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Replacement of the catalytic nucleophile Asp481 by glycine in Schizosaccharomyces pombe α-glucosidase eliminated the hydrolytic activity. The mutant enzyme (D481G) was found to catalyze the formation of an α-glucosidic linkage from β-glucosyl fluoride and 4-nitrophenyl (PNP) α-glucoside to produce two kinds of PNP α-diglucosides, α-isomaltoside and α-maltoside. The two products were not hydrolyzed by D481G, giving 41 and 29% yields of PNP α-isomaltoside and α-maltoside, respectively. PNP monoglycosides, such as α-xyloside, α-mannoside, or β-glucoside, acted as the substrate, but PNP α-galactoside and maltose could not. No detectable product was observed in the combination of α-glucosyl fluoride and PNP α-glucoside. This study is the first report on an “α-glycosynthase”-type reaction to form an α-glycosidic linkage.