DCTP deaminase

 

Deoxycytidine triphosphate deaminase (dCTP deaminase) from E. coli catalyses the deamination of dCTP producing ammonia and dUTP. The dUTP can be hydrolysed by dUTPase producing dUMP which is a precursor of dTTP. dCTP deaminase is inhibited by dTTP and by inorganic phosphate. Deamination of dCTP by dCTP deaminase provides about 80% of the dUMP used for dTMP synthesis in E. coli. The substrate of dCTP deaminase is the dCTP.Mg2+ complex but the magnesium does not have a catalytic role and no other metal ions are involved in catalysis.

 

Reference Protein and Structure

Sequence
P28248 UniProt (3.5.4.13) IPR011962 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
1xs1 - dCTP deaminase from Escherichia coli in complex with dUTP (1.8 Å) PDBe PDBsum 1xs1
Catalytic CATH Domains
2.70.40.10 CATHdb (see all for 1xs1)
Click To Show Structure

Enzyme Reaction (EC:3.5.4.13)

water
CHEBI:15377ChEBI
+
dCTP(4-)
CHEBI:61481ChEBI
→
dUTP(4-)
CHEBI:61555ChEBI
+
ammonia
CHEBI:16134ChEBI
Alternative enzyme names: 5-methyl-dCTP deaminase, Deoxycytidine triphosphate deaminase,

Enzyme Mechanism

Introduction

Glu138 abstracts a proton from a water molecule (HOH5) creating a hydroxide ion which attacks C4 of the pyrimidine ring of dCTP forming a tetrahedral intermediate. The amine group on C4 is protonated by another water molecule (HOH251) and the tetrahedral intermediate breaks down releasing a molecule of ammonia. The hydroxide produced from HOH251 is then neutralised by a proton transferred from the intermediate via Glu138.

Catalytic Residues Roles

UniProt PDB* (1xs1)
Ala124 (main-C) Ala124A (main-C) Helps stabilise the hydroxide ion in the active site. electrostatic stabiliser
Arg126 Arg126A Prevents the dCTP deaminase from also functioning as a dUTPase by taking up the space in which a nucleophilic water molecule could otherwise be located. This suggestion comes from a comparison of the E. coli dCTPdeaminase with the E. coli dUTPase. Arg126 also forms a salt bridge with Asp128. steric role
Glu138 Glu138A Glu138 deprotonates water molecule HOH5 to create the hydroxide ion which acts as the nucleophile and protonate the hydroxide formed from the deprotonation of HOH251 by the C4 amino group. proton acceptor, proton donor
Ser111 Ser111C Stabilises and positions the hydroxide ion in the active site. electrostatic stabiliser
Arg115 Arg115C Arg115 forms a hydrogen bond to Ser111 and prevents it's deprotonation by the hydroxide ion. The positive charge on Arg115 may also polarise the substrate for attack by the hydroxide ion. 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, overall reactant used, bimolecular nucleophilic addition, intermediate formation, unimolecular elimination by the conjugate base, intermediate collapse, overall product formed, keto-enol tautomerisation, native state of enzyme regenerated

References

  1. Johansson E et al. (2005), J Biol Chem, 280, 3051-3059. Structures of dCTP Deaminase from Escherichia coli with Bound Substrate and Product: REACTION MECHANISM AND DETERMINANTS OF MONO- AND BIFUNCTIONALITY FOR A FAMILY OF ENZYMES. DOI:10.1074/jbc.m409534200. PMID:15539408.
  2. Thymark M et al. (2008), Arch Biochem Biophys, 470, 20-26. Mutational analysis of the nucleotide binding site of Escherichia coli dCTP deaminase. DOI:10.1016/j.abb.2007.10.013. PMID:17996716.

Step 1. Glu138 abstracts a proton from a water (HOH5).

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg126A steric role
Ala124A (main-C) electrostatic stabiliser
Ser111C electrostatic stabiliser
Arg115C electrostatic stabiliser
Glu138A proton acceptor

Chemical Components

proton transfer, overall reactant used

Step 2. The hydroxide ion nucleophilically attacks the C4 carbon which results in double bond rearrangement as dCTP acts as an electron sink.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ala124A (main-C) electrostatic stabiliser
Ser111C electrostatic stabiliser
Arg115C electrostatic stabiliser
Arg126A steric role

Chemical Components

ingold: bimolecular nucleophilic addition, intermediate formation

Step 3. The electrons are fed back resulting in the cleavage of the amino group which accepts a proton from another water (HOH251)

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ala124A (main-C) electrostatic stabiliser
Ser111C electrostatic stabiliser
Arg115C electrostatic stabiliser
Arg126A steric role

Chemical Components

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

Step 4. The hydroxide accepts a proton from Glu138 and there is a tautomerisation which results in changes the C4 enol to a keto.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ala124A (main-C) electrostatic stabiliser
Ser111C electrostatic stabiliser
Arg115C electrostatic stabiliser
Arg126A steric role
Glu138A proton donor

Chemical Components

proton transfer, keto-enol tautomerisation, overall product formed, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. Glu138 abstracts a proton from a water (HOH5).

