Conversion of inverting glycoside hydrolases into catalysts for synthesizing glycosides employing a glycosynthase strategy

Reducing-end xylose-releasing exo-oligoxylanase (Rex, EC. 3.2.1.156) is an inverting xylanolytic enzyme, belonging to the glycoside hydrolase (GH) family 8, which hydrolyzes xylooligosaccharides to release xylose (Xi) from its reducing end. Rex hydrolyzes α-xylobiosyl fluoride (α-X ₂F) to yield xylobiose (X₂) only in the presence of X ₁, confirming the Hehre resynthesis-hydrolysis mechanism. A library of mutant Rex at the catalytic base (D263) was constructed by saturation mutagenesis, in which D263C accumulated the highest level of xylotriose (X ₃) from α-X₂F and X₁. However, F ^- releasing activities of the mutants were much less than that of the wild type. Next, Y198 residue of Rex that forms a hydrogen bond with nucleophilic water was substituted with phenylalanine, causing a marked decrease in hydrolytic activity and a small increase in the F^- releasing activity from α-X₂F in the presence of X₁. Y198F of Rex accumulated more product during the glycosynthase reaction than D263C. Recently, an inverting α-1,2-fucosidase belonging to GH95 was converted into glycosynthase by mutating a catalytic base residue. In both cases, the catalytic base should be intact.