Cellulase (GH9)

 

Cellulase E4 from Thermomonospora is both exo- and endo- cellulases, catalysing the hydrolysis of cellulose. It contains both a family 9 catalytic domain, exhibiting an (alpha/apha)6 barrel fold and a family III cellulose binding domain, having an antiparallel beta-sandwich fold.

 

Reference Protein and Structure

Sequence
P26221 UniProt (3.2.1.4) IPR001701 (Sequence Homologues) (PDB Homologues)
Biological species
Thermobifida fusca (Bacteria) Uniprot
PDB
1js4 - ENDO/EXOCELLULASE:CELLOBIOSE FROM THERMOMONOSPORA (2.0 Å) PDBe PDBsum 1js4
Catalytic CATH Domains
1.50.10.10 CATHdb (see all for 1js4)
Click To Show Structure

Enzyme Reaction (EC:3.2.1.4)

water
CHEBI:15377ChEBI
+
cellotetraose
CHEBI:62974ChEBI
→
cellobiose
CHEBI:17057ChEBI
Alternative enzyme names: 9.5 cellulase, Beta-1,4-endoglucan hydrolase, Beta-1,4-glucanase, Alkali cellulase, Avicelase, Celluase A, Celludextrinase, Cellulase A 3, Cellulosin AP, Endo-1,4-beta-D-glucanase, Endoglucanase D, Pancellase SS, Endo-1,4-beta-glucanase, Carboxymethyl cellulase, Endoglucanase, Endo-1,4-beta-D-glucanohydrolase, 1,4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase,

Enzyme Mechanism

Introduction

Same as the other members of family 9 cellulase, E4 endo/exocellulase cleaves cullulose with inversion of configuration at the anomeric carbon. In the mechanism, an acid protonates the glycosidic oxygen and a base extracts a nucleophilic water that attacks the anomeric carbon. Here, Glu470 is the acid. Both Asp 104 and Asp 101 hydrogen bond to the attacking water and both of them appear to contribute to the activation of the water molecule. Asp 104 appears better oriented to accept a proton from the water than Asp 101, however both residues may be able to act as the base.

Catalytic Residues Roles

UniProt PDB* (1js4)
Glu470 Glu424A It acts as an acid to protonate the glycosidic oxygen. promote heterolysis, proton acceptor, proton donor
Tyr252 Tyr206A Hydrogen bonds to be Asp101 contributing to the activation of water. electrostatic stabiliser
Asp104 Asp58A It deprotonates the water molecule, which nucleophillically attacks the glycosidic bond of the substrate in hydrolysis. activator, increase nucleophilicity, proton acceptor, proton donor
Asp101 Asp55A It forms a hydrogen bond to the water molecule, which nucleophillically attacks the glycosidic bond of the substrate in hydrolysis, to activate it. Some evidence suggests this residue may be also be the base. activator, increase nucleophilicity, electrostatic stabiliser
*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

hydrolysis, overall product formed, overall reactant used, bimolecular nucleophilic substitution, proton transfer, inferred reaction step, native state of enzyme regenerated

References

  1. Sakon J et al. (1997), Nat Struct Biol, 4, 810-818. Structure and mechanism of endo/exocellulase E4 from Thermomonospora fusca. DOI:10.1038/nsb1097-810. PMID:9334746.
  2. Zhou W et al. (2004), Biochemistry, 43, 9655-9663. Kinetic studies of Thermobifida fusca Cel9A active site mutant enzymes. DOI:10.1021/bi049394n. PMID:15274620.

Step 1. Either Asp101 or Asp104 deprotonates the water activating it for nucleophilic attack on the anomeric carbon. (Tyr252 hydrogen bonds to Asp101 which contributes to the activation of water.) This causes the glycosidic bond to be broken upon protonation by Glu470.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp55A electrostatic stabiliser, activator
Asp58A activator
Tyr206A electrostatic stabiliser
Asp55A increase nucleophilicity
Asp58A increase nucleophilicity
Glu424A promote heterolysis
Asp58A proton acceptor
Glu424A proton donor

Chemical Components

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

Step 2. Inferred step- active site is regenerated.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu424A proton acceptor
Asp58A proton donor

Chemical Components

inferred reaction step, proton transfer, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. Either Asp101 or Asp104 deprotonates the water activating it for nucleophilic attack on the anomeric carbon. (Tyr252 hydrogen bonds to Asp101 which contributes to the activation of water.) This causes the glycosidic bond to be broken upon protonation by Glu470.

Step 2. Inferred step- active site is regenerated.

Products.

Contributors

Mei Leung, Gemma L. Holliday, James Willey