Utilization of Fluorescently Tagged Synthetic Acceptor Molecules for In Vitro Characterization of a Dual-Domain Glycosyltransferase Enzyme, KpsC, from Escherichia coli

  • Liam Doyle
  • Olga G. Ovchinnikova
  • Chris WhitfieldEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1954)


The incorporation of fluorescent tags into synthetic acceptor molecules for in vitro biochemical assays allows quick and easy detection of enzyme activity. Reaction products can be separated via thin-layer chromatography and visualized under UV light for rapid detection of reaction progress. Subsequent structural analysis of these reaction products through the use of NMR spectroscopy and mass spectrometry allows for complete functional characterization of enzyme activity. Here we describe an application of this technique which was previously used to functionally characterize a dual-domain glycosyltransferase enzyme, KpsC, involved in capsular polysaccharide biosynthesis in Escherichia coli.

Key words

Microbial glycobiology Capsular polysaccharide Glycosyltransferase In vitro biochemical reaction Fluorescently-tagged synthetic acceptor 



We thank Bo-Shun Huang, Matthew S. Kimber, and Todd L. Lowary for their contributions to this project originally published as reference 6. This work was supported by operating funding from the Canadian Institutes of Health Research, the National Science and Engineering Research Council of Canada, and the Canadian Glycomics Network (GlycoNet, National Centres of Excellence Program).


  1. 1.
    Montoya-Peleaz PJ, Riley JG, Szarek WA et al (2005) Identification of a UDP-gal: GlcNAc-R galactosyltransferase activity in Escherichia coli VW187. Bioorg Med Chem Lett 15:1205–1211CrossRefGoogle Scholar
  2. 2.
    Clarke BR, Richards MR, Greenfield LK et al (2011) In vitro reconstruction of the chain termination reaction in biosynthesis of the Escherichia coli O9a O-polysaccharide. J Biol Chem 286:41391–41401CrossRefGoogle Scholar
  3. 3.
    Ovchinnikova OG, Mallette E, Koizumi A et al (2016) Bacterial β-Kdo glycosyltransferases represent a new glycosyltransferase family (GT99). Proc Natl Acad Sci 113:E3120–E3129CrossRefGoogle Scholar
  4. 4.
    Whitfield C (2006) Biosynthesis and assembly of capsular polysaccharides in Escherichia coli. Annu Rev Biochem 75:39–68CrossRefGoogle Scholar
  5. 5.
    Willis LM, Whitfield C (2013) KpsC and KpsS are retaining 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) transferases involved in synthesis of bacterial capsules. Proc Natl Acad Sci U S A 110:20753–20758CrossRefGoogle Scholar
  6. 6.
    Ovchinnikova OG, Doyle L, Huang BS et al (2017) Biochemical characterization of bifunctional 3-deoxy-β-D-manno-oct-2-ulosonic acid (β-Kdo) transferase KpsC from Escherichia coli involved in capsule biosynthesis. J Biol Chem 291:21519–21530CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Liam Doyle
    • 1
  • Olga G. Ovchinnikova
    • 1
  • Chris Whitfield
    • 1
    Email author
  1. 1.Department of Molecular and Cellular BiologyUniversity of GuelphGuelphCanada

Personalised recommendations