Glucan endo-1,3-beta-D-glucosidase

 

Two plant beta-glucan endohydrolases with distinct substrate specificities, thought to have evolved from the same higher enzyme, which both hydrolyse 1,3-beta-D-glucosidic linkages in 1,3-beta-D-glucans. Chain A is the isoenzyme GII, (1->3)-beta-glucanase with EC:3.2.1.39 and chain B is the isoenzyme EII (1->3, 1->4)- beta glucanase with EC:3.2.1.73. Isoenzyme GII can hydrolyse beta-glucans of fungal cell walls and may therefore contribute to plant defence strategies, whereas isoenzyme GII function in plant cell wall hydrolysis during mobilisation of the endosperm or in germinating grain during the growth of vegetative tissue. Both glucanases have essentially identical alpha/beta barrel folds, with a recognisable cleft where the active site is located in the same position with identical catalytic residues, however beyond the conserved patch that surrounds the catalytic amino acid residues, there are different amino acid substitutions along the substrate-binding cleft leading to different substrate specificities. This protein is a member of glycoside hydrolase family 17.

 

Reference Protein and Structure

Sequence
P15737 UniProt (3.2.1.39) IPR000490 (Sequence Homologues) (PDB Homologues)
Biological species
Hordeum vulgare (Barley) Uniprot
PDB
1ghs - THE THREE-DIMENSIONAL STRUCTURES OF TWO PLANT BETA-GLUCAN ENDOHYDROLASES WITH DISTINCT SUBSTRATE SPECIFICITIES (2.3 Å) PDBe PDBsum 1ghs
Catalytic CATH Domains
3.20.20.80 CATHdb (see all for 1ghs)
Click To Show Structure

Enzyme Reaction (EC:3.2.1.39)

water
CHEBI:15377ChEBI
+
D-glucopyranosyl-(1->3)-D-mannopyranose
CHEBI:52997ChEBI
→
D-glucopyranose
CHEBI:4167ChEBI
+
D-mannopyranose
CHEBI:4208ChEBI
Alternative enzyme names: (1->3)-beta-glucan 3-glucanohydrolase, (1->3)-beta-glucan endohydrolase, 1,3-beta-D-glucan 3-glucanohydrolase, Beta-1,3-glucanase, Callase, Endo-(1,3)-beta-D-glucanase, Endo-(1->3)-beta-D-glucanase, Endo-1,3-beta-D-glucanase, Endo-1,3-beta-glucanase, Endo-1,3-beta-glucosidase, Kitalase, Laminaranase, Laminarinase, Oligo-1,3-glucosidase, 1,3-beta-D-glucan glucanohydrolase,

Enzyme Mechanism

Introduction

Hydrolysis of the glycosidic bond takes place via general acid catalysis leading to retention of configuration. The general acid protonated Glu288 protonates the glycosidic oxygen aided by Lys282 and Glu279, followed by nucleophilic attack by Glu231, on the oxy-carbonium ion intermediate. This is then broken down via deprotonation of a water by Glu288, a second nucleophilic attack of the deprotonated water molecule and regeneration of Glu231 results.

Catalytic Residues Roles

UniProt PDB* (1ghs)
Glu259 Glu231A Acts as a catalytic nucleophile. covalently attached, nucleofuge, nucleophile, electrostatic stabiliser
Glu307, Lys310 Glu279A, Lys282A Acts to activate Glu288. activator, modifies pKa
Glu316 Glu288A Acts as a general acid/base. activator, increase nucleophilicity, proton acceptor, proton donor
*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

proton transfer, overall reactant used, heterolysis, bimolecular nucleophilic addition, intermediate formation, bimolecular nucleophilic substitution, overall product formed, native state of enzyme regenerated, intermediate terminated, hydrolysis

References

  1. Varghese JN et al. (1994), Proc Natl Acad Sci U S A, 91, 2785-2789. Three-dimensional structures of two plant beta-glucan endohydrolases with distinct substrate specificities. DOI:10.2210/pdb1ghs/pdb. PMID:8146192.
  2. Høj PB et al. (1995), Plant J, 7, 367-379. Molecular evolution of plant beta-glucan endohydrolases. DOI:10.1046/j.1365-313x.1995.7030367.x. PMID:7757111.

Step 1. Glu288 protonates the glycosidic oxygen. Glu279 and Lys282 activate Glu288. The oxy-carbonium ion is stabilized by Glu231.

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Catalytic Residues Roles

Residue Roles
Glu279A activator
Lys282A activator
Glu279A modifies pKa
Lys282A modifies pKa
Glu231A electrostatic stabiliser
Glu288A proton donor

Chemical Components

proton transfer, overall reactant used

Step 2. The glycosidic bond is cleaved.

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Catalytic Residues Roles

Residue Roles
Glu231A electrostatic stabiliser

Chemical Components

heterolysis

Step 3. Glu231 performs a nucleophilic attack on the oxy-carbonium ion forming an enzyme-substrate intermediate.

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Catalytic Residues Roles

Residue Roles
Glu231A covalently attached, nucleophile

Chemical Components

ingold: bimolecular nucleophilic addition, intermediate formation

Step 4. Glu288 now acts as a base activating a water molecule which hydrolyses the intermediate and regenerates the active site.

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Catalytic Residues Roles

Residue Roles
Glu288A activator, increase nucleophilicity
Glu279A modifies pKa
Lys282A modifies pKa
Glu288A proton acceptor
Glu231A nucleofuge

Chemical Components

proton transfer, ingold: bimolecular nucleophilic substitution, overall product formed, native state of enzyme regenerated, intermediate terminated, hydrolysis
Download: Image, Marvin File

Step 1. Glu288 protonates the glycosidic oxygen. Glu279 and Lys282 activate Glu288. The oxy-carbonium ion is stabilized by Glu231.

Step 2. The glycosidic bond is cleaved.

Step 3. Glu231 performs a nucleophilic attack on the oxy-carbonium ion forming an enzyme-substrate intermediate.

Step 4. Glu288 now acts as a base activating a water molecule which hydrolyses the intermediate and regenerates the active site.

Products.

Contributors

Anna Waters, Craig Porter, Gemma L. Holliday, James Willey