DB code: S00276

RLCP classification 5.202.1504000.1 : Elimination
4.1504.3900000.53 : Addition
4.162.20000.1 : Addition
5.1204.671000.4100 : Elimination
CATH domain 3.30.572.10 : Thymidylate Synthase; Chain A Catalytic domain
E.C. 2.1.2.8
CSA
M-CSA
MACiE

CATH domain Related DB codes (homologues)

Uniprot Enzyme Name
UniprotKB Protein name Synonyms RefSeq Pfam
P08773 Deoxycytidylate 5-hydroxymethyltransferase
Deoxycytidylate hydroxymethylase
EC 2.1.2.8
dCMP hydroxymethylase
dCMP HMase
NP_049659.1 (Protein)
NC_000866.4 (DNA/RNA sequence)
PF00303 (Thymidylat_synt)
[Graphical View]

KEGG enzyme name
deoxycytidylate 5-hydroxymethyltransferase
dCMP hydroxymethylase
d-cytidine 5'-monophosphate hydroxymethylase
deoxyCMP hydroxymethylase
deoxycytidylate hydroxymethylase
deoxycytidylic hydroxymethylase

UniprotKB: Accession Number Entry name Activity Subunit Subcellular location Cofactor
P08773 DCHM_BPT4 5,10-methylenetetrahydrofolate + H(2)O + deoxycytidylate = tetrahydrofolate + 5- hydroxymethyldeoxycytidylate.

KEGG Pathways
Map code Pathways E.C.
MAP00240 Pyrimidine metabolism
MAP00670 One carbon pool by folate

Compound table
Substrates Products Intermediates
KEGG-id C00143 C00001 C00239 C00101 C03997
E.C.
Compound 5,10-Methylenetetrahydrofolate H2O Deoxycytidylate Tetrahydrofolate 5-Hydroxymethyldeoxycytidylate
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
ChEBI 15377
15918
15635
20506
16952
PubChem 439175
22247451
962
13945
5460413
91443
440189
1b49A Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Unbound Unbound Unbound
1b49C Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Unbound Unbound Unbound
1b5dA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Bound:DCM Unbound Unbound
1b5dB Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Bound:DCM Unbound Unbound
1b5eA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Bound:DCM Unbound Unbound
1b5eB Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Bound:DCM Unbound Unbound

Reference for Active-site residues
resource references E.C.
Swiss-prot;P08773, literature [1], [3], [5] & [6]

Active-site residues
PDB Catalytic residues Cofactor-binding residues Modified residues Main-chain involved in catalysis Comment
1b49A Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain GLU 60;TYR 96;CYS 148;ASP 179
1b49C Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain GLU 60;TYR 96;CYS 148;ASP 179
1b5dA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain GLU 60;TYR 96;CYS 148;ASP 179
1b5dB Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain GLU 60;TYR 96;CYS 148;ASP 179
1b5eA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain GLU 60;TYR 96;CYS 148;ASP 179
1b5eB Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain GLU 60;TYR 96;CYS 148;ASP 179

References for Catalytic Mechanism
References Sections No. of steps in catalysis
[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]
Resource
Comments
Medline ID
PubMed ID 1420151
Journal Biochemistry
Year 1992
Volume 31
Pages 10315-21
Authors Graves KL, Butler MM, Hardy LW
Title Roles of Cys148 and Asp179 in catalysis by deoxycytidylate hydroxymethylase from bacteriophage T4 examined by site-directed mutagenesis.
Related PDB
Related UniProtKB
[2]
Resource
Comments
Medline ID
PubMed ID 8068692
Journal Biochemistry
Year 1994
Volume 33
Pages 10521-6
Authors Butler MM, Graves KL, Hardy LW
Title Evidence from 18O exchange studies for an exocyclic methylene intermediate in the reaction catalyzed by T4 deoxycytidylate hydroxymethylase.
Related PDB
Related UniProtKB
[3]
Resource
Comments
Medline ID
PubMed ID 7947710
Journal Biochemistry
Year 1994
Volume 33
Pages 13049-56
Authors Graves KL, Hardy LW
Title Kinetic and equilibrium alpha-secondary tritium isotope effects on reactions catalyzed by dCMP hydroxymethylase from bacteriophage T4.
Related PDB
Related UniProtKB
[4]
Resource
Comments
Medline ID
PubMed ID 7599133
Journal Biochemistry
Year 1995
Volume 34
Pages 8422-32
Authors Hardy LW, Graves KL, Nalivaika E
Title Electrostatic guidance of catalysis by a conserved glutamic acid in Escherichia coli dTMP synthase and bacteriophage T4 dCMP hydroxymethylase.
Related PDB
Related UniProtKB
[5]
Resource
Comments
Medline ID
PubMed ID 7574499
Journal Annu Rev Biochem
Year 1995
Volume 64
Pages 721-62
Authors Carreras CW, Santi DV
Title The catalytic mechanism and structure of thymidylate synthase.
Related PDB
Related UniProtKB
[6]
Resource
Comments X-ray crystallography
Medline ID
PubMed ID 10064578
Journal EMBO J
Year 1999
Volume 18
Pages 1104-13
Authors Song HK, Sohn SH, Suh SW
Title Crystal structure of deoxycytidylate hydroxymethylase from bacteriophage T4, a component of the deoxyribonucleoside triphosphate-synthesizing complex.
Related PDB 1b49 1b5d 1b5e
Related UniProtKB
[7]
Resource
Comments
Medline ID
PubMed ID 10216306
Journal Acta Crystallogr D Biol Crystallogr
Year 1999
Volume 55
Pages 1061-3
Authors Sohn SH, Song HK, Min K, Cho SJ, Moon J, Lee JY, Ahn HJ, Chang C, Kim HJ, Suh SW
Title Crystallization and preliminary X-ray crystallographic analysis of deoxycytidylate hydroxymethylase from bacteriophage T4.
Related PDB
Related UniProtKB

Comments
(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.
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
###
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.

Created Updated
2002-09-23 2009-02-26