Biochemical Characterization of UDP-Gal:GlcNAc-Pyrophosphate-Lipid β-1,4-Galactosyltransferase WfeD, a New Enzyme fromShigella boydiiType 14 That Catalyzes the Second Step in O-Antigen Repeating-Unit Synthesis

Abstract

enhanced the enzyme activity. Mutational analysis showed that the Glu101 residue within a DxD motif is essential for activity, possibly by forming the catalytic nucleophile. The Lys211 residue was also shown to be required for activity and may be involved in the binding of the negatively charged acceptor substrate. Our study revealed that the ␤4-GalT WfeD is a novel enzyme that has virtually no sequence similarity to mammalian ␤4-GalT, although it catalyzes a similar reaction.Lipopolysaccharides (LPSs) consist of O-polysaccharide (Oantigenic) side chains covalently linked to a core polysaccharide and lipid A. LPSs are found in the outer membranes of Gram-negative bacteria, where they contribute to the structural integrity of the membrane and interact with the external environment (9, 10, 15). In the complex and dynamic microbial ecosystem of the human intestine, the communication between microorganisms and the gastrointestinal (GI) epithelium involves O-antigen and LPS binding molecules. Thus, the elimination of the O antigen may reduce virulence (2,16,21). Shigella is a genus of highly adapted bacterial pathogens that cause gastrointestinal disease, such as bacillary dysentery or shigellosis. A recent survey showed that shigellosis causes approximately 165 million cases of severe dysentery and more than 1 million deaths per year, mostly in children from developing countries (10). Shigella strains are categorized into four groups: S. boydii, S. dysenteriae, S. flexneri, and S. sonnei, each containing multiple subgroups of different serotypes, based on structural variations in their O antigens.O antigens consist of repeating units of oligosaccharides that are assembled individually, followed by the polymerization of units to form O antigens of different lengths. The glycosyltransferases involved in the biosynthesis of O antigens play a critical role in determining O-antigen structural diversity. The pentasaccharide repeating unit of S. boydii type 14 (B14) has the structure [36-D-Galp␣134-D-GlcpA␤136-D-Galp␤134-D-Galp␤134-D-GlcpNAc␤13]n (12), suggesting the existence of five specific glycosyltransferases: a GlcNAc-phosphotransferase (WecA), three Gal-transferases, and a glucuronosyltransferase.Three distinct processes for the synthesis and translocation of O antigens have been described: the Wzx/Wzy-dependent pathway, the ATP binding cassette transporter-dependent process, and the synthase-dependent process (20,25,26). The biosynthesis of the S. boydii B14 O antigen that contains a variety of different sugar residues is expected to utilize the * Corresponding author. Mailing address for Lei

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

“…Mixtures were incubated for 10 min at 37°C, and reactions were quenched by the addition of 700 l of ice-cold water and freezing. Enzyme reaction product was isolated using Sep-Pak C 18 columns, eluted first in water and then in MeOH, and quantified by scintillation counting as described previously (22,25). High-pressure liquid chromatography (HPLC) separations were carried out as described previously (24), using a C 18 column and acetonitrile-water as the mobile phase.…”

“…There are many positively charged amino acid residues in WbwC ECO104 and WbwC ECO5 that may possibly be involved in the binding of the negatively charged substrates, and we previously identified an essential Lys residue in Gal-transferase WfeD (25). The inclusion of 0.2 mM HPG in the assay mixture (without DTT) for purified WbwC enzymes showed 22% inhibition of WbwC ECO104 activity and minimal inhibition of WbwC ECO5 activity (Fig.…”

“…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

“…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.…”

Identification and Biochemical Characterization of the Novel α2,3-Sialyltransferase WbwA from Pathogenic Escherichia coli Serotype O104

“…WbwC O104 is the second enzyme in the pathway of O104 antigen synthesis and requires the diphosphate in the acceptor (GalNAc-PP-PhU). Thus far, the second enzymes in the repeating unit assembly sequence have all been shown to have an absolute requirement for at least one phosphate group in the acceptor (20,23,(31)(32)(33)(34). WbwA O104 is the first example of the third enzyme in the pathway and of a eukaryotic or bacterial SiaT that has a requirement for diphosphate in the acceptor.…”