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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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.
Original languageEnglish
Pages (from-to)2423-2430
Number of pages8
JournalBiochimie
Volume94
Issue number11
DOIs
Publication statusPublished - 1 Nov 2012

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inulinase
Aspergillus
Glycoside Hydrolases
Catalytic Domain
Hydrolases
Crystal structure
Inulin
Enzymes
Fructans
Molecular modeling
Propellers
Structural analysis
Data structures
Digestion
Molecules
Substrates

Keywords

  • Endo-inulase
  • Glycoside hydrolase
  • crystal structure
  • Enzyme mechanism
  • Molecular modelling

Cite this

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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 {\AA}. 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 {\AA} of INU2 complexed with fructosyl molecules, experimental digestion data and molecular modelling studies support these hypotheses.",
keywords = "Endo-inulase, Glycoside hydrolase, crystal structure, Enzyme mechanism, Molecular modelling",
author = "J. Pouyez and A. Mayard and A.-M. Vandamme and G. Roussel and E.A. Perp{\`e}te and J. Wouters and I. Housen and C. Michaux",
note = "Copyright 2012 Elsevier B.V., All rights reserved.",
year = "2012",
month = "11",
day = "1",
doi = "10.1016/j.biochi.2012.06.020",
language = "English",
volume = "94",
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T1 - First crystal structure of an endo-inulinase, INU2, from Aspergillus ficuum

T2 - Discovery of an extra-pocket in the catalytic domain responsible for its endo-activity

AU - Pouyez, J.

AU - Mayard, A.

AU - Vandamme, A.-M.

AU - Roussel, G.

AU - Perpète, E.A.

AU - Wouters, J.

AU - Housen, I.

AU - Michaux, C.

N1 - Copyright 2012 Elsevier B.V., All rights reserved.

PY - 2012/11/1

Y1 - 2012/11/1

N2 - 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.

AB - 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.

KW - Endo-inulase

KW - Glycoside hydrolase

KW - crystal structure

KW - Enzyme mechanism

KW - Molecular modelling

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