Fructan beta-fructosidase

 

Exo-inulase from Aspergillus awamori (a fungus) is a member of the glycoside hydrolase family 32. It catalyses the hydrolysis of terminal, non-reducing 2,1- and 2,6-linked beta-D-fructofuranose residues in fructans. Fructans are used as food storage for some plant species, and exo-inulase is involved in their breakdown into fructose.

 

Reference Protein and Structure

Sequence
Q96TU3 UniProt (3.2.1.80) IPR001362 (Sequence Homologues) (PDB Homologues)
Biological species
Aspergillus awamori (Fungus) Uniprot
PDB
1y9m - Crystal structure of exo-inulinase from Aspergillus awamori in spacegroup P212121 (1.89 Å) PDBe PDBsum 1y9m
Catalytic CATH Domains
2.115.10.20 CATHdb (see all for 1y9m)
Click To Show Structure

Enzyme Reaction (EC:3.2.1.80)

water
CHEBI:15377ChEBI
+
Levanbiose
CHEBI:6435ChEBI
→
D-fructofuranose
CHEBI:37721ChEBI
+
D-fructofuranose
CHEBI:37721ChEBI
Alternative enzyme names: Exo-beta-D-fructosidase, Exo-beta-fructosidase, Fructan exohydrolase, Polysaccharide beta-fructofuranosidase, Fructanase,

Enzyme Mechanism

Introduction

Exo-inulase catalyses a double displacement mechanism, resulting in retention of configuration. Asp 41 acts as a nucleophile, attacking the carbon atom of the glycosidic bond. As the glycosidic bond is broken, Glu 241 acts as a general acid by donating a proton to the leaving group oxygen atom. Glu 241 then acts as a general base, deprotonating a water molecule, and activating it for nucleophilic attack on the carbon atom. As HO- nucleophilically attacks, Asp 41 is the leaving group.

Catalytic Residues Roles

UniProt PDB* (1y9m)
Asp41 Asp41(22)A Asp acts as a nucleophile, and attacks the carbon atom of the glycosidic bond. covalently attached, nucleofuge, nucleophile
Glu241 Glu241(222)A Glu 241 donates a proton to the leaving group oxygen atom. It also deprotonates a water molecule, activating it for nucleophilic attack. proton acceptor, proton donor, activator, increase nucleophilicity, promote heterolysis
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

overall product formed, overall reactant used, proton transfer, bimolecular nucleophilic substitution, intermediate formation, intermediate terminated, hydrolysis

References

  1. Nagem RA et al. (2004), J Mol Biol, 344, 471-480. Crystal Structure of Exo-inulinase from Aspergillus awamori: The Enzyme Fold and Structural Determinants of Substrate Recognition. DOI:10.1016/j.jmb.2004.09.024. PMID:15522299.
  2. Holyavka M et al. (2016), Biocatal Biotransformation, 34, 1-17. Structural and functional properties of inulinases: A review. DOI:10.1080/10242422.2016.1196486.
  3. Pouyez J et al. (2012), Biochimie, 94, 2423-2430. 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. DOI:10.1016/j.biochi.2012.06.020. PMID:22750808.

Step 1. Asp41 performs a nucleophilic attack on the glycosidic bond, while Glu241 protonates the leaving group. This leads to the formation of an enzyme-substrate intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp41(22)A covalently attached
Glu241(222)A promote heterolysis, proton donor
Asp41(22)A nucleophile

Chemical Components

overall product formed, overall reactant used, proton transfer, ingold: bimolecular nucleophilic substitution, intermediate formation

Step 2. Glu241 activates a water molecule which hydrolyses the intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu241(222)A activator, increase nucleophilicity, proton acceptor
Asp41(22)A nucleofuge

Chemical Components

overall product formed, intermediate terminated, ingold: bimolecular nucleophilic substitution, hydrolysis, proton transfer
Download: Image, Marvin File

Step 1. Asp41 performs a nucleophilic attack on the glycosidic bond, while Glu241 protonates the leaving group. This leads to the formation of an enzyme-substrate intermediate.

Step 2. Glu241 activates a water molecule which hydrolyses the intermediate.

Products.

Contributors

Ellie Wright, Gemma L. Holliday, James Willey