EzCatDB: S00276

DB codeS00276
RLCP classification5.202.1504000.1
4.1504.3900000.53
4.162.20000.1
5.1204.671000.4100
CATH domainDomain 13.30.572.10Catalytic domain
E.C.2.1.2.8


Enzyme Name
Swiss-protKEGG

P08773
Protein nameDeoxycytidylate 5-hydroxymethyltransferasedeoxycytidylate 5-hydroxymethyltransferase
dCMP hydroxymethylase
d-cytidine 5'-monophosphate hydroxymethylase
deoxyCMP hydroxymethylase
deoxycytidylate hydroxymethylase
deoxycytidylic hydroxymethylase
SynonymsDeoxycytidylate hydroxymethylase
EC 2.1.2.8
dCMP hydroxymethylase
dCMP HMase

KEGG pathways
MAP codePathways
MAP00240Pyrimidine metabolism
MAP00670One carbon pool by folate

Swiss-prot:Accession NumberP08773
Entry nameDCHM_BPT4
Activity5,10-methylenetetrahydrofolate + H(2)O + deoxycytidylate = tetrahydrofolate + 5- hydroxymethyldeoxycytidylate.
Subunit
Subcellular location
Cofactor


SubstratesProducts
KEGG-idC00143C00001C00239C00101C03997
Compound5,10-MethylenetetrahydrofolateH2ODeoxycytidylateTetrahydrofolate5-Hydroxymethyldeoxycytidylate
Typeamino acids,amide group,amine group,aromatic ring (only carbon atom),aromatic ring (with nitrogen atoms),carboxyl groupH2Oamine group,nucleotideamino acids,amide group,amine group,aromatic ring (only carbon atom),aromatic ring (with nitrogen atoms),carboxyl groupamine group,carbohydrate,nucleotide
1b49AUnbound
UnboundUnboundUnbound
1b49CUnbound
UnboundUnboundUnbound
1b5dAUnbound
Bound:DCMUnboundUnbound
1b5dBUnbound
Bound:DCMUnboundUnbound
1b5eAUnbound
Bound:DCMUnboundUnbound
1b5eBUnbound
Bound:DCMUnboundUnbound

Active-site residues
resource
Swiss-prot;P08773, literature [1], [3], [5] & [6]
pdbCatalytic residues
1b49AGLU 60;TYR 96;CYS 148;ASP 179
1b49CGLU 60;TYR 96;CYS 148;ASP 179
1b5dAGLU 60;TYR 96;CYS 148;ASP 179
1b5dBGLU 60;TYR 96;CYS 148;ASP 179
1b5eAGLU 60;TYR 96;CYS 148;ASP 179
1b5eBGLU 60;TYR 96;CYS 148;ASP 179

References for Catalytic Mechanism
ReferencesSectionsNo. of steps in catalysis
[1]Scheme 1, p.10320-103215
[2]Scheme 1, Scheme 2, p.10525-105265
[3]Scheme 1, Scheme 2, p.130505
[4]Scheme 1, Scheme 3, Scheme 5, p.84235
[6]Fig.5, p.1110-11112

references
[1]
PubMed ID1420151
JournalBiochemistry
Year1992
Volume31
Pages10315-21
AuthorsGraves KL, Butler MM, Hardy LW
TitleRoles of Cys148 and Asp179 in catalysis by deoxycytidylate hydroxymethylase from bacteriophage T4 examined by site-directed mutagenesis.
[2]
PubMed ID8068692
JournalBiochemistry
Year1994
Volume33
Pages10521-6
AuthorsButler MM, Graves KL, Hardy LW
TitleEvidence from 18O exchange studies for an exocyclic methylene intermediate in the reaction catalyzed by T4 deoxycytidylate hydroxymethylase.
[3]
PubMed ID7947710
JournalBiochemistry
Year1994
Volume33
Pages13049-56
AuthorsGraves KL, Hardy LW
TitleKinetic and equilibrium alpha-secondary tritium isotope effects on reactions catalyzed by dCMP hydroxymethylase from bacteriophage T4.
[4]
PubMed ID7599133
JournalBiochemistry
Year1995
Volume34
Pages8422-32
AuthorsHardy LW, Graves KL, Nalivaika E
TitleElectrostatic guidance of catalysis by a conserved glutamic acid in Escherichia coli dTMP synthase and bacteriophage T4 dCMP hydroxymethylase.
[5]
PubMed ID7574499
JournalAnnu Rev Biochem
Year1995
Volume64
Pages721-62
AuthorsCarreras CW, Santi DV
TitleThe catalytic mechanism and structure of thymidylate synthase.
[6]
CommentsX-ray crystallography
PubMed ID10064578
JournalEMBO J
Year1999
Volume18
Pages1104-13
AuthorsSong HK, Sohn SH, Suh SW
TitleCrystal structure of deoxycytidylate hydroxymethylase from bacteriophage T4, a component of the deoxyribonucleoside triphosphate-synthesizing complex.
Related PDB1b49,1b5d,1b5e
[7]
PubMed ID10216306
JournalActa Crystallogr D Biol Crystallogr
Year1999
Volume55
Pages1061-3
AuthorsSohn SH, Song HK, Min K, Cho SJ, Moon J, Lee JY, Ahn HJ, Chang C, Kim HJ, Suh SW
TitleCrystallization and preliminary X-ray crystallographic analysis of deoxycytidylate hydroxymethylase from bacteriophage T4.

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
###
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.

createdupdated
2002-09-232009-02-26


Copyright: Nozomi Nagano, JST & CBRC-AIST
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