Scanning tunnelling microscopy of Aspergillus niger glucoamylases

Kramer, G.F.H.; Gunning, A. Patrick; Morris, Victor J.; Belshaw, Nigel J.; Williamson, Gary

doi:10.1039/ft9938902595

Cited by 25 publications

(21 citation statements)

References 21 publications

(2 reference statements)

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“…The approximate distance of 10 nm between the centers of masses for the CD and SBD, which is also revealed by the pairwise distance distribution function of GA1 (Fig. 2B), corresponds well with scanning tunneling microscopy analysis (14). Kramer and co-workers (14), however, do not exclude the existence of a flexible linker in solution, because scanning tunneling microscopy requires dehydration of the protein, which possibly results in changes conferring non-natural facets to protein structure.…”

Section: Glucoamylase Has a Rigid Extended Conformation In Solution-mentioning

confidence: 67%

“…2B), corresponds well with scanning tunneling microscopy analysis (14). Kramer and co-workers (14), however, do not exclude the existence of a flexible linker in solution, because scanning tunneling microscopy requires dehydration of the protein, which possibly results in changes conferring non-natural facets to protein structure. One of the great advantages of SAXS analysis is that there is no need for specific experimental conditions, allowing the data to be collected e.g.…”

Section: Glucoamylase Has a Rigid Extended Conformation In Solution-mentioning

confidence: 91%

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Small Angle X-ray Studies Reveal That Aspergillus niger Glucoamylase Has a Defined Extended Conformation and Can Form Dimers in Solution

Jørgensen

Nøhr

Kastrup

et al. 2008

Journal of Biological Chemistry

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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...

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Section: Glucoamylase Has a Rigid Extended Conformation In Solution-mentioning

confidence: 67%

Section: Glucoamylase Has a Rigid Extended Conformation In Solution-mentioning

confidence: 91%

Small Angle X-ray Studies Reveal That Aspergillus niger Glucoamylase Has a Defined Extended Conformation and Can Form Dimers in Solution

Jørgensen

Nøhr

Kastrup

et al. 2008

Journal of Biological Chemistry

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show abstract

“…This region is known to be structurally rigid (Ito et al 1991) and there is no evidence of proteolytic cleavage indicating that such a linker would not be susceptible to rapid proteolysis. Other, semi-rigid linkers have been reported, such as that of glucoamylase 1 (Kramer et al 1993), which could be used to ful®l a similar role. The oc-IDD86/go/cpti construct directed expression of a fusion protein that remained primarily intact as a ca.…”

Section: Transgenementioning

confidence: 98%

Enhanced transgenic plant resistance to nematodes by dual proteinase inhibitor constructs

Urwin

McPherson

Atkinson

1998

Planta

148

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Plant defence strategies usually involve the action of several gene products. Transgenic resistance strategies are likely to have enhanced efficacy when they involve more than one transgene. Here we explore possible mechanisms for the co-delivery of multiple effectors via a single transgene. As an example we report the co-delivery of two distinct proteinase inhibitors in Arabidopsis thaliana (L.) Heynh. to examine resistance against plant parasitic nematodes. A cysteine and serine proteinase inhibitor have been joined as translational fusions by one of two peptide linkers. One linker, part of the spacer region of a plant metallothionein-like protein (PsMTa), was selected to be cleaved in planta. A second linker, derived from the fungal enzyme galactose oxidase (GO) was chosen to be refractory to cleavage in planta. Western blot analysis of cell extracts confirmed the expected pattern of predominantly single inhibitors derived from the PsMTa construct and a primarily dual inhibitor from the GO construct. Analysis of cyst and root-knot nematodes recovered from transgenic Arabidopsis expressing inhibitors as single or dual molecules revealed the uptake of inhibitors with the exception of those linked by the PsMTa linker. This unexpected result may be due to residues of the PsMTa linker interacting with cell membranes. Despite lack of ingestion, PsMTa-linked cowpea trypsin inhibitor (CpTI) affected the sexual development of the cyst nematodes, indicating an external site of action. The engineered cystatin (Oc-I delta D86) component from the PsMTa constuct had no effect, indicating that ingestion is necessary for the cystatin to be effective. The delivery of dual inhibitors linked by the GO linker showed a clear additive effect over either inhibitor delivered singly. The application of this technology to other plant pathogens is discussed.

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“…Three domains have been identified: the N-terminal catalytic domain (residues 1 to 470, 55 kDa), the C-terminal granular starch binding domain (SBD; residues 509 to 616, 12 kDa), and a short but bulky linker (residues 471 to 508, 13 kDa) which is heavily O-glycosylated at the abundant serine and threonine residues (Svensson et al, 1983). The linker joins the two main domains giving the enzyme an overall dumbbell shape which has been observed clearly by scanning tunnelling microscopy (Kramer et al, 1993).…”

Section: Introductionmentioning

confidence: 93%

Solution Structure of the Granular Starch Binding Domain of Glucoamylase fromAspergillus nigerby Nuclear Magnetic Resonance Spectroscopy

Sorimachi

Jacks

Gal-Coeffet

et al. 1996

Journal of Molecular Biology

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125

113

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Scanning tunnelling microscopy of Aspergillus niger glucoamylases

Cited by 25 publications

References 21 publications

Small Angle X-ray Studies Reveal That Aspergillus niger Glucoamylase Has a Defined Extended Conformation and Can Form Dimers in Solution

Small Angle X-ray Studies Reveal That Aspergillus niger Glucoamylase Has a Defined Extended Conformation and Can Form Dimers in Solution

Enhanced transgenic plant resistance to nematodes by dual proteinase inhibitor constructs

Solution Structure of the Granular Starch Binding Domain of Glucoamylase fromAspergillus nigerby Nuclear Magnetic Resonance Spectroscopy

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