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

J Biol Chem. 2011 Dec 2;286(48):41391-41401. doi: 10.1074/jbc.M111.295857. Epub 2011 Oct 11.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Carbohydrate Conformation
  • Carbohydrate Metabolism / physiology*
  • Carbohydrate Sequence
  • Escherichia coli / genetics
  • Escherichia coli / metabolism*
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism
  • Magnetic Resonance Spectroscopy
  • Methylation
  • Methyltransferases / genetics
  • Methyltransferases / metabolism
  • Organophosphates / metabolism
  • Phosphorylation / physiology
  • Polysaccharides, Bacterial / biosynthesis*
  • Polysaccharides, Bacterial / genetics


  • Escherichia coli Proteins
  • Organophosphates
  • Polysaccharides, Bacterial
  • methylphosphate
  • Methyltransferases