Characterization of the UDP-Glc/GlcNAc 4-epimerase AglW from the archaeon Methanococcus maripaludis using glycosyltransferase coupled assays
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Authors
Sharma, Sulav
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Type
thesis
Language
eng
Keyword
UDP-hexose 4-epimerase , Methanococcus maripaludis , AglW
Alternative Title
Abstract
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.
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ProQuest PhD and Master's Theses International Dissemination Agreement
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This publication is made available by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner.