Characterization of the UDP-Glc/GlcNAc 4-epimerase AglW from the archaeon Methanococcus maripaludis using glycosyltransferase coupled assays
Archaea are ubiquitous single-celled microorganisms that have often adapted to harsh conditions and play important roles in biogeochemical cycles with potential applications in biotechnology. Methanococcus maripaludis, a methane-producing archaeon, is motile through multiple archaella on its cell surface. The major structural proteins (archaellins) of the archaellum are glycoproteins, modified with the N-linked tetrasaccharide: 4-deoxy-5-O-Me-ManNAc β1-4 ManNAc3NAmA6Thr β1-4 GlcNAc3NAcA β1-3 GalNAc β-Asn, where the sugar 4-deoxy-5-O-Me-ManNAc is found exclusively in this species. The attachment of the N-linked tetrasaccharide to archaellins is required for proper motility of M. maripaludis. The aglW gene encoding the putative UDP-Glc/GlcNAc 4-epimerase AglW plays a key role in the biosynthesis of the unique terminal sugar residue found in the N-linked tetrasaccharide. The goal of this study was to biochemically demonstrate the 4-epimerase activity of AglW, and to develop glycosyltransferase (GT) coupled assays to determine its substrate specificity and properties. We carried out coupled assays using UDP-Gal, UDP-Glc, UDP-GlcNAc, UDP-GalNAc and GlcNAc/GalNAc-diphosphate – lipid as substrates. We established that AglW is active without the addition of divalent metal ions or NAD+ and has a broad specificity towards UDP-sugars. We also showed that tyrosine-151 within a conserved YxxxK sequon is essential for the 4-epimerase function of AglW. The GT-coupled assays are generally useful for the identification and specificity studies of novel 4-epimerases. The results of this work provide insight into the synthesis of complex N-glycans in M. maripaludis.