title: Characterization of the alpha-1,2-N-acetylglucosaminyltransferase of Neisseria gonorrhoeae, a key control point in lipooligosaccharide biosynthesis.
abstract: The biosynthesis of the lipooligosaccharide (LOS) in Neisseria meningitidis has a control point that regulates the extension of the alpha-chain on heptose (I) of the LOS. The gene that encodes the protein responsible for this control had been identified elsewhere, but the enzyme encoded by the gene was not characterized. We have now shown that this same control mechanism operates in the related species, Neisseria gonorrhoeae, using a gene knockout and subsequent characterization of the LOS species produced. We also cloned and expressed the enzyme from both of these pathogens. Using a synthetic acceptor substrate, we have shown unequivocally that the enzyme is an alpha-1,2-N-acetylglucosaminyltransferase. Experiments with both the core oligosaccharide and the synthetic acceptors suggests that the addition of the alpha-1,2-N-acetylglucosamine moiety on the heptose (II) residue precedes the addition of the ethanolamine phosphate at the O3 position on this heptose (II), and that in the absence of the alpha-1,2-N-acetylglucosamine moiety leads to the addition of an extra ethanolamine phosphate on the heptose (II) residue. Our data do not support the hypothesis that ethanolamine phosphate at O3 of heptose (II) is added and is then required for the addition of the N-acetylglucosamine at O2 by the LgtK enzyme. This enzyme represents a control point in the biosynthesis of the LOS of this pathogen and is a potential target for therapeutic intervention.
authors.affiliation: National Research Council of Canada, Institute for Biological Sciences, 100 Sussex Drive, Ottawa, Ontario, K1A 0R6 Canada. warren.wakarchuk@nrc-cnrc.gc.ca
authors.forename: Warren
authors.initials: W
authors.lastname: Wakarchuk
authors.forename: Melissa J
authors.initials: MJ
authors.lastname: Schur
authors.forename: Frank
authors.initials: F
authors.lastname: St  Michael
authors.forename: Jinjuan
authors.initials: J
authors.lastname: Li
authors.forename: Eva
authors.initials: E
authors.lastname: Eichler
authors.forename: Dennis
authors.initials: D
authors.lastname: Whitfield
mesh_terms: D002236:Carbohydrate Conformation; D002855:Chromatography, Thin Layer; D008070:Lipopolysaccharides / Q000096:biosynthesis* / Q000737:chemistry; D013058:Mass Spectrometry; D017351:N-Acetylglucosaminyltransferases / Q000737:chemistry* / Q000378:metabolism; D009344:Neisseria gonorrhoeae / Q000201:enzymology*; D019906:Nuclear Magnetic Resonance, Biomolecular; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D008070:Lipopolysaccharides; D011994:Recombinant Proteins; D017351:N-Acetylglucosaminyltransferases