Delta N-123-glucan-binding domain-catalytic domain 2 (Delta N-123-GBD-CD2) is a truncated form of the bifunctional glucansucrase DSR-E from Leuconostoc mesenteroides NRRL B-1299. It was constructed by rational truncation of GBD-CD2, which harbors the second catalytic domain of DSR-E. Like GBD-CD2, this variant displays alpha-(1-->2) branching activity when incubated with sucrose as glucosyl donor and (oligo-)dextran as acceptor, transferring glucosyl residues to the acceptor via a ping-pong bi-bi mechanism. This allows the formation of prebiotic molecules containing controlled amounts of alpha-(1-->2) linkages. The crystal structure of the apo alpha-(1-->2) branching sucrase Delta N-123-GBD-CD2 was solved at 1.90 angstrom resolution. The protein adopts the unusual U-shape fold organized in five distinct domains, also found in GTF180-Delta N and GTF-SI glucansucrases of glycoside hydrolase family 70. Residues forming subsite -1, involved in binding the glucosyl residue of sucrose and catalysis, are strictly conserved in both GTF180-Delta N and Delta N-123-GBD-CD2. Subsite +1 analysis revealed three residues (Ala-2249, Gly-2250, and Phe-2214) that are specific to Delta N-123-GBD-CD2. Mutation of these residues to the corresponding residues found in GTF180-Delta N showed that Ala-2249 and Gly-2250 are not directly involved in substrate binding and regiospecificity. In contrast, mutant F2214N had lost its ability to branch dextran, although it was still active on sucrose alone. Furthermore, three loops belonging to domains A and B at the upper part of the catalytic gorge are also specific to Delta N-123-GBD-CD2. These distinguishing features are also proposed to be involved in the correct positioning of dextran acceptor molecules allowing the formation of alpha-(-->2) branches.