Glucansucrases are large enzymes belonging to glycoside hydrolase family 70, which catalyze the cleavage of sucrose into fructose and glucose, with the concomitant transfer of the glucose residue to a growing α-glucan polymer. Among others, plaque-forming oral bacteria secrete these enzymes to produce α-glucans, which facilitate the adhesion of the bacteria to the tooth enamel. We determined the crystal structure of a fully active, 1,031-residue fragment encompassing the catalytic and C-terminal domains of GTF180 from Lactobacillus reuteri 180, both in the native state, and in complexes with sucrose and maltose. These structures show that the enzyme has an α-amylase-like ðβ∕αÞ 8 -barrel catalytic domain that is circularly permuted compared to the catalytic domains of members of glycoside hydrolase families 13 and 77, which belong to the same GH-H superfamily. In contrast to previous suggestions, the enzyme has only one active site and one nucleophilic residue. Surprisingly, in GTF180 the peptide chain follows a "U"-path, such that four of the five domains are made up from discontiguous N-and C-terminal stretches of the peptide chain. Finally, the structures give insight into the factors that determine the different linkage types in the polymeric product.crystal structure complexes | exopolysaccharide | Lactobacillus reuteri | dental caries

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The bacterium Microbacterium aurum strain B8.A, originally isolated from a potato plant wastewater facility, is able to degrade different types of starch granules. Here we report the characterization of an unusually large, multidomain M. aurum B8.A ␣-amylase enzyme (MaAmyA). MaAmyA is a 1,417-amino-acid (aa) protein with a predicted molecular mass of 148 kDa. Sequence analysis of MaAmyA showed that its catalytic core is a family GH13_32 ␣-amylase with the typical ABC domain structure, followed by a fibronectin (FNIII) domain, two carbohydrate binding modules (CBM25), and another three FNIII domains. Recombinant expression and purification yielded an enzyme with the ability to degrade wheat and potato starch granules by introducing pores. Characterization of various truncated mutants of MaAmyA revealed a direct relationship between the presence of CBM25 domains and the ability of MaAmyA to form pores in starch granules, while the FNIII domains most likely function as stable linkers. At the C terminus, MaAmyA carries a 300-aa domain which is uniquely associated with large multidomain amylases; its function remains to be elucidated. We concluded that M. aurum B8.A employs a multidomain enzyme system to initiate degradation of starch granules via pore formation. Starch is an excellent carbon and energy source for many microorganisms, which employ a dedicated set of proteins for extracellular hydrolysis of this polysaccharide, uptake of shorter oligosaccharides into the cell, and further degradation into glucose. Most studies on degradation of starch by microbial enzymes have focused on soluble starch. This has resulted in the identification and characterization of a large variety of enzymes cleaving either ␣(1¡4) or ␣(1¡6) linkages in amylose and amylopectin. Most of these enzymes belong to the glycoside hydrolase 13 (GH13) family (1). Sequence diversity is such that, at the moment, the GH13 family contains a total of 40 subfamilies (1). Most of the new members in subfamilies are identified in DNA sequencing projects, and biochemical information about the activity and specificity of these potentially new enzymes is highly lagging.Many plants produce starch in a granular form for the storage of carbohydrates. The crystallinity of such granules varies with the plant source. Potato starch granules have a relatively high degree of crystallinity, making them notoriously resistant to bacterial and fungal degradation (2-4). Nevertheless, some microorganisms have been reported to employ enzymes that are able to digest granular starch (5, 6).Amylases found to be involved in granular starch degradation are often multidomain enzymes that include one or more carbohydrate binding modules (CBMs), which aid in the binding of the enzyme to the granular substrate (7-10).In previous work, various bacteria able to grow on potato starch granules as a carbon source were isolated, and their enzymatic degradation mechanism was evaluated. Initially, this resulted in the identification of an enzyme mechanism involving peeling off layer afte...

Glucansucrases are large extracellular transglycosidases secreted by lactic acid bacteria. Using sucrose as a substrate they synthesize high molecular mass a-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 threedimensional 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 Å resolution, respectively. Enzyme activity assays, pH and temperature optima show comparable values for both the full-length and the N-terminally truncated GTF180.

Glucansucrases from lactic acid bacteria convert sucrose into various α‐glucans that differ greatly with respect to the glucosidic bonds present (e.g. dextran, mutan, alternan and reuteran). This study aimed to identify the structural features of the reuteransucrase from Lactobacillus reuteri 121 (GTFA) that determine its reaction specificity. We here report a detailed mutational analysis of a conserved region immediately next to the catalytic Asp1133 (putative transition‐state stabilizing) residue in GTFA. The data show that Asn1134 is the main determinant of glucosidic bond product specificity in this reuteransucrase. Furthermore, mutations at this position greatly influenced the hydrolysis/transglycosylation ratio. Changes in this amino acid expands the range of glucan and gluco‐oligosaccharide products synthesized from sucrose by mutant GTFA enzymes.