Preparation of a glycosynthase from the beta-glycosidase of the Archaeon Pyrococcus horikoshii

The beta-glycosidase from the hyperthermophilic Archaeon Pyrococcus horikoshii (Phobeta-gly) is a monomeric enzyme with wide substrate specificity belonging to family 1 of glycoside hydrolases classification. Inspection of the three-dimensional structure of the enzyme, recently resolved, showed that Phobeta-gly is membrane bound and that the residues putatively involved in the catalytic activity are Glu155 and Glu324 working as the general acid/base and the nucleophile of the reaction, respectively. We show here that mutation of the latter completely eliminated the activity of the enzyme and that it could be reactivated in the presence of sodium formate. Analysis of the products obtained in the presence of sodium formate buffer pH 4.0 at 75°C showed that the Glu324Gly mutant acts as a hyperthermophilic glycosynthase.