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| Enzyme Name | | Swiss-prot | KEGG |
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| P08773 |
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| Protein name | Deoxycytidylate 5-hydroxymethyltransferase | deoxycytidylate 5-hydroxymethyltransferasedCMP hydroxymethylased-cytidine 5'-monophosphate hydroxymethylasedeoxyCMP hydroxymethylasedeoxycytidylate hydroxymethylasedeoxycytidylic hydroxymethylase |
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| Synonyms | Deoxycytidylate hydroxymethylaseEC 2.1.2.8dCMP hydroxymethylasedCMP HMase |
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| KEGG pathways | | MAP code | Pathways |
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| MAP00240 | Pyrimidine metabolism | | MAP00670 | One carbon pool by folate |
| Swiss-prot:Accession Number | P08773 |
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| Entry name | DCHM_BPT4 |
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| Activity | 5,10-methylenetetrahydrofolate + H(2)O + deoxycytidylate = tetrahydrofolate + 5- hydroxymethyldeoxycytidylate. |
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| Subunit |
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| Subcellular location |
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| Cofactor |
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| Substrates | Products |
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| KEGG-id | C00143 | C00001 | C00239 | C00101 | C03997 |
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| Compound | 5,10-Methylenetetrahydrofolate | H2O | Deoxycytidylate | Tetrahydrofolate | 5-Hydroxymethyldeoxycytidylate |
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| Type | amino acids,amide group,amine group,aromatic ring (only carbon atom),aromatic ring (with nitrogen atoms),carboxyl group | H2O | amine group,nucleotide | amino acids,amide group,amine group,aromatic ring (only carbon atom),aromatic ring (with nitrogen atoms),carboxyl group | amine group,carbohydrate,nucleotide |
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| 1b49A |  | Unbound |
| Unbound | Unbound | Unbound |
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| 1b49C | | Unbound |
| Unbound | Unbound | Unbound |
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| 1b5dA | | Unbound |
| Bound:DCM | Unbound | Unbound |
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| 1b5dB | | Unbound |
| Bound:DCM | Unbound | Unbound |
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| 1b5eA | | Unbound |
| Bound:DCM | Unbound | Unbound |
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| 1b5eB | | Unbound |
| Bound:DCM | Unbound | Unbound |
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| References for Catalytic Mechanism | | References | Sections | No. of steps in catalysis |
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| [1] | Scheme 1, p.10320-10321 | 5 | | [2] | Scheme 1, Scheme 2, p.10525-10526 | 5 | | [3] | Scheme 1, Scheme 2, p.13050 | 5 | | [4] | Scheme 1, Scheme 3, Scheme 5, p.8423 | 5 | | [6] | Fig.5, p.1110-1111 | 2 |
| references | | [1] |
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| PubMed ID | 1420151 |
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| Journal | Biochemistry |
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| Year | 1992 |
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| Volume | 31 |
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| Pages | 10315-21 |
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| Authors | Graves KL, Butler MM, Hardy LW |
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| Title | Roles of Cys148 and Asp179 in catalysis by deoxycytidylate hydroxymethylase from bacteriophage T4 examined by site-directed mutagenesis. |
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| [2] |
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| PubMed ID | 8068692 |
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| Journal | Biochemistry |
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| Year | 1994 |
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| Volume | 33 |
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| Pages | 10521-6 |
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| Authors | Butler MM, Graves KL, Hardy LW |
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| Title | Evidence from 18O exchange studies for an exocyclic methylene intermediate in the reaction catalyzed by T4 deoxycytidylate hydroxymethylase. |
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| [3] |
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| PubMed ID | 7947710 |
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| Journal | Biochemistry |
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| Year | 1994 |
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| Volume | 33 |
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| Pages | 13049-56 |
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| Authors | Graves KL, Hardy LW |
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| Title | Kinetic and equilibrium alpha-secondary tritium isotope effects on reactions catalyzed by dCMP hydroxymethylase from bacteriophage T4. |
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| [4] |
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| PubMed ID | 7599133 |
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| Journal | Biochemistry |
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| Year | 1995 |
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| Volume | 34 |
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| Pages | 8422-32 |
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| Authors | Hardy LW, Graves KL, Nalivaika E |
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| Title | Electrostatic guidance of catalysis by a conserved glutamic acid in Escherichia coli dTMP synthase and bacteriophage T4 dCMP hydroxymethylase. |
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| [5] |
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| PubMed ID | 7574499 |
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| Journal | Annu Rev Biochem |
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| Year | 1995 |
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| Volume | 64 |
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| Pages | 721-62 |
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| Authors | Carreras CW, Santi DV |
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| Title | The catalytic mechanism and structure of thymidylate synthase. |
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| [6] |
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| Comments | X-ray crystallography |
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| PubMed ID | 10064578 |
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| Journal | EMBO J |
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| Year | 1999 |
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| Volume | 18 |
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| Pages | 1104-13 |
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| Authors | Song HK, Sohn SH, Suh SW |
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| Title | Crystal structure of deoxycytidylate hydroxymethylase from bacteriophage T4, a component of the deoxyribonucleoside triphosphate-synthesizing complex. |
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| Related PDB | 1b49,1b5d,1b5e |
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| [7] |
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| PubMed ID | 10216306 |
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| Journal | Acta Crystallogr D Biol Crystallogr |
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| Year | 1999 |
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| Volume | 55 |
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| Pages | 1061-3 |
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| Authors | Sohn SH, Song HK, Min K, Cho SJ, Moon J, Lee JY, Ahn HJ, Chang C, Kim HJ, Suh SW |
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| Title | Crystallization and preliminary X-ray crystallographic analysis of deoxycytidylate hydroxymethylase from bacteriophage T4. |
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| comments | This enzyme belongs to the thymidylate synthase family.
