O antigen biosynthesis in Gram-negative bacteria: Characterization of alpha1,4-glucosyltransferase WclY from Escherichia coli O117 and development of glycosyltransferase inhibitors.
Gram-negative bacteria have a polysaccharide coat (O antigen) consisting of repeating units of oligosaccharides that is assembled by glycosyltransferases. Many of these bacteria are associated with disease, and the polysaccharides are functionally important for immune responses. In light of increasing antibiotic resistance it is critical to develop new antibacterial strategies and gain knowledge of the mechanisms by which bacteria assemble their virulence factors. We hypothesize that inhibition of the glycosyltransferases responsible for O antigen synthesis in pathogenic Gram-negative bacteria can lead to the attenuation of bacteria’s virulence. The O antigens in O117 and O107 serotypes of Escherichia coli, differ by a single sugar residue. The glucose (Glc) residue found in the O antigen of E. coli O117 [D-GalNAcβ1-3-L-Rhaα1-4-D-Glcα1-4-D-Galβ1-3-D-GalNAc]n is replaced by N-acetylglucosamine (GlcNAc) in the O antigen of E. coli O107 (GalNAc is N-acetylgalactosamine, Rha is Rhamnose, Gal is galactose). The glycosyltransferases that create the Glc/GlcNAcα1-4Gal linkage are WclY(O117) and WclY(O107), and differ in only 3 amino acid residues. We have expressed WclY(O117) in a soluble and active form in the Lemo21 (DE3) expression system. We developed an activity assay for Glc-transferase WclY using a synthetic analog of its natural acceptor substrate and subsequently characterized the enzyme. WclY(O117) is a retaining Glc-transferase (sugar of the donor substrate retains α/β stereochemistry post-transfer) of the Carbohydrate Active Enzymes (CAZy) GT4 family; it has a strict substrate specificity requiring Galβ1-3GalNAcα-diphosphate lipid as the acceptor. In contrast, the donor substrate specificity is more relaxed. The structures of reaction products were confirmed using Electrospray Ionization-Negative ion mode Mass Spectrometry as well as Nuclear Magnetic Resonance Spectroscopy. Mutants were expressed to provide insight into the catalytic mechanism of WclY. Mutants transferred sugars from UDP-Glc, UDP-GlcNAc, UDP-Gal or UDP-GalNAc at varying levels. By mutating Arg194 we were able to convert the Glc-transferase to a GlcNAc-transferase. Protein modeling confirmed the role of Arg194 and other amino acids in the catalytic activity of WclY. Characterization of the enzyme has opened the door for testing of compounds with the potential to act as antimicrobial and antiseptic agents.