Tuning the transglycosylation reaction of a GH11 xylanase by a delicate enhancement of its thumb flexibility.

Glycoside hydrolases (GH) are attractive tools for multiple biotechnological applications. In conjunction with their hydrolytic function, GH can perform transglycosylation reaction under specific conditions. In nature, oligosaccharides synthesis is performed by glycosyltransferase (GT). However, the industrial utilization of GT is limited by their instability in solution. A key difference between GT and GH is the flexibility of their binding sites architecture. In this report, we used the xylanase from  Bacillus circulans  (BCX) to study the interplay between active site flexibility and the transglycosylation reaction. Residues of the BCX thumb were substituted to increase the flexibility of the enzyme binding site. Replacement of the highly conserved residue P116 with glycine shifted the balance of the BCX enzymatic reaction toward transglycosylation. The effects of this point mutation on the structure and dynamics of BCX were investigated by NMR spectroscopy. The P116G mutation induces subtle changes in the configuration of the thumb and enhances the millisecond dynamics of the active site. Based on our findings, we propose the remodeling of the GH enzymes glycon site flexibility as a strategy to improve the transglycosylation efficiency of these biotechnologically important catalysts.