Edited by Stuart FergusonKeywords: Dextran glucosidase a-GlucosidaseOligo-1,6-glucosidase Substrate specificity Glycoside hydrolase family 13 a b s t r a c t Glycoside hydrolase family 13 contains exo-glucosidases specific for a-(1 ? 4)-and a-(1 ? 6)-linkages including a-glucosidase, oligo-1,6-glucosidase, and dextran glucosidase. The a-(1 ? 6)-linkage selectivity of Streptococcus mutans dextran glucosidase was altered to a-(1 ? 4)-linkage selectivity through site-directed mutations at Val195, Lys275, and Glu371. V195A showed 1300-fold higher k cat /K m for maltose than wild-type, but its k cat /K m for isomaltose remained 2-fold higher than for maltose. K275A and E371A combined with V195A mutation only decreased isomaltase activity. V195A/K275A, V195A/E371A, and V195A/K275A/E371A showed 27-, 26-, and 73-fold higher k cat /K m for maltose than for isomaltose, respectively. Consequently, the three residues are structural elements for recognition of the a-(1 ? 6)-glucosidic linkage.