Although this enzyme is classified into transferases (E.C. 2.-.-.-), it does not catalyze transfer reaction.
According to the literature [1], [2], [3], [4] & [5], this enzyme catalyzes several reactions as follows:
For 5,10-Methylenetetrahydrofolate, the following reaction occurs (see [5]):
(A) Activation of methylene group by iminium ion formation; Elimination of N-10 amino group from the methylene, giving CH2=THF intermediate.
For Deoxycytidylate (dCMP), the following reactions occur succesively, where reactions (E) and (D) occurs concertedly :
(B) Addition of Cys148 to C6 atom of dCMP, forming a covalent intermediate.
(C) Addition of the C5 atom of the covalent intermediate to the carbon atom of the iminium ion of CH2=THF, giving a dCMP-CH2-THF intermediate.
(D) Elimination of THF from the dCMP-CH2-THF intermediate, giving a dCMP=CH2 intermediate.
(E) Concerted addition & elimination; Addition of water to the attached methylene group of dCMP=CH2. Elimination of Cys148 from the dCMP=CH2 or Hydride transfer to Cys148
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The detailed catalytic mechanisms are as follows:
(A) Elimination of N-10 amino group from the methylene, giving CH2=THF intermediate.
(A1) According to the literature [4] & [5], which discussed the mechanism of the homologous enzyme, thymidylate synthase (TS; E.C. 2.1.1.45, S00275 in EzCatDB), constraints of the 5-membered ring of methyle-THF could be distorted by the interaction with the enzyme. This distortion may facilitate the bond cleavage between N10 and the methylene. This reaction probably proceeds via E1 mechanism, suggested by the bond lengthening (see [5]).
(A2) Glu60, which is conserved in this enzyme and the homologue, TS, acts as a general acid to protonate the eliminated N10-amine group, to complete the elimination.
(B) Addition of Cys148 to C6 atom of dCMP (see [1] & [2]).
(B1) Cys148 makes a nucleophilic attack on the C6 atom of dCMP, forming a covalent bond with the substrate.
(B2) Asp179 acts as a general acid to protonate the N3 atom (protonation site) of dCMP.
(C) Addition of the C5 (sp2 carbon) atom to the carbon atom of the iminium ion (see [4]).
(C1) Asp179 acts as a general base, which activates the nucleophile, the C5 atom of the cytosine ring, by deprotonating the N3 atom.
(C2) The activated nucleophile, the C5 atom, makes a nucleophilic attack on the activated methylene group of CH2=THF. This reaction leaves a proton at the C5 atom (sp3 carbon).
(C3) Substrate-assisted base, the N5 atom of THF, deprotonates the proton at the C5 atom, through a water.
(C4) Asp179 acts as a general acid to protonate the N3 atom.
(D) Elimination of THF from the dCMP-CH2-THF intermediate (see [4]).
(D1) Glu60 may modulate and facilitate Asp179, by destabilizing the negative charge on Asp179 with its own negative charge.
(D2) Asp179 acts as a general base to deprotonate the N3 atom (deprotonation site) of dCMP-CH2-THF, which leads to the bond cleavage between the methylene carbon and the N5 atom of THF, and to the formation of double bond between the carbon and the C5 carbon.
(E) Concerted addition & elimination; Addition of water & Elimination of Cys148.
(E1) Tyr96 probably acts as a general base to activate the water molecule.
(E2) The activated water molecule makes a nucleophilic attack on the C7 methylene atom.
(E3) The reaction leads to the bond cleavage between the C6 atom and Cys148, forming a double-bond between the C5 and C6 atoms.
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| created | updated |
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| 2002-09-23 | 2009-02-26 |
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