GH52 XynB2 and GH10 XynA Xylosynthases: Kinetic and Structural Studies for Efficient Synthesis of Xylooligosaccharides

Glycosynthases are retaining glycoside hydrolases (GH) in which the native catalytic nucleophile is replaced by a small non-nucleophilic residue. In the presence of an activated glycosyl fluoride donor with the opposite anomeric configuration from the original substrate of the parental GH, these mutants are able to catalyze the transfer of the glycosyl donor to a suitable acceptor sugars, thus synthesizing oligo- and polysaccharides. This enzymatic synthesis of saccharides provides an attractive alternative to the classical chemical methods, since it enables a complete control over the newly generated anomeric centers, and allows the reaction to be carried out 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, rigorous kinetic analyses and crystal structures of the obtained mutants, an improved variant of XynB2 glycosynthase was isolated. This variant contained two crucial amino acid substitutions, F206L and T343P, enhancing the glycosynthetic activity by a 100-fold compared to XynB2 E335G, with k_cat values of 85 sec^-1 and 0.8 sec^-1, respectively.
GH10 XynA E265G is a nucleophile deficient xylanase that in the presence of α-D-xylobiosyl fluoride can synthesize xylo-oligosaccharides ranging from 6 up to over 100 xylose units. The kinetic constants of XynA E265G were determined to be 0.2 sec^-1 and 15 mM for k_cat and K_M, respectively. By combining the two glycosynthases in the same reaction xylo-oligosaccharides were obtained from α-D-xylopyranosyl fluoride.