Abstract
The outer membrane of gram-negative bacteria is stabilized by lipopolysaccharides (LPS). The O-antigenic polysaccharides of LPS are composed of repeating units that are exposed to and can interact with the environment. The glycosyltransferases that assemble these repeating units are encoded by the O-antigen gene cluster and utilize undecaprenol-phosphate-linked intermediates as natural acceptor substrates, and nucleotide sugars as donor substrates on the cytoplasmic face of the inner membrane. Many of the glycosyltransferase genes are known but the enzymatic functions of most of them remain to be identified. We describe here how the function of a recombinant glucosyltransferase WbdN from Escherichia coli O157 can be determined by NMR analysis of the enzyme product, using a synthetic acceptor substrate analog. A fluorescent acceptor substrate analog can be used in highly sensitive enzyme assays that allow the characterization of enzyme activity without the use of radioactive nucleotide sugar donor substrates.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Raetz CRH, Whitfield C (2002) Lipopolysaccharide endotoxins. Annu Rev Biochem 71:635–700
Duerr CU, Zenk SF, Chassin C, Pott J, Gutle D, Hensel M, Hornef MW (2009) O-antigen delays lipopolysaccharide recognition and impairs antibacterial host defense inmurine intestinal epithelial cells. PLoS Pathog 5:e1000567
Sheng H, Lim JY, Watkins MK, Minnich SA, Hovde CJ (2008) Characterization of an Escherichia coli O157:H7 O-antigen deletion mutant and effect of the deletion on bacterial persistence in the mouse intestine and colonization at the bovine terminal rectal mucosa. Appl Environ Microbiol 74:5015–5022
Whitfield C (1995) Biosynthesis of lipopolysaccharide O antigens. Trends Microbiol 3:178–185
Riley JG, Menggad M, Montoya-Peleaz P, Szarek WA, Marolda CL, Valvano MA, Schutzbach JS, Brockhausen I (2005) The wbbD gene of E. coli strain VW187 (O7:K1) encodes a UDP-Gal: GlcNAc(alpha)-pyrophosphate-R (beta)1,3-galactosyltransferase involved in the biosynthesis of O7-specific lipopolysaccharide. Glycobiology 15:605–613
Brockhausen I, Hu B, Liu B, Lau K, Szarek WA, Wang L, Feng L (2008) Characterization of two UDP-GlcNAc: β1,3-glucosyltransferases from the Escherichia coli serotypes O56 and O152. J Bacteriol 190:4922–4932
Xu C, Liu B, Hu B, Han Y, Feng L, Allingham JS, Szarek WA, Wang L, Brockhausen I (2011) Biochemical characterization of UDP-Gal:GlcNAc-pyrophosphate-lipid β-1,4-Galactosyltransferase WfeD, a new enzyme from Shigella boydii typH 14 that catalyzes the second step in O-antigen repeating-unit synthesis. J Bacteriol 193:449–459
Riley JG, Xu C, Brockhausen I (2010) Synthesis of acceptor substrate analogs for the study of glycosyltransferases involved in the second step of the biosynthesis of O antigen repeating units. Carbohydr Res 345:586–597
Montoya-Peleaz PJ, Riley JG, Szarek WA, Valvano MA, Schutzbach JS, Brockhausen I (2005) Identification of a UDP-Gal: GlcNAc-R galactosyltransferase activity in Escherichia coli VW187. Bioorg Med Chem Lett 15:1205–1211
Gao Y, Liu B, Strum S, Schutzbach JS, Druzhinina TN, Utkina NS, Torgov VI, Danilov LL, Veselovsky VV, Vlahakis JZ, Szarek WA, Wang L, Brockhausen I (2012) Biochemical characterization of WbdN, a β1,3-glucosyltransferase involved in O-antigen synthesis in enterohemorrhagic Escherichia coli O157. Glycobiology 22:1092–1102
Utkina NS, Danilov LL, Druzhinina TN, Veselovsky VV (2010) Simple synthesis of P1-(11-phenoxyundecyl)-P2-(2-acetamido-2-deoxy-α-D-galactopyranosyl)diphosphate. Russian J Bioorg Chem 36:783–785
Vinnikova AN, Druzhinina TN, Danilov LL, Utkina NS, Torgov VI, Veselovsky VV, Wang S, Liu B, Wang L, Brockhausen I (2013) Synthesis of a fluorescent acceptor substrate for glycosyltransferases involved in the assembly of O-antigens of enterohemorrhagic Escherichia coli O157 and O5. Carbohydr Res 366:17–24
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media New York
About this protocol
Cite this protocol
Gao, Y., Vinnikova, A., Brockhausen, I. (2013). Functional Identification of Bacterial Glucosyltransferase WbdN. In: Brockhausen, I. (eds) Glycosyltransferases. Methods in Molecular Biology, vol 1022. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-465-4_16
Download citation
DOI: https://doi.org/10.1007/978-1-62703-465-4_16
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-464-7
Online ISBN: 978-1-62703-465-4
eBook Packages: Springer Protocols