Chitinase (GH18, class II)

 

Chitinase A1 from Bacillus circulans catalyses the hydrolysis of the 1,4-beta linkages of N-acetyl-D glucosamine polymers of chitin. For bacteria, this hydrolysis is utilised primarily as carbon and energy sources. Chitinase A1 is proposed to be very similar to hen egg-white lysozyme, differing only in that it undergoes a 'substrate assisted' mechanism.

 

Reference Protein and Structure

Sequence
P20533 UniProt (3.2.1.14) IPR011583 (Sequence Homologues) (PDB Homologues)
Biological species
Bacillus circulans (Bacteria) Uniprot
PDB
1itx - Catalytic Domain of Chitinase A1 from Bacillus circulans WL-12 (1.1 Å) PDBe PDBsum 1itx
Catalytic CATH Domains
3.20.20.80 CATHdb (see all for 1itx)
Click To Show Structure

Enzyme Reaction (EC:3.2.1.14)

N-acetyl-beta-D-glucosaminyl-(1->4)-N-acetyl-beta-D-glucosamine
CHEBI:50670ChEBI
+
water
CHEBI:15377ChEBI
→
N-acetyl-D-glucosamine
CHEBI:506227ChEBI
+
N-acetyl-D-glucosamine
CHEBI:506227ChEBI
Alternative enzyme names: 1,4-beta-poly-N-acetylglucosaminidase, Beta-1,4-poly-N-acetyl glucosamidinase, Chitodextrinase, Poly-beta-glucosaminidase, Poly(1,4-(N-acetyl-beta-D-glucosaminide)) glycanohydrolase, ChiC,

Enzyme Mechanism

Introduction

The nitrogen atom of the substrate pushes electrons up toward the acetyl group, causing the oxygen atom of the acetyl to nucleophilically attack the C1 atom of the substrate. This breaks the C1-O bond, and the leaving group oxygen atom is protonated by Glu 204. Asp 202 and Tyr 279 distort the substrate to encourage the intiatial nucleophilic attack. Asp 200, Asp202 and Tyr279 stabilise the positive charge on the substrate. Glu 204 then acts as a general base, deprotonating a water molecule and activating it for nucleophilic attack on the substrate C1 atom. The N-acetyl group is reformed, and the product leaves.

Catalytic Residues Roles

UniProt PDB* (1itx)
Asp202, Tyr279 Asp202(170)A, Tyr279(247)A Stabilise the positive charge of the intermediate and distort the substrate to encourage the formation of the intermediate. electrostatic stabiliser, steric role
Asp200 Asp200(168)A The side chain of Asp 200 stabilises the positively charged intermediate. electrostatic stabiliser
Glu204 Glu204(172)A Glu 204 acts as a general acid/base by protonating the leaving group, and by deprotonating a water molecule to activate it. 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, intramolecular nucleophilic substitution, cyclisation, native state of enzyme regenerated, decyclisation, bimolecular nucleophilic substitution, hydrolysis

References

  1. Watanabe T et al. (1993), J Biol Chem, 268, 18567-18572. Identification of glutamic acid 204 and aspartic acid 200 in chitinase A1 of Bacillus circulans WL-12 as essential residues for chitinase activity. PMID:8103047.
  2. Synstad B et al. (2004), Eur J Biochem, 271, 253-262. Mutational and computational analysis of the role of conserved residues in the active site of a family 18 chitinase. DOI:10.1046/j.1432-1033.2003.03923.x.
  3. Tews I et al. (1997), J Am Chem Soc, 119, 7954-7959. Substrate-Assisted Catalysis Unifies Two Families of Chitinolytic Enzymes. DOI:10.1021/ja970674i.

Step 1. The substrate is distorted in the active site this causes the nitrogen of the N-acetyl group to push electrons into the carbonyl oxygen which then attacks C1 of the glycosidic bond. This causes the bond to be broken upon protonation from Glu204 and a cyclic oxazolinium ion to be formed. This is stabilized by Asp200, Asp202 and Tyr279.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp200(168)A electrostatic stabiliser
Asp202(170)A electrostatic stabiliser
Tyr279(247)A electrostatic stabiliser, steric role
Glu204(172)A promote heterolysis
Asp202(170)A steric role
Glu204(172)A proton donor

Chemical Components

overall product formed, overall reactant used, proton transfer, ingold: intramolecular nucleophilic substitution, cyclisation

Step 2. Glu204 activates a water molecule which hydrolyses the cyclic intermediate and the product is formed.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp200(168)A electrostatic stabiliser
Asp202(170)A electrostatic stabiliser
Tyr279(247)A electrostatic stabiliser
Glu204(172)A activator, increase nucleophilicity, proton acceptor

Chemical Components

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

Step 1. The substrate is distorted in the active site this causes the nitrogen of the N-acetyl group to push electrons into the carbonyl oxygen which then attacks C1 of the glycosidic bond. This causes the bond to be broken upon protonation from Glu204 and a cyclic oxazolinium ion to be formed. This is stabilized by Asp200, Asp202 and Tyr279.

Step 2. Glu204 activates a water molecule which hydrolyses the cyclic intermediate and the product is formed.

Products.

Contributors

Ellie Wright, Gemma L. Holliday, James Willey