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 15377	15918 15918	15635 20506 15635 20506	16952 16952
PubChem	439175 439175	22247451 962 22247451 962	13945 13945	5460413 91443 5460413 91443	440189 440189

1b49A	Unbound		Unbound	Unbound	Unbound
1b49C	Unbound		Unbound	Unbound	Unbound
1b5dA	Unbound		Bound:DCM	Unbound	Unbound
1b5dB	Unbound		Bound:DCM	Unbound	Unbound
1b5eA	Unbound		Bound:DCM	Unbound	Unbound
1b5eB	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	GLU 60;TYR 96;CYS 148;ASP 179
1b49C	GLU 60;TYR 96;CYS 148;ASP 179
1b5dA	GLU 60;TYR 96;CYS 148;ASP 179
1b5dB	GLU 60;TYR 96;CYS 148;ASP 179
1b5eA	GLU 60;TYR 96;CYS 148;ASP 179
1b5eB	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.

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