Journal of Biological Chemistry
Volume 288, Issue 46, 15 November 2013, Pages 32991-33005
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Protein Structure and Folding
Aspergillus niger β-Glucosidase Has a Cellulase-like Tadpole Molecular Shape: INSIGHTS INTO GLYCOSIDE HYDROLASE FAMILY 3 (GH3) β-GLUCOSIDASE STRUCTURE AND FUNCTION*

https://doi.org/10.1074/jbc.M113.479279Get rights and content
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Aspergillus niger is known to secrete large amounts of β-glucosidases, which have a variety of biotechnological and industrial applications. Here, we purified an A. niger β-glucosidase (AnBgl1) and conducted its biochemical and biophysical analyses. Purified enzyme with an apparent molecular mass of 116 kDa forms monomers in solution as judged by native gel electrophoresis and has a pI value of 4.55, as found for most of the fungi of β-glucosidases. Surprisingly, the small angle x-ray experiments reveal that AnBgl1 has a tadpole-like structure, with the N-terminal catalytic domain and C-terminal fibronectin III-like domain (FnIII) connected by the long linker peptide (∼100 amino acid residues) in an extended conformation. This molecular organization resembles the one adopted by other cellulases (such as cellobiohydrolases, for example) that frequently contain a catalytic domain linked to the cellulose-binding module that mediates their binding to insoluble and polymeric cellulose. The reasons why AnBgl1, which acts on the small soluble substrates, has a tadpole molecular shape are not entirely clear. However, our enzyme pulldown assays with different polymeric substrates suggest that AnBgl1 has little or no capacity to bind to and to adsorb cellulose, xylan, and starch, but it has high affinity to lignin. Molecular dynamics simulations suggested that clusters of residues located in the C-terminal FnIII domain interact strongly with lignin fragments. The simulations showed that numerous arginine residues scattered throughout the FnIII surface play an important role in the interaction with lignin by means of cation-π stacking with the lignin aromatic rings. These results indicate that the C-terminal FnIII domain could be operational for immobilization of the enzyme on the cell wall and for the prevention of unproductive binding of cellulase to the biomass lignin.

Enzyme Mechanisms
Enzyme Structure
Enzymes
Fungi
Glycobiology
Aspergillus niger
SAXS
β-Glucosidase
Glycoside Hydrolase Family 3
Multidomain Proteins

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*

This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo Grants 08/56255-9, 09/54035-4, and 10/16542-9], Conselho Nacional de Desenvolvimento Científico e Tecnológico Grant 490022/2009-0, Coordenação de Aperfeiçoamento de Pessoal de Nivel Superior and Instituto Nacional de Ciência e Tecnologia do Bioetanol.

This article contains supplemental Figs. S1 and S2 and Table S1.