Kinetic and structural studies reveal two crucial residues enhancing GH52 XynB2 glycosynthetic activity by a 100-fold

Glycosynthases are engineered mutants of retaining glycoside hydrolases (GH) lacking the native hydrolytic activity that are able to synthesize polysaccharides. In these enzymes, the catalytic nucleophile is replaced by a small residue and in the presence of an activated glycosyl fluoride donor, with the opposite anomeric configuration from the original substrate of the parental GH, they are able to catalyze the transfer of the glycosyl donor to a suitable acceptor sugars. Enzymatic synthesis of oligosaccharides provides an attractive alternative to the classical synthetic chemical methods, since it enables a complete control over newly generated anomeric centers, and the reaction can be performed in aqueous solution under mild conditions.

The β-xylosidase GH52 XynB2 nucleophilic mutant (E335G) from Geobacillus stearothermophilus has already proven to be useful for glycosynthesis applications. This enzyme can catalyze self-condensation reaction of α-D-xylopyranosyl fluoride, providing mainly α-D-xylobiosyl fluoride. By using directed evolution, two improved variants (V27, V29) of XynB2 (E335G) containing in total ten mutations were isolated. The crystal structures of XynB2 E335G and the improved variants, with the glycosyl-fluoride donor and the product bound to the active site, were obtained. These structures, together with rigorous kinetic analysis of selected mutations allowed to pinpoint two crucial amino acid substitutions, F206L and T343P, contributing to a 100-fold improvement in glycosynthase activity compared to XynB2 E335G, with k_cat values of 85 sec^-1 and 0.8 sec^-1, respectively.

α-D-xylobiosyl fluoride produced by XynB2 (E335G) can be further utilized as a substrate by two GH10 xylansynthases, XynA (E265G) and XynA2 (E241G), from G. stearothermophilus to produce xylo-oligosaccharides ranging from 6 up to over 100 xylose units.