Arabinogalactan endo-1,4-beta-galactosidase

 

Asperfillus aculeatus beta-1,4-Galactanase (AAGAL) is an enzyme involved in pectin degradation. It belongs to the glycoside hydrolase family 53 (GH-53) in clan GH-A. It catalyses the endohydrolysis of beta-1,4-linked galactan and type I arabinogalactan to galactose and galactose oligomers.

Galactan and arabinogalactan are components of pectin which attaches to the C4 position of rhamnose. They forms the side-chains of the 'hairy' region of pectin. Degradation and modification of the galatan and arabinogalactan side chain has many industrial applications, so AAGAL has potential industrial uses.

 

Reference Protein and Structure

Sequence
P48842 UniProt (3.2.1.89) IPR011683 (Sequence Homologues) (PDB Homologues)
Biological species
Aspergillus aculeatus (Fungus) Uniprot
PDB
1fhl - CRYSTAL STRUCTURE OF BETA-1,4-GALACTANASE FROM ASPERGILLUS ACULEATUS AT 293K (2.3 Å) PDBe PDBsum 1fhl
Catalytic CATH Domains
3.20.20.80 CATHdb (see all for 1fhl)
Click To Show Structure

Enzyme Reaction (EC:3.2.1.89)

water
CHEBI:15377ChEBI
+
beta-(1->6)-galactotriose
CHEBI:61765ChEBI
→
beta-D-galactose
CHEBI:27667ChEBI
+
beta-(1->6)-galactobiose
CHEBI:61770ChEBI
Alternative enzyme names: Arabinogalactanase, Endo-1,4-beta-galactanase, Galactanase, GanB (gene name),

Enzyme Mechanism

Introduction

Glu136 protonates the glycosidic oxygen while Glu246 acts as a nucleophilic to attack the anomeric carbon to form a covalent enzyme-substrate intermediate. The deprotonated Glu136 then activates a water molecule to hydrolyse the covalent enzyme-substrate intermediate. Arg45 hydrogen bonds with Glu246 to ensure that it is deprotonated even at relatively low pH value to allow it to act as a catalytic nucleophile.

Catalytic Residues Roles

UniProt PDB* (1fhl)
Glu152 Glu136A Protonates glycosyl oxygen to facilitate nucleophilic attack on the anomeric carbon resulting in the glycosyl-enzyme intermediate. Then activates water to allow hydrolysis of the intermediate. increase nucleophilicity, activator, proton acceptor, proton donor
Glu262 Glu246A Attacks anomeric carbon to form the glycosyl-enzyme intermediate which subsequently is hydrolysed to give the products. covalently attached, hydrogen bond acceptor, nucleofuge, nucleophile
Arg61 Arg45A Hydrogen bonds with Glu 246 and alters the pKa of Glu 246 to keep it deprotonated even at relatively low pH value to allow it to act as a nucleophile to attack the anomeric carbon of the glycosidic bond. modifies pKa, increase nucleophilicity, hydrogen bond donor, 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

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

References

  1. Ryttersgaard C et al. (2002), Biochemistry, 41, 15135-15143. Aspergillus aculeatusβ-1,4-Galactanase:  Substrate Recognition and Relations to Other Glycoside Hydrolases in Clan GH-A†. DOI:10.1021/bi026238c. PMID:12484750.

Step 1. Glu246 performs a nucleophilic attack on the glycosidic linkage while Glu136 protonates the leaving group. This leads to an enzyme-substrate intermediate being formed.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu246A covalently attached
Arg45A modifies pKa, increase nucleophilicity, electrostatic stabiliser, hydrogen bond donor
Glu246A hydrogen bond acceptor, nucleophile
Glu136A proton donor

Chemical Components

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

Step 2. Glu136 activates a water for nucleophilic attack, which leads to the intermediate being hydrolysed and the active site being regenerated.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu136A increase nucleophilicity, activator
Glu246A nucleofuge
Glu136A proton acceptor

Chemical Components

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

Step 1. Glu246 performs a nucleophilic attack on the glycosidic linkage while Glu136 protonates the leaving group. This leads to an enzyme-substrate intermediate being formed.

Step 2. Glu136 activates a water for nucleophilic attack, which leads to the intermediate being hydrolysed and the active site being regenerated.

Products.

Contributors

Peter Sarkies, Gemma L. Holliday, James Willey