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 reuteransucrase GTFA from Lactobacillus reuteri 121, which belongs to glycosyl hydrolase family GH70, synthesizes branched α-glucans with both α-1,6- and α-1,4-glycosidic linkages (reuteran) from sucrose. The crystal structure of GTFA-ΔN, a 118 kDa fragment of GTFA comprising residues 745-1763 and including the catalytic domain, was determined at 3.6 Å resolution by molecular replacement. The crystals have large solvent channels and an unusually high solvent content of 85%. GTFA-ΔN has the same domain arrangement and domain topologies as observed in previously determined GH70 glucansucrase structures. The architecture of the GTFA-ΔN active site and binding pocket confirms that glucansucrases have a conserved substrate specificity for sucrose. However, this first crystal structure of an α-1,6/α-1,4-specific glucansucrase shows that residues from conserved sequence motif IV (1128-1136 in GTFA-ΔN) contribute to the acceptor-binding subsites and that they display differences compared with other structurally characterized glucansucrases. In particular, the structure clarifies the importance of residues following the transition-state stabilizer for product specificity, and especially residue Asn1134, which is in a position to interact with sugar units in acceptor subsite +2.

SummaryGlycan utilization plays a key role in modulating the composition of the gut microbiota, but molecular insight into oligosaccharide uptake by this microbial community is lacking. Arabinoxylo-oligosaccharides (AXOS) are abundant in the diet, and are selectively fermented by probiotic bifidobacteria in the colon. Here we show how selectivity for AXOS uptake is established by the probiotic strain Bifidobacterium animalis subsp. lactis Bl-04. The binding protein BlAXBP, which is associated with an ATP-binding cassette (ABC) transporter that mediates the uptake of AXOS, displays an exceptionally broad specificity for arabinosyl-decorated and undecorated xylooligosaccharides, with preference for tri-and tetrasaccharides. Crystal structures of BlAXBP in complex with four different ligands revealed the basis for this versatility. Uniquely, the protein was able to recognize oligosaccharides in two opposite orientations, which facilitates the optimization of interactions with the various ligands. Broad substrate specificity was further enhanced by a spacious binding pocket accommodating decorations at different mainchain positions and conformational flexibility of a lid-like loop. Phylogenetic and genetic analyses show that BlAXBP is highly conserved within Bifidobacterium, but is lacking in other gut microbiota members. These data indicate niche adaptation within Bifidobacterium and highlight the metabolic syntrophy (cross-feeding) among the gut microbiota.

PDB References: native A. niger -amylase, 2guy, r2guysf; maltose-bound A. niger -amylase, 2gvy, r2gvysf.Aspergillus niger -amylase catalyses the hydrolysis of -1,4-glucosidic bonds in starch. It shows 100% sequence identity to the A. oryzae homologue (also called TAKA-amylase), three crystal structures of which have been published to date. Two of them belong to the orthorhombic space group P2 1 2 1 2 1 with one molecule per asymmetric unit and one belongs to the monoclinic space group P2 1 with three molecules per asymmetric unit. Here, the purification, crystallization and structure determination of A. niger -amylase crystallized in the monoclinic space group P2 1 with two molecules per asymmetric unit in complex with maltose at 1.8 Å resolution is reported. Furthermore, a novel 1.6 Å resolution orthorhombic crystal form (space group P2 1 2 1 2) of the native enzyme is presented. Four maltose molecules are observed in the maltose--amylase complex. Three of these occupy active-site subsites À2 and À1, +1 and +2 and the hitherto unobserved subsites +4 (Asp233, Gly234) and +5 (Asp235). The fourth maltose molecule binds at the distant binding sites d1 (Tyr382) and d2 (Trp385), also previously unobserved. Furthermore, it is shown that the activesite groove permits different binding modes of sugar units at subsites +1 and +2. This flexibility of the active-site cleft close to the catalytic centre might be needed for a productive binding of substrate chains and/or release of products.

Glucansucrase enzymes synthesize high-molecular-mass extracellular aglucan polysaccharides from sucrose. Previously, the crystal structure of truncated glucansucrase glucosyltransferase (GTF)180-DN from Lactobacillus reuteri 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 a-1,6/a-1,3 specific glucansucrase GTF180-DN 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 Xray 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~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-DN (and likely also the full-length GTF180) shows conformational flexibility; this may be a general feature of GH70 glucansucrases.

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.

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