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Functional Characterization of Xyloglucan Glycosynthases from GH7, GH12, and GH16 Scaffolds

TitleFunctional Characterization of Xyloglucan Glycosynthases from GH7, GH12, and GH16 Scaffolds
Publication TypeJournal Article
Year of Publication2009
AuthorsGullfot, F, Ibatullin, FM, Sundqvist, G, Davies, GJ, Brumer, H

Glycosynthases, hydrolytically inactive mutant glycosidases that catalyze glycosylation reactions using glycosyl fluoride donor substrates, are emerging as useful tools for the synthesis of large, complex polysaccharides [Faijes, M.; Planas, A. Carbohydr. Res. 2007, 342, 1581-1594]. Guided by wild-type xyloglucanase activity, we have produced and characterized new glycosynthases for the synthesis of xyloglucan oligo- and polysaccharides, based on family GH7, GH12, and GH16 scaffolds. The Humicola insolens GH7 glycosynthase, HiCe17B E197S, is capable of synthesizing nongalactosylated, XXXG-based homoxyloglucan up to M(w) 60000 [G = Glc beta(1 -> 4); X = Xyl alpha(1 -> 6)Glc beta(1 -> 4); L = Gal beta(1 -> 2)Xyl alpha(1 -> 6)Glc beta(1 -> 4)], which is among the largest products so far obtained with glycosynthase technology. Novel glycosynthases based on the GH16 xyloglucan hydrolase from Tropaeolum majus (nasturtium), TmNXG1, are capable of synthesizing XLLG-based xyloglucan oligosaccharides at rates feasible for preparative synthesis, thus providing an essential expansion of product range. Finally, a new glycosynthase based on the recently characterized GH12 xyloglucanase from Bacillus licheniformis, BlXG12 E155A, can perform the condensation of xyloglucosyl fluorides, albeit at poor rates. Altogether, the high catalytic efficiency demonstrated by HiCe17B E197S and the extended product range provided by TmNXG1 E94A are key achievements toward a robust and versatile method for the preparative synthesis of homogeneous xyloglucans with regular substitution patterns not available in nature. Such compounds enable in vitro experimental studies regarding the role of particular structural elements for xyloglucan properties and its interaction with cellulose.