title: In vitro reconstruction of the chain termination reaction in biosynthesis of the Escherichia coli O9a O-polysaccharide: the chain-length regulator, WbdD, catalyzes the addition of methyl phosphate to the non-reducing terminus of the growing glycan.
abstract: 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-(33)P]ATP. Methylation of the acceptor was dependent on phosphorylation and was demonstrated by radiolabeling using S-[methyl-(3)H]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.
authors: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
authors: Bradley R
authors: BR
authors: Clarke
authors: Alberta Innovates Centre for Carbohydrate Science and Department of Chemistry, The University of Alberta, Edmonton, Alberta T6G 2G2, Canada.
authors: Michele R
authors: MR
authors: Richards
authors: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
authors: Laura K
authors: LK
authors: Greenfield
authors: Alberta Innovates Centre for Carbohydrate Science and Department of Chemistry, The University of Alberta, Edmonton, Alberta T6G 2G2, Canada.
authors: Dianjie
authors: D
authors: Hou
authors: Alberta Innovates Centre for Carbohydrate Science and Department of Chemistry, The University of Alberta, Edmonton, Alberta T6G 2G2, Canada.
authors: Todd L
authors: TL
authors: Lowary
authors: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada. Electronic address: cwhitfie@uoguelph.ca.
authors: Chris
authors: C
authors: Whitfield
mesh_terms: D002236:Carbohydrate Conformation; D050260:Carbohydrate Metabolism / Q000502:physiology*; D002240:Carbohydrate Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008745:Methylation; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism; D010755:Organophosphates / Q000378:metabolism; D010766:Phosphorylation / Q000502:physiology; D011135:Polysaccharides, Bacterial / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D029968:Escherichia coli Proteins; D010755:Organophosphates; D011135:Polysaccharides, Bacterial; C027265:methylphosphate; D008780:Methyltransferases