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Glucansucrase enzymes synthesize high-molecular-mass extracellular -glucan polysaccharides from sucrose. Previously, the crystal structure of truncated glucansucrase glucosyltransferase (GTF)180-N from Lactobacillusreuteri 180 (lacking the N-terminal domain) revealed an elongated overall structure with two remote domains (IV and V) extending away from the core. By contrast, a new crystal form of the -1,6/-1,3 specific glucansucrase GTF180-N shows an approximate 120(o) rotation of domain V about a hinge located between domains IV and V, giving a much more compact structure than before. Positional variability of domain V in solution is confirmed by small angle X-ray scattering experiments and rigid-body ensemble calculations. In addition, small angle X-ray scattering measurements of full-length GTF180 also provide the first structural data for a full-length glucansucrase, showing that the enzyme has an almost symmetric boomerang-like molecular shape, with a bend likely located between domains IV and V. The similar to 700-residue N-terminal domain, which is not present in the crystal structures, extends away from domain V and the catalytic core of the enzyme. We conclude that, as a result of the hinge region, in solution, GTF180-N (and likely also the full-length GTF180) shows conformational flexibility; this may be a general feature of GH70 glucansucrases.Database center dot Structural data for GTF180-N II have been deposited in the Protein Data Bank under accession code .

Glucansucrases are large extracellular transglycosidases secreted by lactic acid bacteria. Using sucrose as a substrate they synthesize high molecular mass alpha-glucans or, in the presence of suitable acceptor molecules, low molecular mass oligosaccharides. Although about 60 glucansucrases have been classified in glycoside hydrolase family GH70, no three-dimensional structure has been reported for any. With the aim of solving the first structure of a GH70 glucansucrase, purification and crystallization experiments were performed with a fully active, 117 kDa N-terminally truncated fragment of glucansucrase GTF180 from Lactobacillus reuteri 180 (residues 742-1772). Crystallization experiments yielded crystals that belong to two different triclinic crystal forms (space group P1) and one orthorhombic crystal form (space group P2(1)2(1)2(1)). Native data sets for both triclinic and the orthorhombic crystals were collected at 1.7 and 2.0 angstrom resolution, respectively. Enzyme activity assays, pH and temperature optima show comparable values for both the full-length and the N-terminally truncated GTF180.