Creatininase

 

Creatininase (creatinine amidohydrolase) from Pseudomonas putida catalyses the conversion of creatinine to creatine. It is a member of the urease-related amidohydrolase superfamily. The enzyme enables the bacteria to use creatinine as a source of carbon and nitrogen. The biological molecule is trimer of dimers. The active site of each monomer contains a binuclear metal centre located at the bottom of a cleft. In the native form of the enzyme both metal sites are occupied by Zn2+ ions.

 

Reference Protein and Structure

Sequence
P83772 UniProt (3.5.2.10) IPR031034 (Sequence Homologues) (PDB Homologues)
Biological species
Pseudomonas putida (Bacteria) Uniprot
PDB
1j2u - Creatininase Zn (1.85 Å) PDBe PDBsum 1j2u
Catalytic CATH Domains
3.40.50.10310 CATHdb (see all for 1j2u)
Cofactors
Zinc(2+) (2)
Click To Show Structure

Enzyme Reaction (EC:3.5.2.10)

water
CHEBI:15377ChEBI
+
creatinine
CHEBI:16737ChEBI
→
creatine zwitterion
CHEBI:57947ChEBI
Alternative enzyme names: Creatinine hydrolase, Creatinine amidohydrolase,

Enzyme Mechanism

Introduction

A water molecule bridging the two metal ions is likely to form a hydroxide ion which acts as the nucleophile. The hydroxide attacks the carbonyl carbon of the creatinine substrate and is then deprotonated by His 178. The tetrahedral intermediate is stabilised by coordination to the two metal ions. Ring opening occurs simultaneously with the protonation of the amide nitrogen on the intermediate by His 178.

Catalytic Residues Roles

UniProt PDB* (1j2u)
Glu34, His120, Asp45, His36, Glu183 Glu34A, His120A, Asp45A, His36A, Glu183A Forms zinc binding site. metal ligand
Glu122 Glu122A Glu122 forms a hydrogen bond to the water molecule part of the zinc coordination sphere and likely protonates creatine to make the kinetically favourable product. electrostatic stabiliser
His178 His178A Deprotonates the hydroxide that is part of the oxyanion intermediate and then protonates the amide group to initiate an elimination. 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

bimolecular nucleophilic addition, intermediate formation, overall reactant used, proton transfer, unimolecular elimination by the conjugate base, intermediate collapse, overall product formed

References

  1. Yoshimoto T et al. (2004), J Mol Biol, 337, 399-416. Crystal Structures of Creatininase Reveal the Substrate Binding Site and Provide an Insight into the Catalytic Mechanism. DOI:10.1016/j.jmb.2004.01.022. PMID:15003455.
  2. Jitonnom J et al. (2017), Biochemistry, 56, 6377-6388. Quantum Mechanics/Molecular Mechanics Simulations Identify the Ring-Opening Mechanism of Creatininase. DOI:10.1021/acs.biochem.7b01032. PMID:29140090.

Step 1. The zinc-bound water performs a nucleophilic attack attack on the carbon of the carbonyl bond.

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Catalytic Residues Roles

Residue Roles
Glu122A electrostatic stabiliser
Glu34A metal ligand
His36A metal ligand
Asp45A metal ligand
His120A metal ligand
Glu183A metal ligand

Chemical Components

ingold: bimolecular nucleophilic addition, intermediate formation, overall reactant used

Step 2. His178 deprotonates the hydroxyl group.

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Catalytic Residues Roles

Residue Roles
Glu34A metal ligand
His36A metal ligand
Asp45A metal ligand
His120A metal ligand
Glu183A metal ligand
Glu122A electrostatic stabiliser
His178A proton acceptor

Chemical Components

proton transfer, intermediate formation

Step 3. The oxyanion initiates an elimination which results in the cleavage of the amide bond and the nitrogen accepts a proton from His178.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu34A metal ligand
His36A metal ligand
Asp45A metal ligand
His120A metal ligand
Glu183A metal ligand
Glu122A electrostatic stabiliser
His178A proton donor

Chemical Components

proton transfer, ingold: unimolecular elimination by the conjugate base, intermediate collapse, overall product formed
Download: Image, Marvin File

Step 1. The zinc-bound water performs a nucleophilic attack attack on the carbon of the carbonyl bond.

Step 2. His178 deprotonates the hydroxyl group.

Step 3. The oxyanion initiates an elimination which results in the cleavage of the amide bond and the nitrogen accepts a proton from His178.

Products.

Contributors

Gemma L. Holliday, Charity Hornby, Noa Marson