically distinct structure:

CCCCCCCCCCCOP(=O)(O)OP(=O)(O)O[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]6O[C@H]6O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]6O[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]8O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]8O[C@H]8O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]8O[C@H]8O[C@H](CO)[C@@H](O)[C@H](O[C@H]9O[C@H](CO)[C@@H](O)[C@H](O[C@H]%10O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]%10O[C@H]%10O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]%10O[C@H]%10O[C@H](CO)[C@@H](O)[C@H](O[C@H]%11O[C@H](CO)[C@@H](O)[C@H](O[C@H]%12O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]%12O[C@H]%12O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]%12OP(=O)(O)OC)[C@@H]%11O)[C@@H]%10O)[C@@H]9O)[C@@H]8O)[C@@H]7O)[C@@H]6O)[C@@H]5O)[C@@H]4O)[C@@H]3O)[C@@H]2O)[C@H]1NC(C)=O

SVG MW SMILES 3D mol Ionize

Warning = sub-repeat ending at aDManp: unknown unit count was assumed to be 3