Xyn52B2 Glycosynthase: Kinetic and Structural Studies Reveal Crucial Residues Important for Glycosynthetic Activity

Glycosynthases are catalytic mutants of retaining glycoside hydrolases that catalyze the synthesis of oligosaccharides from their corresponding glycosyl-fluoride donors and suitable acceptors. In these enzymes, the nucleophile is replaced by a smaller non-nucleophilic residue. 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 Xyn52B2 nucleophilic mutant (E335G) from Geobacillus stearothermophilus has already proven to be useful for glycosynthesis applications. The enzyme can catalyze the self-condensation reaction of
α-D-xylopyranosyl fluoride, providing mainly α-D-xylobiosyl fluoride (1).
By using two cycles of directed evolution, an improved variant of Xyn52B2-E335G was isolated containing in total ten random mutations (2). Recently, the crystal structures of Xyn52B2-E335G and its improved variant, with the glycosyl-fluoride donor and the product bound to the active site, were obtained. These 3D structures, together with rigorous kinetic analysis of selected mutations (out of the ten random ones) allowed to pinpoint two crucial amino acid substitutions, F206L and T343P, contributing to a 100-fold improvement in glycosynthase activity compared to Xyn52B2-E335G.

1. A. Ben-David et al., Glycosynthase Activity of Geobacillus stearothermophilus GH52 β-Xylosidase: Efficient Synthesis of Xylooligosaccharides from a-D-Xylopyranosyl Fluoride, ChemBioChem, 2007, Vol. 8, 2145-2151
2. A. Ben-David, G. Shoham, Y. Shoham, A Universal Screening Assay for Glycosynthases: Directed Evolution of Glycosynthase XynB2(E335G) Suggests a General Path to Enhance Activity, Chemistry and Biology, 2008, Vol. 15, 546-551