Deoxycytidylate 5-hydroxymethyltransferase

 

Deoxycytidylate (dCMP) 5-hydroxymethylase (CH) of bacteriophage T4 catalyses the addition of a protective hydroxymethyl group to cytosine monophosphate as a method of avoiding digestion by the host restriction systems. It requires the presence of methylenetetrahydrofolate (CH2THF).

 

Reference Protein and Structure

Sequence
P08773 UniProt (2.1.2.8) IPR014619 (Sequence Homologues) (PDB Homologues)
Biological species
Enterobacteria phage T4 (Virus) Uniprot
PDB
1b5d - DCMP Hydroxymethylase from T4 (Intact) (2.2 Å) PDBe PDBsum 1b5d
Catalytic CATH Domains
3.30.572.10 CATHdb (see all for 1b5d)
Click To Show Structure

Enzyme Reaction (EC:2.1.2.8)

(6R)-5,10-methylenetetrahydrofolate(2-)
CHEBI:15636ChEBI
+
2'-deoxycytosine 5'-monophosphate(2-)
CHEBI:57566ChEBI
+
water
CHEBI:15377ChEBI
→
5-hydroxymethyldeoxycytidylate(2-)
CHEBI:57962ChEBI
+
(6S)-5,6,7,8-tetrahydrofolate(2-)
CHEBI:57453ChEBI
Alternative enzyme names: D-cytidine 5'-monophosphate hydroxymethylase, dCMP hydroxymethylase, DeoxyCMP hydroxymethylase, Deoxycytidylate hydroxymethylase, Deoxycytidylic hydroxymethylase, Deoxycytidylate hydroxymethyltransferase,

Enzyme Mechanism

Introduction

Glu60 acts through a water molecule to donate a proton to N10 of CH2THF allowing the formation of an imminium ion at the N5 position. This later permits the donation of a methyl group to dCMP. The sulphur atom of Cys148 undergoes nucleophilic attack on the cytosine ring with the aid of Asp179 which donates a proton to the ring. Asp179 subsequently accepts the proton back, initiating attack on the carbon of the imminium ion by C5 of cytosine forming a bridging methyl group between the C5 of cytosine and N5 of THF. Cytosine loses a proton from the C5 position to reform the double bond and at the same time accepts a proton from Asp179. Loss of this proton to Asp179 causes a double bond to be formed at the new bridging methyl with concomitant lysis of the C-THF bond. The pi system is then attacked by water at the new carbon which causes the lysis of the bond to cystine. A loss of a proton from the water results in formation of the product .

Catalytic Residues Roles

UniProt PDB* (1b5d)
Cys148 Cys148A Holds dCMP in position during the reaction and activates the C5 position. Allows the final addition of water. covalently attached, nucleofuge, nucleophile
Asp179 Asp179A Acts as a proton acceptor and donor to dCMP proton acceptor, electrostatic stabiliser, proton donor
Glu60 Glu60A Promotes the formation of the imminium ion form of C2THF through a water. proton acceptor, electrostatic stabiliser, 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

proton transfer, proton relay, overall reactant used, decyclisation, bimolecular nucleophilic addition, enzyme-substrate complex formation, intermediate formation, elimination (not covered by the Ingold mechanisms), overall product formed, aromatic unimolecular elimination by the conjugate base, intermediate collapse, heterolysis, bimolecular elimination, enzyme-substrate complex cleavage, native state of enzyme regenerated, inferred reaction step

References

  1. Song HK et al. (1999), EMBO J, 18, 1104-1113. Crystal structure of deoxycytidylate hydroxymethylase from bacteriophage T4, a component of the deoxyribonucleoside triphosphate-synthesizing complex. DOI:10.1093/emboj/18.5.1104. PMID:10064578.
  2. Hardy LW et al. (1995), Biochemistry, 34, 8422-8432. Electrostatic guidance of catalysis by a conserved glutamic acid in Escherichia coli dTMP synthase and bacteriophage T4 dCMP hydroxymethylase. DOI:10.1021/bi00026a025. PMID:7599133.
  3. Butler MM et al. (1994), Biochemistry, 33, 10521-10526. Evidence from 18O Exchange Studies for an Exocyclic Methylene Intermediate in the Reaction Catalyzed by T4 Deoxycytidylate Hydroxymethylase. DOI:10.1021/bi00200a038. PMID:8068692.

Step 1. N10 of CH2THF is protonated by a water molecule which is subsequently reprotonated by Glu60.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp179A electrostatic stabiliser
Glu60A proton donor

Chemical Components

proton transfer, proton relay, overall reactant used

Step 2. The 5 membered ring of CH2THF collapses.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu60A electrostatic stabiliser
Asp179A electrostatic stabiliser

Chemical Components

decyclisation

Step 3. Cys148 acts as a nucleophile to attack the cytosine ring of dCMP. dCMP then accepts a proton from Asp179.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Cys148A covalently attached
Asp179A electrostatic stabiliser, proton donor
Cys148A nucleophile

Chemical Components

ingold: bimolecular nucleophilic addition, enzyme-substrate complex formation, intermediate formation, overall reactant used, proton transfer

Step 4. Asp179 abstracts a the proton from the nitrogen of dCMP which triggers the nucleophilic attack on the CH2THF intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu60A electrostatic stabiliser
Cys148A covalently attached
Asp179A proton acceptor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic addition, intermediate formation

Step 5. There is loss of a proton from the intermediate. This is likely to be lost to the solvent. The intermediate accepts a proton from Asp179.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Cys148A covalently attached
Glu60A electrostatic stabiliser
Asp179A proton donor

Chemical Components

proton transfer, elimination (not covered by the Ingold mechanisms)

Step 6. Asp179 abstracts the proton from the nitrogen of the cytosine intermediate. This causes a double bond to be formed at the new bridging methyl with concomitant lysis of the C-THF bond.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Cys148A covalently attached
Glu60A electrostatic stabiliser
Asp179A proton acceptor

Chemical Components

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

Step 7. Water attacks the newly formed double bond which causes the elimination of Cys148.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu60A electrostatic stabiliser
Asp179A electrostatic stabiliser
Cys148A covalently attached, nucleofuge

Chemical Components

ingold: bimolecular elimination, ingold: bimolecular nucleophilic addition, enzyme-substrate complex cleavage

Step 8. In an inferred step the hydroxyl group is deprotonated to form the product and Glu60 is reprotonated to regenerate the native state of the enzyme.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu60A proton acceptor

Chemical Components

proton transfer, native state of enzyme regenerated, overall product formed, inferred reaction step
Download: Image, Marvin File

Step 1. N10 of CH2THF is protonated by a water molecule which is subsequently reprotonated by Glu60.

Step 2. The 5 membered ring of CH2THF collapses.

Step 3. Cys148 acts as a nucleophile to attack the cytosine ring of dCMP. dCMP then accepts a proton from Asp179.

Step 4. Asp179 abstracts a the proton from the nitrogen of dCMP which triggers the nucleophilic attack on the CH2THF intermediate.

Step 5. There is loss of a proton from the intermediate. This is likely to be lost to the solvent. The intermediate accepts a proton from Asp179.

Step 6. Asp179 abstracts the proton from the nitrogen of the cytosine intermediate. This causes a double bond to be formed at the new bridging methyl with concomitant lysis of the C-THF bond.

Step 7. Water attacks the newly formed double bond which causes the elimination of Cys148.

Step 8. In an inferred step the hydroxyl group is deprotonated to form the product and Glu60 is reprotonated to regenerate the native state of the enzyme.

Products.

Contributors

Gemma L. Holliday, Amelia Brasnett