Information on EC 6.3.3.2 - 5-Formyltetrahydrofolate cyclo-ligase

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The expected taxonomic range for this enzyme is: Eukaryota, Bacteria

EC NUMBER
COMMENTARY hide
6.3.3.2
-
RECOMMENDED NAME
GeneOntology No.
5-Formyltetrahydrofolate cyclo-ligase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ATP + 5-formyltetrahydrofolate = ADP + phosphate + 5,10-methenyltetrahydrofolate
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
heteroatomic ring closure
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
folate transformations I
-
-
folate transformations II
-
-
Metabolic pathways
-
-
N10-formyl-tetrahydrofolate biosynthesis
-
-
One carbon pool by folate
-
-
tetrahydrofolate metabolism
-
-
SYSTEMATIC NAME
IUBMB Comments
5-Formyltetrahydrofolate cyclo-ligase (ADP-forming)
-
CAS REGISTRY NUMBER
COMMENTARY hide
37318-64-2
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
strain BA4489
Uniprot
Manually annotated by BRENDA team
strain BA4489
Uniprot
Manually annotated by BRENDA team
cow
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
E0IWA2
UniProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
Mus musculus 129/Sny
strain 129/Sny
Uniprot
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
inhibition of MTHFS in human MCF-7 breast cancer cells arrests the growth of cells
metabolism
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
araATP + 5-formyltetrahydrofolate
araADP + phosphate + 5,10-methylenetetrahydrofolate
show the reaction diagram
-
-
-
-
-
ATP + (6S)-5-formyltetrahydrofolate
ADP + 5,10-methenyltetrahydrofolate + phosphate
show the reaction diagram
-
-
-
?
ATP + 5-formyltetrahydrofolate
?
show the reaction diagram
ATP + 5-formyltetrahydrofolate
ADP + 5,10-methenyltetrahydrofolate + phosphate
show the reaction diagram
ATP + 5-formyltetrahydrofolate
ADP + phosphate + 5,10-methenyltetrahydrofolate
show the reaction diagram
ATP + 5-formyltetrahydrofolate
ADP + phosphate + 5,10-methylenetetrahydrofolate
show the reaction diagram
ATP + 5-formyltetrahydrofolate-pentaglutamate
ADP + phosphate + 5,10-methylenetetrahydrofolate-pentaglutamate
show the reaction diagram
preferred substrate to the monoglutamyl form
-
ir
ATP + 5-formyltetrahydrohomofolate
?
show the reaction diagram
-
-
-
-
-
ATP + 5-formyltetrahydropteroyl-pentaglutamate
?
show the reaction diagram
ATPgammaS + 5-formyltetrahydrofolate
ADPgammaS + phosphate + 5,10-methylenetetrahydrofolate
show the reaction diagram
-
-
-
-
-
CTP + 5-formyltetrahydrofolate
CDP + phosphate + 5,10-methylenetetrahydrofolate
show the reaction diagram
dATP + 5-formyltetrahydrofolate
dADP + phosphate + 5,10-methylenetetrahydrofolate
show the reaction diagram
-
-
-
-
-
dTTP + 5-formyltetrahydrofolate
dTDP + phosphate + 5,10-methylenetetrahydrofolate
show the reaction diagram
-
-
-
-
-
GTP + 5-formyltetrahydrofolate
GDP + phosphate + 5,10-methylenetetrahydrofolate
show the reaction diagram
ITP + 5-formyltetrahydrofolate
IDP + phosphate + 5,10-methylenetetrahydrofolate
show the reaction diagram
-
-
-
-
-
UTP + 5-formyltetrahydrofolate
UDP + phosphate + 5,10-methylenetetrahydrofolate
show the reaction diagram
additional information
?
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
ATP + 5-formyltetrahydrofolate
?
