Taxonomic group: bacteria / Proteobacteria
(Phylum: Proteobacteria)
Associated disease: infection due to Escherichia coli [ICD11:
XN6P4 
]
The structure was elucidated in this paperNCBI PubMed ID: 21990359Publication DOI: 10.1074/jbc.M111.295857Journal NLM ID: 2985121RPublisher: 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, Alberta Innovates Centre for Carbohydrate Science and Department of Chemistry, The University of Alberta, Edmonton, AB, Canada
The Escherichia coli O9a O-polysaccharide (O-PS) represents a model system for glycan biosynthesis and export by the ATP-binding cassette (ABC) transporter-dependent pathway. The polymannose O9a O-PS is synthesized using an undecaprenol-diphosphate-linked acceptor by mannosyltransferases located at the cytoplasmic membrane. An ABC-transporter subsequently exports the polymer to the periplasm where it is assembled onto lipopolysaccharide prior to translocation to the cell surface. The chain length of the O9a O-PS is regulated by the dual kinase/methyltransferase activity of the WbdD enzyme and modification of the polymer is crucial for binding and export by the ABC-transporter. Previous biochemical data provided evidence for phosphorylation/methylation at the non-reducing end of the O9a O-PS but the structure of the terminus has not been determined. Here, we describe the exploitation of a synthetic O9a O-PS repeating unit carrying a fluorescent tag as an acceptor for in vitro phosphorylation and methylation by a purified soluble form of WbdD. Phosphorylation of the acceptor was evident by both a mobility shift in thin layer chromatography and radiolabeling of the acceptor using [γ-33P]ATP. Methylation of the acceptor was dependent on phosphorylation and was demonstrated by radiolabeling using S-[methyl 3H]adenosyl-methionine as a substrate, in the presence of ATP. NMR spectroscopic and mass spectrometric methods were used to determine the precise structure of the terminal modification, leading to the conclusion that WbdD catalyzes the addition of a novel methyl phosphate group to the 3-position of the non-reducing terminal mannose of the O9a O-PS repeating unit.
biosynthesis, polysaccharide, bacteria, glycosyltransferases, ABC transporter, carbohydrate structure, polymerization, export, membrane enzymes, chain termination
Structure type: oligomer
Location inside paper: p.41394, scheme 1, fig.S1
Trivial name: synthetic repeating unit acceptor
Contained glycoepitopes: IEDB_115576,IEDB_130701,IEDB_136104,IEDB_140116,IEDB_143632,IEDB_144983,IEDB_152206,IEDB_164174,IEDB_76933,IEDB_983930,SB_136,SB_196,SB_197,SB_44,SB_67,SB_72
Methods: 13C NMR, 1H NMR, methylation, NMR-2D, DNA techniques, ESI-MS, NMR-1D, genetic methods, biochemical methods, phosphorylation
Enzymes that release or process the structure: WbdD (dual kinase-methyltransferase)
Biosynthesis and genetic data: biochemical data,genetic data
Synthetic data: enzymatic
Comments, role: synthetic structure
NCBI Taxonomy refs (TaxIDs): 1010797
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There is only one chemically distinct structure:
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