Dihydroorotase

 

Dihyrdoorotase catalyses the reversible interconversion of carbamoyl aspartate and dihydroorotate. This reaction is an essential part of the pathway for the biosynthesis of pyrimidine nucleotides. Sequence comparisons show that there are two general classes of dihydroorotases. Class II enzymes are all monofunctional proteins from Gram-negative bacteria and yeast. Class I enzymes, found in higher organisms, are much larger and typically contain several enzyme activities as exemplified by CAD, a multifunctional enzyme found in mammals, insects and moulds. This protein combines the first three enzymes of the pyrimidine biosynthesis pathway: carbamoyl phosphate synthetase, aspartate carbamoylase, and dihydroorotase. Sequence homology within each class of enzymes is quite high (>40%) while that between the two classes is much lower. Both classes employ the same mechanism involving a binuclear zinc centre.

 

Reference Protein and Structure

Sequence
P05020 UniProt (3.5.2.3) IPR004721 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
1j79 - Molecular Structure of Dihydroorotase: A Paradigm for Catalysis Through the Use of a Binuclear Metal Center (1.7 Å) PDBe PDBsum 1j79
Catalytic CATH Domains
3.20.20.140 CATHdb (see all for 1j79)
Cofactors
Zinc(2+) (2)
Click To Show Structure

Enzyme Reaction (EC:3.5.2.3)

water
CHEBI:15377ChEBI
+
(S)-dihydroorotate
CHEBI:30864ChEBI
→
N-carbamoyl-L-aspartate(2-)
CHEBI:32814ChEBI
+
hydron
CHEBI:15378ChEBI
Alternative enzyme names: Dihydroorotate hydrolase, Carbamoylaspartic dehydrase, DHOase,

Enzyme Mechanism

Introduction

Dihydroorotase uses a bi-nuclear zinc centre to catalyse the reversible formation and hydrolysis of dihydroorotate. In the hydrolysis reaction, the two zinc ions (alpha and beta) coordinate a bridging hydroxide ion that attacks the substrate carbonyl to form a tetrahedral intermediate. This process is facilitated by Asp 250 acting as a general base to deprotonate the attacking hydroxide, and by polarisation of the carbonyl group through coordination to the beta zinc ion. The tetrahedral intermediate and associated transition state is stabilised by coordination to both zinc ions. Collapse of this intermediate occurs with Asp 250 acting as a general acid to protate the departing amide nitrogen.

Catalytic Residues Roles

UniProt PDB* (1j79)
His17, His19, Lys103, His140, His178 His16A, His18A, Lys102A, His139A, His177A Forms Zinc binding site metal ligand
Asp251 Asp250A Acts as a general base to deprotonate the attacking bridging hydroxide. Later acts as a general acid to protonate the amide nitrogen leaving group. metal ligand, 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, heterolysis, intermediate collapse, overall product formed, rate-determining step

References

  1. Thoden JB et al. (2001), Biochemistry, 40, 6989-6997. Molecular structure of dihydroorotase: a paradigm for catalysis through the use of a binuclear metal center. DOI:10.2210/pdb1j79/pdb. PMID:11401542.
  2. Liao RZ et al. (2008), Chemistry, 14, 4287-4292. Theoretical investigation of the reaction mechanism of the dinuclear zinc enzyme dihydroorotase. DOI:10.1002/chem.200701948. PMID:18366031.
  3. Porter TN et al. (2004), Biochemistry, 43, 16285-16292. Mechanism of the Dihydroorotase Reaction†. DOI:10.1021/bi048308g. PMID:15610022.

Step 1. The zinc coordinated hydroxide nucleophilically attacks the carbon of the carbonyl group of dihydroorotate.

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Catalytic Residues Roles

Residue Roles
His16A metal ligand
His18A metal ligand
Lys102A metal ligand
His139A metal ligand
His177A metal ligand
Asp250A metal ligand

Chemical Components

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

Step 2. Asp251 deprotonates the hydroxyl group.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His16A metal ligand
His18A metal ligand
Lys102A metal ligand
His139A metal ligand
His177A metal ligand
Asp250A metal ligand
Asp250A proton acceptor

Chemical Components

proton transfer, intermediate formation

Step 3. The amide group is protonated by Asp51 which initiates an elimination from the oxyanion and results in the cleavage of the amide bond.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His16A metal ligand
His18A metal ligand
Lys102A metal ligand
His139A metal ligand
His177A metal ligand
Asp250A metal ligand
Asp250A proton donor

Chemical Components

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

Step 1. The zinc coordinated hydroxide nucleophilically attacks the carbon of the carbonyl group of dihydroorotate.

Step 2. Asp251 deprotonates the hydroxyl group.

Step 3. The amide group is protonated by Asp51 which initiates an elimination from the oxyanion and results in the cleavage of the amide bond.

Products.

Contributors

Steven Smith, Gemma L. Holliday, Charity Hornby