Biochemical characterization of WbdN, a β1,3-glucosyltransferase involved in O-antigen synthesis in enterohemorrhagic Escherichia coli O157

Abstract

The enterohemorrhagic O157 strain of Escherichia coli, which is one of the most well-known bacterial pathogens, has an O-antigen repeating unit structure with the sequence [-2-d-Rha4NAcα1-3-l-Fucα1-4-d-Glcβ1-3-d-GalNAcα1-]. The O-antigen gene cluster of E. coli O157 contains the genes responsible for the assembly of this repeating unit and includes wbdN. In spite of cloning many O-antigen genes, biochemical characterization has been done on very few enzymes involved in O-antigen synthesis. In this work, we expressed the wbdN gene in E. coli BL21, and the His-tagged protein was purified. WbdN activity was characterized using the donor substrate UDP-[(14)C]Glc and the synthetic acceptor substrate GalNAcα-O-PO(3)-PO(3)-(CH(2))(11)-O-Ph. The enzyme product was isolated by high pressure liquid chromatography, and mass spectrometry showed that one Glc residue was transferred to the acceptor by WbdN. Nuclear magnetic resonance analysis of the product structure indicated that Glc was β1-3 linked to GalNAc. WbdN contains a conserved DxD motif and requires divalent metal ions for full activity. WbdN activity has an optimal pH between 7 and 8 and is highly specific for UDP-Glc as the donor substrate. GalNAcα derivatives lacking the diphosphate group were inactive as substrates, and the enzyme did not transfer Glc to GlcNAcα-O-PO(3)-PO(3)-(CH(2))(11)-O-Ph. Our results illustrate that WbdN is a specific UDP-Glc:GalNAcα-diphosphate-lipid β1,3-Glc-transferase. The enzyme is a target for the development of inhibitors to block O157-antigen synthesis.

Characterization of Two UDP-Gal:GalNAc-Diphosphate-Lipid β1,3-Galactosyltransferases WbwC from Escherichia coli Serotypes O104 and O5

“…For Gal-transferase assays, bacterial homogenates were prepared by sonication as described before (23), or purified enzyme was used as indicated. The standard assay mixtures contained, in a total of 40 l, 0.1 mM acceptor substrate GalNAc-PP-PhU (compound 8 in Fig.…”

“…Human core 1 Gal-transferase (C1GalT1), which also forms a Gal␤1-3GalNAc linkage, shows very low sequence identity to WbwC ECO104 (12%) and to WbwC ECO5 (11%). WbwC is predicted to form a GT-A glycosyltransferase fold and has been classified in the CAZy GT2 family, which includes many other bacterial and mammalian inverting ␤-glycosyltransferases (22)(23)(24)(37)(38)(39). All of the WbwC homologs have a DxD sequence that might be involved in catalysis (40).…”

“…We have characterized several Gal and Glc transferases that catalyze the second step in O antigen repeating-unit synthesis (22)(23)(24)(25). The synthesis of a natural substrate analog, GlcNAc␣-diphosphate phenylundecyl [GlcNAc␣-O-PO 3 -PO 3 -(CH 2 ) 11 -O-Ph, GlcNAc-PP-PhU], numbered 14 in Fig.…”

Biosynthesis of the Common Polysaccharide Antigen of Pseudomonas aeruginosa PAO1: Characterization and Role of GDP- d -Rhamnose:GlcNAc/GalNAc-Diphosphate-Lipid α1,3- d -Rhamnosyltransferase WbpZ

“…We presented evidence that the second step catalyzed by WbpZ can be inhibited by a number of bis-imidazolium salts having aliphatic spacer chains of between 18 and 22 carbons. The inhibition is not as strong as that of other GTs (27,35), which may be due to the fact that homogenates containing WbpZ were used. Inhibition of this critical step for LPS synthesis in P. aeruginosa could be a basis for drug development to prevent or treat P. aeruginosa infections.…”

“…It is not surprising that WbpZ, the second enzyme in the repeating-unit pathway, would require the diphosphate in the acceptor substrate (34,35). However, the dual acceptor specificity of WbpZ is unusual and suggests that the adaptor could contain a GlcNAc or a GalNAc residue.…”