The wclY gene of Escherichia coli serotype O117 encodes an α1, 4-glucosyltransferase with strict acceptor specificity but broad donor specificity. (18th May 2020)

• Record Type: Journal Article
• Authors: Kocev, Alexander; Melamed, Jacob; Torgov, Vladimir; Danilov, Leonid; Veselovsky, Vladimir; Brockhausen, Inka
• Is Part Of: Glycobiology. Volume 30:Number 12(2020)
• Page Start: 9003
• Page End: 9014
• Publication Date: 2020-05-18
• Subjects: E. coli O117 -- GlcNAc-transferase -- Glc-transferase -- mutations -- structural analysis; Glycoproteins -- Periodicals; Glycolipids -- Periodicals; Glycoconjugates -- Periodicals; 572.567
• DOI: 10.1093/glycob/cwaa045
• Languages: English
• ISSNs: 0959-6658

Abstract

Abstract: The O antigen of enterotoxigenic Escherichia coli serotype O117 consists of repeating units with the structure [-D-GalNAcβ1-3-L-Rhaα1-4-D-Glcα1-4-D-Galβ1-3-D-GalNAcα1-4]n . A related structure is found in E. coli O107 where Glc is replaced by a GlcNAc residue. The O117 and O107 antigen biosynthesis gene clusters are homologous and reveal the presence of four putative glycosyltransferase (GT) genes, wclW, wclX, wclY and wclZ, but the enzymes have not yet been biochemically characterized. We show here that the His6 -tagged WclY protein expressed in E. coli Lemo21(DE3) cells is an α1, 4-Glc-transferase that transfers Glc to the Gal moiety of Galβ1-3GalNAcα-OPO3 -PO3 -phenoxyundecyl as a specific acceptor and that the diphosphate moiety of this acceptor is required. WclY utilized UDP-Glc, TDP-Glc, ADP-Glc, as well as UDP-GlcNAc, UDP-Gal or UDP-GalNAc as donor substrates, suggesting an unusual broad donor specificity. Activity using GDP-Man suggested the presence of a novel Man-transferase in Lemo21(DE3) cells. Mutations of WclY revealed that both Glu residues of the Ex7 E motif within the predicted GT domain are essential for activity. High GlcNAc-transferase (GlcNAc-T) activities of WclY were created by mutating Arg194 to Cys. A triple mutant identical to WclY in E. coli O107 was identified as an α1, 4 GlcNAc-T. The characterization of WclY opens the door for the development of antibacterial approaches.