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 ␣-1,4 and ␣-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.Glucoamylase (GA, 3 1,4-␣-D-glucan glucohydrolase; EC 3.2.1.3; glycoside hydrolase family 15) is an exo-acting glycosidase that cleaves ␣-1,4 and, less efficiently, ␣-1,6 linkages at non-reducing ends of starch and related oligo-and polysaccharides (1, 2). GA is industrially important in production of bioethanol, glucose, and fructose syrups. The glucoamylase 1 form (GA1) from Aspergillus niger has an N-terminal catalytic domain (CD) and a C-terminal starch binding domain (SBD) connected by a 69-amino acid-long linker that is decorated by short, predominantly mannose-containing O-glycosylations corresponding to a minimum content of 63 mol of hexose attached to about 32 serines and threonines (2-4) (see the schematic in Fig. 1). The functional role of the linker region is not fully understood. The isolated CD and the GA2 form (Fig. 1), which includes the linker and lacks the SBD, are both able to hydrolyze soluble substrates, but not starch granules, in contrast to GA1 (4 -6). Moreover, it has been shown that the addition of free SBD to a mixture of GA2 and starch granules increases the rate of hydrolysis (7). The SBD is, therefore, suggested to enhance the substrate accessibility by disentangling ␣-glucan helices on the surface of the starch granule rather than to act as an intramolecular guide directing the substrate chain to the active site pocket of the CD (7, 8). Conformational constraints on the linker are not required in this function and engineered linker variants, low-glycosylated or shortened ones inclusive, behave very similarly to wild-type GA1. Thus, the specific sequence of the linker seems to have a modest if any effect on the action of GA1 (9). This is much in contr...

Maize AKINβγ dimerizes through the KIS/CBM domain and assembles into SnRK1 complexes

“…In this context, homodimerization of the CBM domain could represent a regulatory mechanism to facilitate or prevent the interaction with carbohydrates, which might in turn modulate the activity of the SnRK1 complex. This is in accordance with biochemical analyses showing that a KIS/CBM-related domain present in glucoamylase from Aspergillus niger establishes transient dimeric associations that are critical for binding and hydrolysis of starch [26].…”

Raw starch conversion by Saccharomyces cerevisiae expressing Aspergillus tubingensis amylases

“…The delay may be ascribed to the need for dimerization of the glucoamylase prior to its functioning on insoluble starch, as was described for the A . niger glucoamylase [35]. …”

The carbohydrate‐binding module family 20 – diversity, structure, and function

“…The rather compact conformation and the orientation of the SBD relative to the catalytic domain suggests that the SBD is important in directing the enzyme to regions where the starch granular structure is disrupted. By contrast, the low‐resolution structure of the intact GA1 from A. niger in solution, recently determined with the aid of small‐angle X‐ray scattering, reveals an extended conformation where the highly O ‐glycosylated polypeptide linker separates the two domains of the enzyme [63]. Interestingly, the linker of the A. niger GA is 22 amino acids longer than that of H. jecorina .…”

“…Hence, some CBM20s are true modules separated from catalytic domains by O‐glycosylated polypeptide linkers, as in the case of A. niger GA and several other fungal GAs. There is still a debate about the conformations of such enzymes in solution, with data on the H. jecorina GA structure [62] supporting a compact conformation, and small‐angle X‐ray scattering studies showing that A. niger GA has an extended conformation [63].…”