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.
Highlights
High resolution structures are available of the individual domains, the catalytic domain (CD) [11], and the starch binding domain (SBD) [12], but structural information on full-length glucoamylase 1 form (GA1) or the linker region is very limited since the highly glycosylated linker [2, 4, 13] renders analysis of GA1 or either of the GA domains while attached to the linkerregion inherently difficult
Scanbidentate ligand consisting of acarbose and -cyclodextrin covalently coupled via an S-glycosidic linkage; molecular masses (MMs), molecular mass; radii of gyrations (Rg), radius of gyration; small-angle x-ray scattering (SAXS), small-angle X-ray scattering; IFT, indirect Fourier transformations
Further adding to the debate, in an attempt to estimate the distance between the CD and SBD sugar binding sites, synthetic heterobidentate GA inhibitors consisting of acarbose coupled either directly with -cyclodextrin or via ethylene glycol linkers with varying lengths decreased the hydrodynamic radius of GA1 by complexation [15]
Summary
Present joint SAXS and calorimetry analysis of GA1, GA2, a low-glycosylated GA1 (dgGA) as well as complexes with the heterobidentate inhibitor L0 of GA1 and dgGA, respectively. The crystal structure of the CD shows its C-terminal 31-amino acidlong segment, which constitutes the most N-terminal part of the linker region in GA1, to wrap around the globular domain in a well defined manner [11]. If this conformation recurs in the solution structure of full-length GA1, a distance of 9.5 nm between the CD and the SBD domain centers would require the remaining part of the linker to be essentially fully extended.
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