title: Small angle X-ray studies reveal that Aspergillus niger glucoamylase has a defined extended conformation and can form dimers in solution.
abstract: The industrially important glucoamylase 1 is an exo-acting glycosidase with substrate preference for alpha-1,4 and alpha-1,6 linkages at non-reducing ends of starch. It consists of a starch binding and a catalytic domain interspersed by a highly glycosylated polypeptide linker. The linker function is poorly understood and structurally undescribed, and data regarding domain organization and intramolecular functional cooperativity are conflicting or non-comprehensive. Here, we report a combined small angle x-ray scattering and calorimetry study of Aspergillus niger glucoamylase 1, glucoamylase 2, which lacks a starch binding domain, and an engineered low-glycosylated variant of glucoamylase 1 with a short linker. Low resolution solution structures show that the linker adopts a compact structure rendering a well defined extended overall conformation to glucoamylase. We demonstrate that binding of a short heterobidentate inhibitor simultaneously directed toward the catalytic and starch binding domains causes dimerization of glucoamylase and not, as suggested previously, an intramolecular conformational rearrangement mediated by linker flexibility. Our results suggest that glucoamylase functions via transient dimer formation during hydrolysis of insoluble substrates and address the question of the cooperative effect of starch binding and hydrolysis.
authors: Department of Medicinal Chemistry, University of Copenhagen, Copenhagen, Denmark.
authors: Anders Dysted
authors: AD
authors: Jørgensen
authors: Jane
authors: J
authors: Nøhr
authors: Jette Sandholm
authors: JS
authors: Kastrup
authors: Michael
authors: M
authors: Gajhede
authors: Bent W
authors: BW
authors: Sigurskjold
authors: Jørgen
authors: J
authors: Sauer
authors: Dmitri I
authors: DI
authors: Svergun
authors: Birte
authors: B
authors: Svensson
authors: Bente
authors: B
authors: Vestergaard
mesh_terms: D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics; D055592:Biophysical Phenomena; D001703:Biophysics; D002152:Calorimetry, Differential Scanning; D002240:Carbohydrate Sequence; D003432:Cross-Linking Reagents / Q000737:chemistry; D018360:Crystallography, X-Ray; D019281:Dimerization; D004791:Enzyme Inhibitors / Q000737:chemistry; D005087:Glucan 1,4-alpha-Glucosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011489:Protein Denaturation; D020836:Protein Structure, Quaternary; D012996:Solutions; D013696:Temperature
chemical_list: D003432:Cross-Linking Reagents; D004791:Enzyme Inhibitors; D012996:Solutions; D005087:Glucan 1,4-alpha-Glucosidase