Small angle X-ray studies reveal that Aspergillus niger glucoamylase has a defined extended conformation and can form dimers in solution

TY - JOUR

T1 - Small angle X-ray studies reveal that Aspergillus niger glucoamylase has a defined extended conformation and can form dimers in solution

AU - Jørgensen, Anders Dysted

AU - Nøhr, Jane

AU - Kastrup, Jette Sandholm

AU - Gajhede, Michael

AU - Sigurskjold, Bent Walther

AU - Sauer, Jørgen

AU - Svergun, Dmitri I.

AU - Svensson, Birte

AU - Vestergaard, Bente

N1 - Keywords: Amino Acid Sequence; Aspergillus niger; Biophysical Phenomena; Biophysics; Calorimetry, Differential Scanning; Carbohydrate Sequence; Cross-Linking Reagents; Crystallography, X-Ray; Dimerization; Enzyme Inhibitors; Glucan 1,4-alpha-Glucosidase; Glycosylation; Models, Molecular; Molecular Sequence Data; Mutation; Protein Denaturation; Protein Structure, Quaternary; Solutions; Temperature

PY - 2008

Y1 - 2008

N2 - 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.

AB - 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.

KW - Former Faculty of Pharmaceutical Sciences

U2 - 10.1074/jbc.M801709200

DO - 10.1074/jbc.M801709200

M3 - Journal article

C2 - 18378674

SN - 0021-9258

VL - 283

SP - 14772

EP - 14780

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 21

ER -