Cytosine deaminase (bacterial)

 

Cytosine deaminase (CD) catalyses the deamination of cytosine to uracil and ammonia. It is an enzyme produced by bacteria and yeast, but not mammalian cells, and is a potential gene therapy agent. CD deaminates 5-fluorocytosine to potent antimetabolite 5-fluorouracil, which can block DNA replication in cells. Bacterial CD (bCD) is capable of deaminating a wide range of cytosine derivatives with varying efficiency. It is dependent of Fe2+ for maximal catalytic activity.

 

Reference Protein and Structure

Sequence
P25524 UniProt (3.5.4.-, 3.5.4.1) IPR013108 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
1ra0 - Bacterial cytosine deaminase D314G mutant bound to 5-fluoro-4-(S)-hydroxy-3,4-dihydropyrimidine. (1.12 Å) PDBe PDBsum 1ra0
Catalytic CATH Domains
3.20.20.140 CATHdb (see all for 1ra0)
Cofactors
Zinc(2+) (1)
Click To Show Structure

Enzyme Reaction (EC:3.5.4.1)

water
CHEBI:15377ChEBI
+
cytosine
CHEBI:16040ChEBI
→
ammonia
CHEBI:16134ChEBI
+
uracil
CHEBI:17568ChEBI
Alternative enzyme names: Isocytosine deaminase, Cytosine aminohydrolase,

Enzyme Mechanism

Introduction

Glu217 initially deprotonates the Zn bound and then after some structural movements transfers the proton to the N3 nitrogen. The hydroxyl can then attack the C4 carbon and then the amino group accepts a proton from the hydroxyl which initiates the collapse of the tetrahedral intermediate which releases the products: ammonia and uracil.

Catalytic Residues Roles

UniProt PDB* (1ra0)
Asp314, His62, His64, His215 Asp313(317)A, His61(65)A, His63(67)A, His214(218)A Bind the metal ion. metal ligand
Glu218 Glu217(221)A Glu217 acts as a catalytic acid/base residue. It donates a proton to the N3 nitrogen, increasing the susceptibility of the C4 carbon to nucleophilic attack by weakening the N3 to C4 double bond character. proton acceptor, proton donor
Gln157 Gln156(160)A Gln156 forms hydrogen bonds with the N1 nitrogen and O2 oxygen of the pyrimidine ring, helping to orientate the substrate, and polarise the aromatic structure of the pyrimidine ring, also making the C4 carbon more reactive towards an addition by the activated water molecule. 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, overall reactant used, bimolecular nucleophilic addition, cofactor used, unimolecular elimination by the conjugate base, decoordination from a metal ion, heterolysis, intermediate collapse, native state of enzyme regenerated, overall product formed

References

  1. Ireton GC et al. (2002), J Mol Biol, 315, 687-697. The structure of Escherichia coli cytosine deaminase. DOI:10.1006/jmbi.2001.5277. PMID:11812140.
  2. Manta B et al. (2014), J Phys Chem B, 118, 5644-5652. Reaction mechanism of zinc-dependent cytosine deaminase from Escherichia coli: a quantum-chemical study. DOI:10.1021/jp501228s. PMID:24833316.
  3. Mahan SD et al. (2004), Protein Eng Des Sel, 17, 625-633. Random mutagenesis and selection of Escherichia coli cytosine deaminase for cancer gene therapy. DOI:10.1093/protein/gzh074. PMID:15381761.
  4. Porter DJ et al. (1993), J Biol Chem, 268, 24005-24011. Cytosine deaminase. The roles of divalent metal ions in catalysis. PMID:8226944.

Step 1. Glu218 abstracts a proton from the zinc bound water.

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Catalytic Residues Roles

Residue Roles
Gln156(160)A electrostatic stabiliser
His61(65)A metal ligand
His63(67)A metal ligand
His214(218)A metal ligand
Asp313(317)A metal ligand
Glu217(221)A proton acceptor

Chemical Components

proton transfer, intermediate formation, overall reactant used

Step 2. Glu217 has to break its hydrogen bond to the nucleophile, rotate toward the substrate, and create a new hydrogen bond to the N3 nitrogen where it transfers a proton to the N3 nitrogen.

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Catalytic Residues Roles

Residue Roles
Gln156(160)A electrostatic stabiliser
His61(65)A metal ligand
His63(67)A metal ligand
His214(218)A metal ligand
Asp313(317)A metal ligand
Glu217(221)A proton donor

Chemical Components

proton transfer, intermediate formation

Step 3. The Zinc bound hydroxyl nucleophilically attacks the C4 carbon which is more electrophilic due to the protonation at N3.

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Catalytic Residues Roles

Residue Roles
Gln156(160)A electrostatic stabiliser
His61(65)A metal ligand
His63(67)A metal ligand
His214(218)A metal ligand
Asp313(317)A metal ligand

Chemical Components

ingold: bimolecular nucleophilic addition, cofactor used, intermediate formation

Step 4. The amino group bound to C4 accepts a proton from the hydroxyl group.

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Catalytic Residues Roles

Residue Roles
Gln156(160)A electrostatic stabiliser
His61(65)A metal ligand
His63(67)A metal ligand
His214(218)A metal ligand
Asp313(317)A metal ligand

Chemical Components

proton transfer, intermediate formation

Step 5. The protonation of the amino group results in the initiation of an elimination from the oxyanion which leads to the release of ammonia and uracil.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Gln156(160)A electrostatic stabiliser
His61(65)A metal ligand
His63(67)A metal ligand
His214(218)A metal ligand
Asp313(317)A metal ligand

Chemical Components

ingold: unimolecular elimination by the conjugate base, decoordination from a metal ion, heterolysis, intermediate collapse, native state of enzyme regenerated, overall product formed
Download: Image, Marvin File

Step 1. Glu218 abstracts a proton from the zinc bound water.

Step 2. Glu217 has to break its hydrogen bond to the nucleophile, rotate toward the substrate, and create a new hydrogen bond to the N3 nitrogen where it transfers a proton to the N3 nitrogen.

Step 3. The Zinc bound hydroxyl nucleophilically attacks the C4 carbon which is more electrophilic due to the protonation at N3.

Step 4. The amino group bound to C4 accepts a proton from the hydroxyl group.

Step 5. The protonation of the amino group results in the initiation of an elimination from the oxyanion which leads to the release of ammonia and uracil.

Products.

Contributors

Gemma L. Holliday, Charity Hornby