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

Biochimie. 2012 Nov;94(11):2423-30. doi: 10.1016/j.biochi.2012.06.020. Epub 2012 Jun 28.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Aspergillus / enzymology*
  • Biocatalysis
  • Catalytic Domain*
  • Crystallography, X-Ray
  • Glycoside Hydrolases / chemistry*
  • Glycoside Hydrolases / metabolism*
  • Kinetics
  • Molecular Docking Simulation
  • Molecular Sequence Data

Substances

  • Glycoside Hydrolases
  • inulinase