title: 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.
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.
authors: Unité de Chimie Physique Théorique et Structurale, Chemistry Department, University of Namur, 61 rue de Bruxelles, B-5000 Namur, Belgium.
authors: Jenny
authors: J
authors: Pouyez
authors: Aurélie
authors: A
authors: Mayard
authors: Anne-Michèle
authors: AM
authors: Vandamme
authors: Guillaume
authors: G
authors: Roussel
authors: Eric A
authors: EA
authors: Perpète
authors: Johan
authors: J
authors: Wouters
authors: Isabelle
authors: I
authors: Housen
authors: Catherine
authors: C
authors: Michaux
mesh_terms: D000595:Amino Acid Sequence; D001230:Aspergillus / Q000201:enzymology*; D055162:Biocatalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data
chemical_list: D006026:Glycoside Hydrolases; C023086:inulinase