Characterization of the six glycosyltransferases involved in the biosynthesis of Yersinia enterocolitica serotype O:3 lipopolysaccharide outer core

J Biol Chem. 2010 Sep 3;285(36):28333-42. doi: 10.1074/jbc.M110.111336. Epub 2010 Jul 1.


Yersinia enterocolitica (Ye) is a gram-negative bacterium; Ye serotype O:3 expresses lipopolysaccharide (LPS) with a hexasaccharide branch known as the outer core (OC). The OC is important for the resistance of the bacterium to cationic antimicrobial peptides and also functions as a receptor for bacteriophage phiR1-37 and enterocoliticin. The biosynthesis of the OC hexasaccharide is directed by the OC gene cluster that contains nine genes (wzx, wbcKLMNOPQ, and gne). In this study, we inactivated the six OC genes predicted to encode glycosyltransferases (GTase) one by one by nonpolar mutations to assign functions to their gene products. The mutants expressed no OC or truncated OC oligosaccharides of different lengths. The truncated OC oligosaccharides revealed that the minimum structural requirements for the interactions of OC with bacteriophage phiR1-37, enterocoliticin, and OC-specific monoclonal antibody 2B5 were different. Furthermore, using chemical and structural analyses of the mutant LPSs, we could assign specific functions to all six GTases and also revealed the exact order in which the transferases build the hexasaccharide. Comparative modeling of the catalytic sites of glucosyltransferases WbcK and WbcL followed by site-directed mutagenesis allowed us to identify Asp-182 and Glu-181, respectively, as catalytic base residues of these two GTases. In general, conclusive evidence for specific GTase functions have been rare due to difficulties in accessibility of the appropriate donors and acceptors; however, in this work we were able to utilize the structural analysis of LPS to get direct experimental evidence for five different GTase specificities.

MeSH terms

  • Antibodies, Monoclonal / metabolism
  • Bacteriophages / metabolism
  • Catalytic Domain
  • Computational Biology
  • Drug Resistance, Bacterial
  • Galactose / chemistry
  • Galactose / metabolism
  • Glycosyltransferases / chemistry
  • Glycosyltransferases / genetics
  • Glycosyltransferases / metabolism*
  • Lipopolysaccharides / biosynthesis*
  • Lipopolysaccharides / chemistry*
  • Models, Molecular
  • Multigene Family
  • Mutagenesis, Site-Directed
  • O Antigens / chemistry
  • O Antigens / metabolism
  • Oligosaccharides / chemistry
  • Oligosaccharides / metabolism
  • Polymyxin B / pharmacology
  • Yersinia enterocolitica / drug effects
  • Yersinia enterocolitica / enzymology*
  • Yersinia enterocolitica / genetics
  • Yersinia enterocolitica / metabolism


  • Antibodies, Monoclonal
  • Lipopolysaccharides
  • O Antigens
  • Oligosaccharides
  • Glycosyltransferases
  • Polymyxin B
  • Galactose