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1.  Structure of the α-1,6/α-1,4-specific glucansucrase GTFA from Lactobacillus reuteri 121 
A 118 kDa fragment, comprising the catalytic domain and four other domains, of the glucansucrase GTFA from L. reuteri 121, which synthesizes α-glucans with both α-1,6- and α-1,4-glycosidic linkages, was crystallized. The weakly diffracting crystals, which contained 85% solvent, were used to determine the structure at 3.6 Å resolution.
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.
doi:10.1107/S1744309112044168
PMCID: PMC3509963  PMID: 23192022
lactic acid bacteria; glucansucrase; reuteransucrase
2.  Monoclinic crystal form of Aspergillus niger α-­amylase in complex with maltose at 1.8 Å resolution 
Two new crystal structures of A. niger α-amylase are reported, one of which reveals two hitherto unobserved maltose-binding sites.
Aspergillus niger α-amylase catalyses the hydrolysis of α-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 P212121 with one molecule per asymmetric unit and one belongs to the monoclinic space group P21 with three molecules per asymmetric unit. Here, the purification, crystallization and structure determination of A. niger α-amylase crystallized in the monoclinic space group P21 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 P21212) of the native enzyme is presented. Four maltose molecules are observed in the maltose–α-amylase complex. Three of these occupy active-site subsites −2 and −1, +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 active-site 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.
doi:10.1107/S1744309106024729
PMCID: PMC2242925  PMID: 16880540
α-amylase; Aspergillus niger; maltose; Aspergillus oryzae; TAKA-amylase

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