Synthesis of the O antigen repeating units of Escherichia coli serotypes O117 and O107.

Autor:	Falconer D; Department of Biomedical and Molecular Sciences, Queen's University, 18 Stuart Street, Kingston, ON K7L3N6, Canada., Melamed J; Department of Biomedical and Molecular Sciences, Queen's University, 18 Stuart Street, Kingston, ON K7L3N6, Canada., Kocev A; Department of Biomedical and Molecular Sciences, Queen's University, 18 Stuart Street, Kingston, ON K7L3N6, Canada., Bossert M; Department of Biomedical and Molecular Sciences, Queen's University, 18 Stuart Street, Kingston, ON K7L3N6, Canada., Jakeman DL; College of Pharmacy, Dalhousie University, 5968 College Street, Halifax, NS, Canada., Brockhausen I; Department of Biomedical and Molecular Sciences, Queen's University, 18 Stuart Street, Kingston, ON K7L3N6, Canada.
Jazyk:	angličtina
Zdroj:	Glycobiology [Glycobiology] 2024 Dec 10; Vol. 34 (12).
DOI:	10.1093/glycob/cwae074
Abstrakt:	Escherichia coli serotype O117 (ECO117) are pathogenic bacteria that produce Shiga toxin. Repeating units of the O antigen of ECO117 have the pentasaccharide structure [4-D-GalNAcβ1-3-L-Rhaα1-4-D-Glcα1-4-D-Galβ1-3-D-GalNAcα1-]n. The related non-pathogenic serotype (ECO107) contains a GlcNAc residue instead of Glc in the repeating unit, and the biosynthetic enzymes involved are almost identical. We assembled these repeating units based on GalNAcα-diphosphate-phenylundecyl (GalNAcα-PP-PhU), an analog of the natural intermediate GalNAc-diphosphate-undecaprenyl. We previously characterized α1,4-Glc-transferase WclY from ECO117 that transfers the Glc residue to Galβ1-3GalNAcα-PP-PhU and showed that Arg194Cys mutants of WclY are active α1,4-GlcNAc-transferases. In this work, the reaction products of WclY were used as acceptor substrates for the final enzymes in pathway, L-Rha-transferase WclX, and GalNAc-transferase WclW, demonstrating a complete synthesis of the ECO117 and O107 repeating units. WclX transfers L-Rha with high specificity for the WclY enzyme product as the acceptor and for TDP-L-Rha as the donor substrate. A number of highly conserved sequence motifs were identified (DDGSxD, DxDD, and YR). Mutational analysis revealed several Asp residues are essential for the catalysis of L-Rha transfer, while mutations of Asp44 and Arg212 substantially reduced the activity of WclX. WclW is a GT2 enzyme specific for UDP-GalNAc but with broad specificity for the acceptor substrate. Using L-Rhaα-p-nitrophenyl as an acceptor for WclW, the reaction product was analyzed by NMR demonstrating that GalNAc was transferred in a β1-3 linkage to L-Rha. The in vitro synthesis of the repeating units allows the production of vaccine candidates and identifies potential targets for inhibition of O antigen biosynthesis. (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)
Databáze:	MEDLINE
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