Step 2. The hydroxide ion nucleophilically attacks the C4 carbon which results in double bond rearrangement as dCTP acts as an electron sink.

Step 3. The electrons are fed back resulting in the cleavage of the amino group which accepts a proton from another water (HOH251)

Step 4. The hydroxide accepts a proton from Glu138 and there is a tautomerisation which results in changes the C4 enol to a keto.

Products.

Introduction

The reaction is initiated by the deprotonation of a water (HOH5) by Glu138 which then transfers the proton to N3's nitrogen. The hydroxide then nucleophilically attacks the C4 carbon. Glu138 then deprotonates the C4 hydroxyl which initiates an elimination that results in the release dUTP and the amino group which accepts a proton from another water (HOH251) which produces ammonia. The hydroxide then accepts a proton from Glu138 which regenerates the native state of the active site.

Catalytic Residues Roles

UniProt PDB* (1xs1)
Ala124 (main-C) Ala124A (main-C) Helps stabilise the hydroxide ion in the active site. electrostatic stabiliser
Arg126 Arg126A Prevents the dCTP deaminase from also functioning as a dUTPase by taking up the space in which a nucleophilic water molecule could otherwise be located. This suggestion comes from a comparison of the E. coli dCTPdeaminase with the E. coli dUTPase. Arg126 also forms a salt bridge with Asp128. steric role
Glu138 Glu138A Deprotonates HOH5 so it can act as nucleophile and attack the C4 carbon of dCTP. Also transfer a proton to N3 which enables the nucleophilic attack of C4 as it increases C4's electrophilicity. Deprotonates the C4 hydroxyl to initiate an elimination and protonate the HOH251 hydroxide. proton acceptor, proton donor
Ser111 Ser111C Stabilises and positions the hydroxide ion in the active site. electrostatic stabiliser
Arg115 Arg115C Arg115 forms a hydrogen bond to Ser111 and prevents it's deprotonation by the hydroxide ion. The positive charge on Arg115 may also polarise the substrate for attack by the hydroxide ion. 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, overall reactant used, bimolecular nucleophilic addition, intermediate formation, unimolecular elimination by the conjugate base, intermediate collapse, overall product formed, native state of enzyme regenerated

References

  1. Johansson E et al. (2005), J Biol Chem, 280, 3051-3059. Structures of dCTP Deaminase from Escherichia coli with Bound Substrate and Product: REACTION MECHANISM AND DETERMINANTS OF MONO- AND BIFUNCTIONALITY FOR A FAMILY OF ENZYMES. DOI:10.1074/jbc.m409534200. PMID:15539408.
  2. Thymark M et al. (2008), Arch Biochem Biophys, 470, 20-26. Mutational analysis of the nucleotide binding site of Escherichia coli dCTP deaminase. DOI:10.1016/j.abb.2007.10.013. PMID:17996716.

Step 1. Glu138 abstracts a proton from water.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ala124A (main-C) electrostatic stabiliser
Ser111C electrostatic stabiliser
Arg115C electrostatic stabiliser
Arg126A steric role
Glu138A proton acceptor

Chemical Components

proton transfer, overall reactant used

Step 2. The N3 nitrogen accepts a proton from Glu138 and the hydroxide nucleophilically attacks the C3 carbon which is more electrophilic due to protonation at N3.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ala124A (main-C) electrostatic stabiliser
Ser111C electrostatic stabiliser
Arg115C electrostatic stabiliser
Arg126A steric role
Glu138A proton donor

Chemical Components

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

Step 3. Glu138 deprotonates hydroxyl group which initiates an elimination resulting in the release of the amino group which accepts a proton from a water (HOH251).

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ala124A (main-C) electrostatic stabiliser
Ser111C electrostatic stabiliser
Arg115C electrostatic stabiliser
Arg126A steric role
Glu138A proton acceptor

Chemical Components

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

Step 4. The hydroxide accepts a proton from Glu138 to regenerate the active site's native state.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ala124A (main-C) electrostatic stabiliser
Ser111C electrostatic stabiliser
Arg115C electrostatic stabiliser
Arg126A steric role
Glu138A proton donor

Chemical Components

proton transfer, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. Glu138 abstracts a proton from water.

Step 2. The N3 nitrogen accepts a proton from Glu138 and the hydroxide nucleophilically attacks the C3 carbon which is more electrophilic due to protonation at N3.

Step 3. Glu138 deprotonates hydroxyl group which initiates an elimination resulting in the release of the amino group which accepts a proton from a water (HOH251).

Step 4. The hydroxide accepts a proton from Glu138 to regenerate the active site's native state.

Products.

Contributors

Gemma L. Holliday, Charity Hornby