Glucansucrase or glucosyltransferase (GTF) enzymes of lactic acid bacteria display high sequence similarity but catalyze synthesis of different α-glucans (e.g., dextran, mutan, alternan, and reuteran) from sucrose. The variations in glucosidic linkage specificity observed in products of different glucansucrase enzymes appear to be based on relatively small differences in amino acid sequences in their sugar-binding acceptor subsites. This notion was derived from mutagenesis of amino acids of GTFA (reuteransucrase) from Lactobacillus reuteri strain 121 putatively involved in acceptor substrate binding. A triple amino acid mutation (N1134S:N1135E:S1136V) in a region immediately next to the catalytic Asp1133 (putative transition state stabilizing residue) converted GTFA from a mainly α-(1→4) (∼45%, reuteran) to a mainly α-(1→6) (∼80%, dextran) synthesizing enzyme. The subsequent introduction of mutation P1026V:I1029V, involving two residues located in a region next to the catalytic Asp1024 (nucleophile), resulted in synthesis of an α-glucan containing only a very small percentage of α-(1→4) glucosidic linkages (∼5%) and a further increased percentage of α-(1→6) glucosidic linkages (∼85%). This changed glucosidic linkage specificity was also observed in the oligosaccharide products synthesized by the different mutant GTFA enzymes from (iso)maltose and sucrose. Amino acids crucial for glucosidic linkage type specificity of reuteransucrase have been identified in this report. The data show that a combination of mutations in different regions of GTF enzymes influences the nature of both the glucan and oligosaccharide products. The amino acids involved most likely contribute to sugar-binding acceptor subsites in glucansucrase enzymes. © 2005 American Chemical Society. Chemicals / CAS: 1,4 alpha glucan branching enzyme, 9001-97-2; dextransucrase, 9032-14-8; glucosyltransferase, 9031-48-5; maltose, 16984-36-4, 69-79-4; sucrose, 122880-25-5, 57-50-1; Bacterial Proteins; dextransucrase, EC 2.4.1.5; Glucans; Glucose, 50-99-7; Glucosyltransferases, EC 2.4.1.-; Isomaltose, 499-40-1; Maltose, 69-79-4; Sucrase, EC 3.2.1.48; Sucrose, 57-50-1