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Identification and biochemical characterization of WbwB, a novel UDP-Gal: Neu5Ac-R α1,4-galactosyltransferase from the intestinal pathogen Escherichia coli serotype O104

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Abstract

The intestinal pathogen Escherichia coli serotype O104:H4 (ECO104) can cause bloody diarrhea and haemolytic uremic syndrome. The ECO104 O antigen has the unique repeating unit structure [4Galα1–4Neu5,7,9Ac3α2–3Galβ1–3GalNAcβ1-], which includes the mammalian sialyl-T antigen as an internal structure. Previously, we identified WbwC from ECO104 as the β3Gal-transferase that synthesizes the T antigen, and showed that α3-sialyl-transferase WbwA transfers sialic acid to the T antigen. Here we identify the wbwB gene product as a unique α1,4-Gal-transferase WbwB that transfers Gal from UDP-Gal to the terminal sialic acid residue of Neu5Acα2–3Galβ1–3GalNAcα-diphosphate-lipid acceptor. NMR analysis of the WbwB enzyme reaction product indicated that Galα1-4Neu5Acα2–3Galβ1–3GalNAcα-diphosphate-lipid was synthesized. WbwB from ECO104 has a unique acceptor specificity for terminal sialic acid as well as the diphosphate group in the acceptor. The characterization studies showed that WbwB does not require divalent metal ion as a cofactor. Mutagenesis identified Lys243 within an RKR motif and both Glu315 and Glu323 of the fourth EX7E motif as essential for the activity. WbwB is the final glycosyltransferase in the biosynthesis pathway of the ECO104 antigen repeating unit. This work contributes to knowledge of the biosynthesis of bacterial virulence factors.

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Acknowledgements

Mass spectrometry was carried out by J. Wang, Department of Chemistry, Queen’s University. The authors thank F. Sauriol, Department of Chemistry, Queen’s University for NMR analyses and J.S. Schutzbach and J. Allingham for helpful advice. We are grateful to R.Gerardy-Schahn for plasmids encoding SiaD constructs. This work was funded by the Canadian Institutes of Health Research and the Natural Sciences and Engineering Council of Canada.

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  1. Department of Biomedical and Molecular Sciences, Queen’s University, 18 Stuart St., Kingston, ON, K7L3N6, Canada

    Diana Czuchry & Inka Brockhausen

  2. Department of Chemistry, Queen’s University, 90 Bader Lane, Kingston, ON, K7L3N6, Canada

    Walter A. Szarek

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  1. Diana Czuchry
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Correspondence to Inka Brockhausen.

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Czuchry, D., Szarek, W.A. & Brockhausen, I. Identification and biochemical characterization of WbwB, a novel UDP-Gal: Neu5Ac-R α1,4-galactosyltransferase from the intestinal pathogen Escherichia coli serotype O104. Glycoconj J 35, 65–76 (2018). https://doi.org/10.1007/s10719-017-9799-y

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