A novel Leuconostoc mesenteroides NRRL B-1299 dextransucrase gene, dsrE, was isolated, sequenced, and cloned in Escherichia coli, and the recombinant enzyme was shown to be an original glucansucrase which catalyses the synthesis of ␣-1,6 and ␣-1,2 linkages. The nucleotide sequence of the dsrE gene consists of an open reading frame of 8,508 bp coding for a 2,835-amino-acid protein with a molecular mass of 313,267 Da. This is twice the average mass of the glucosyltransferases (GTFs) known so far, which is consistent with the presence of an additional catalytic domain located at the carboxy terminus of the protein and of a central glucan-binding domain, which is also significantly longer than in other glucansucrases. From sequence comparison with family 70 and ␣-amylase enzymes, crucial amino acids involved in the catalytic mechanism were identified, and several original sequences located at some highly conserved regions in GTFs were observed in the second catalytic domain.Glucansucrase enzymes from oral streptococci, Leuconostoc mesenteroides strains, and some Lactobacillus and Neisseria sp. catalyze the transfer of glucosyl residues from sucrose to synthesize ␣-D-glucopyranosyl homopolymers and oligomers. When sucrose is the sole substrate, high-molecular-weight polymers are obtained. Depending on the glucansucrase-producing strain, the synthesized glucans differ in size and structure. When efficient acceptors, such as maltose or isomaltose, are added to the reaction medium, glucansucrases catalyze the synthesis of low-molecular-weight oligosaccharides and the regiospecificity of several dextransucrases (type of linkages) from the Leuconostoc genus is conserved in oligosaccharide synthesis (8,13,41,45).To date, 17 glucosyltransferase (GTF)-encoding genes from Streptococcus spp., 8 glucansucrase-encoding genes from L. mesenteroides, and 1 gene from Lactobacillus reuteri have been cloned (for reviews, see references 3, 16, 32, and 59). Sequence information shows that they are closely related and share a common structure. These genes code for large enzymes with an average molecular mass of 160,000 Da composed of two main domains: an N-terminal conserved catalytic core of about 900 amino acids and a C-terminal domain covering 300 to 400 residues thought to be responsible for glucan binding and constituted by a series of repeated units (32). In addition, biochemical studies revealed that glucansucrases share many mechanistic features with amylolytic enzymes (15). Structural homologies were later confirmed by amino acid sequence comparison with glucoside hydrolases from family 13 (20). In family 13, the catalytic domain is formed of eight ␤-sheets alternating with eight ␣-helices, conferring a (␤/␣) 8 barrel structure (55). Two structure predictions (9, 26) concluded that glucansucrase enzymes also possess a catalytic (␤/␣) 8 barrel domain. However, MacGregor et al. (26) observed that well-recognized sequence segments appear in a different order, which tends to show that the ␤/␣ barrel elements are circularly permutate...