First crystal structure of an endo-inulinase,INU2, from Aspergillus ficuum: Discovery of an extra-pocket in the catalytic domain responsible for its endo-activity |
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Authors: | Jenny Pouyez,Auré lie Mayard,Anne-Michè le Vandamme,Guillaume Roussel,Eric A. Perpè te,Johan Wouters,Isabelle Housen,Catherine Michaux |
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Affiliation: | 1. Unité de Chimie Physique Théorique et Structurale, Chemistry Department, University of Namur, 61 rue de Bruxelles, B-5000 Namur, Belgium;2. Unité de Recherche en Biologie des Microorganismes, Biology Department, University of Namur, Namur, Belgium |
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Abstract: | Endo-inulinase is a member of glycosidase hydrolase family 32 (GH32) degrading fructans of the inulin type with an endo-cleavage mode and is an important class of industrial enzyme. In the present study, we report the first crystal structure of an endo-inulinase, INU2, from Aspergillus ficuum at 1.5 Å. It was solved by molecular replacement with the structure of exo-inulinase as search model. The 3D structure presents a bimodular arrangement common to other GH32 enzymes: a N-terminal 5-fold β-propeller catalytic domain with four β-sheets and a C-terminal β-sandwich domain organized in two β-sheets with five β-strands. The structural analysis and comparison with other GH32 enzymes reveal the presence of an extra pocket in the INU2 catalytic site, formed by two loops and the conserved motif W-M(I)-N-D(E)-P-N-G. This cavity would explain the endo-activity of the enzyme, the critical role of Trp40 and particularly the cleavage at the third unit of the inulin(-like) substrates. Crystal structure at 2.1 Å of INU2 complexed with fructosyl molecules, experimental digestion data and molecular modelling studies support these hypotheses. |
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Keywords: | Endo-inulinase Glycoside hydrolase Crystal structure Enzyme mechanism Molecular modelling |
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