The<i>wclY</i>gene of<i>Escherichia coli</i>serotype O117 encodes an α1,4-glucosyltransferase with strict acceptor specificity but broad donor specificity

The O157:H7 strain of Escherichia coli is responsible for frequent outbreaks of hemorrhagic colitis worldwide. Its lipopolysaccharide is a virulence factor and contains an O antigen having repeating units with the tetrasaccharide structure [2-D-PerNAcα1-3-L-Fucα1-4-D-Glcβ1-3-D-GalNAcα1-]n. Genes encoding glycosyltransferases WbdN, WbdO, and WbdP are responsible for the biosynthesis of this repeating unit. We have previously characterized the second enzyme in the pathway, WbdN, which transfers Glc in β1-3 linkage to GalNAcα-O-PO3-PO3-(CH2)11-O-Ph (GalNAc-PP-PhU). In this work, Fuc-transferase WbdO from E. coli O157:H7 expressed in BL21 bacteria was characterized using the product of WbdN as the acceptor substrate. We showed that WbdO is specific for GDP-β-L-Fuc as the donor substrate. Compounds that contained terminal Glc or Glcβ1-3GalNAc structures but lacked the diphosphate group did not serve as acceptor substrates. The structure of the WbdO product was identified by mass spectrometry and Nuclear magnetic resonance (NMR) as L-Fucα1-4-D-Glcβ1-3-D-GalNAc PP-PhU. WbdO is an unusual bivalent metal ion-dependent Fuc-transferase classified as an inverting GT2 family enzyme that has 2 conserved sequences near the N-terminus. The Asp37 residue within the 36VDGGSTD42 sequence was found to be essential for catalysis. Mutation of Asp68 to Ala within the conserved 67YDAMNK72 sequence resulted in a 3-fold increase in activity. These studies show that WbdOO157 is a highly specific Fuc-transferase with little homology to other characterized Fuc-transferases.

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

Falconer,

Melamed,

Kocev

et al. 2024

Glycobiology

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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 R212 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.

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ThewclYgene ofEscherichia coliserotype O117 encodes an α1,4-glucosyltransferase with strict acceptor specificity but broad donor specificity

Cited by 4 publications

References 41 publications

Biosynthesis of Bacterial Polysaccharides

Biosynthesis of Bacterial Polysaccharides

Biosynthesis of the O antigen of pathogenicEscherichia coliO157:H7. Characterization of α1,4-Fuc-transferase WbdO

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

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