show the reaction diagram
ATP + 5-formyltetrahydrofolate
ADP + 5,10-methenyltetrahydrofolate + phosphate
show the reaction diagram
ATP + 5-formyltetrahydrofolate
ADP + phosphate + 5,10-methenyltetrahydrofolate
show the reaction diagram
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
MgATP2-
-
enzyme is inhibited by salt, therefore MgATP2- is used as cofactor instead of MgCl2 and NaATP-
additional information
-
no activity with nucleoside 5'-mono- or 5'-diphosphates
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Cu2+
-
activates, can substitute for Mg2+
Fe2+
-
activates, can substitute for Mg2+
Zn2+
-
activates, can substitute for Mg2+
additional information
-
the enzyme is dependent on divalent cation, Mg2+ is preferred
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(NH4)2SO4
-
-
10-formylfolic acid
-
-
4-amino-N10-methylpteroyl pentaglutamate
-
i.e. methotrexate pentaglutamate
5-formyl,10-methyltetrahydrofolate
-
weak inhibitor
5-Formyltetrahydrohomofolate
5-methyltetrahydrofolate
AgNO3
-
-
dihydropteroyl pentaglutamate
-
potent
dihydropteroyl-L-Glu
-
weak
folic acid
-
-
folic acid pentaglutamate
-
-
folic acid triglutamate
-
-
lometrexol
LY309887
N-(4-{[2-(2-amino-5-formyl-4-oxo-1,4,5,6,7,8-hexahydropteridin-6-yl)ethyl](methyl)amino}benzoyl)-L-glutamic acid
-
at 37C in 100 mM MES, pH 6.0
NEM
-
-
o-Iodosobenzoate
-
-
p-mercuribenzoate
-
-
pemetrexed
phosphate
succinate
-
-
[6R,S]-10-formyltetrahydrofolate triglutamate
-
-
[6R,S]-10-formyltetrahydrohomofolate
-
-
[6R,S]-10-formyltetrahydropteroate
-
-
[6R,S]-5-formyl,10-methyltetrahydrofolateGlu3
-
-
[6R,S]-5-formyl,11-methyltetrahydrohomofolate
-
-
[6R,S]-5-formyltetrahydrohomofolate
-
-
[6R]-10-formyltetrahydrofolate
additional information
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
5-formyltetrahydrofolate polyglutamate
-
induced recombinant enzyme expression in cancer cell cultures, increasing effect with increasing chain length
dopamine
-
0.02 mM, increases the initial rapid phase of folate turnover by 4%
N-acetyldopamine
-
0.02 mM, accelerates markedly folate turnover
additional information
-
L-dopa does not affect folate turnover rate
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.004
(6R,S)-5-formyltetrahydrofolate
-
-
0.0044 - 0.0047
(6R,S)-5-formyltetrahydropteroyl-Glu
0.002 - 0.005
(6S)-5-formyltetrahydrofolate
0.0008
(6S)-5-formyltetrahydropteroyl pentaglutamate
-
mitochondrial enzyme
0.0006
(6S)-5-formyltetrahydropteroyl-pentaglutamate
0.0002
5-formyl-tetrahydropteroyl-pentaglutamate
-
-
0.0005 - 130
5-formyltetrahydrofolate
0.0056
araATP
-
-
0.00096 - 5
ATP
0.00021
ATPgammaS
-
-
0.0016
CTP
-
-
0.0013
dATP
-
-
0.0066
dTTP
-
-
0.021
GTP
-
-
0.024
ITP
-
-
0.0003 - 0.769
MgATP2-
0.4 - 0.6
MgCTP2-
0.5
MgUTP2-
-
-
0.0064
UTP
-
-
additional information
additional information
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
16.3
(6S)-5-formyltetrahydrofolate
Mus musculus
Q9D110
pH 6.0, 28C, recombinant enzyme
0.02 - 5.3
5-formyltetrahydrofolate
1 - 1.7
ATP
0.99
MgATP2-
Mycoplasma pneumoniae
-
pH 6.0, 37C, recombinant enzyme
additional information
additional information
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.013
10-formylfolic acid
-
at 37C in 100 mM MES, pH 6.0
0.001
5-formyl,10-methyltetrahydrofolate
-
at 37C in 100 mM MES, pH 6.0
0.0007
5-Formyltetrahydrohomofolate
-
at 37C in 100 mM MES, pH 6.0
0.058
folic acid
-
at 37C in 100 mM MES, pH 6.0
0.0001
folic acid pentaglutamate
-
at 37C in 100 mM MES, pH 6.0
0.0017
folic acid triglutamate
-
at 37C in 100 mM MES, pH 6.0
0.009
lometrexol
-
-
0.006
LY309887
-
-
0.048
pemetrexed
-
-
2.12
phosphate
-
pH 6.0, 37C, recombinant enzyme
0.00003
[6R,S]-10-formyltetrahydrofolate triglutamate
-
at 37C in 100 mM MES, pH 6.0
0.0002
[6R,S]-10-formyltetrahydrohomofolate
-
at 37C in 100 mM MES, pH 6.0
0.005
[6R,S]-10-formyltetrahydropteroate
-
at 37C in 100 mM MES, pH 6.0
0.02
[6R,S]-5-formyl,10-methyltetrahydrofolateGlu3
-
at 37C in 100 mM MES, pH 6.0
0.006
[6R,S]-5-formyl,11-methyltetrahydrohomofolate
-
at 37C in 100 mM MES, pH 6.0
0.0007
[6R,S]-5-formyltetrahydrohomofolate
-
at 37C in 100 mM MES, pH 6.0
0.00015
[6R]-10-formyltetrahydrofolate
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.223 - 0.303
lometrexol
0.014 - 0.029
LY309887
0.033 - 0.045
pemetrexed
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.00006
-
placenta
0.00026
-
postweaning, brain
0.00032
-
kidney
0.00036
-
fetal liver
0.0011
-
adult liver
0.0015
-
3 weeks old liver
2.22
purified recombinant enzyme
2.8
-
purified recombinant enzyme
7
-
purified recombinant enzyme
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.8
-
-
7.4
-
assay at
8.5
bicine buffer
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4 - 6
-
4.0: about 50% of maximal activity, 6.0: about 60% of maximal activity
5.5 - 9
linear increase of activity to maximum at pH 8.5
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
24 - 28
assay at
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
-
T-lymphoblast cell line CEM and its methotrexate resistant subline, colon tumor cell line
Manually annotated by BRENDA team
-
CHO, ovary cell line and its methotrexate resistant subline
Manually annotated by BRENDA team
-
moderate enzyme expression
Manually annotated by BRENDA team
-
low enzyme expression
Manually annotated by BRENDA team
-
L210 mouse leukemia cells
Manually annotated by BRENDA team
-
low activity
Manually annotated by BRENDA team
-
moderate enzyme expression
Manually annotated by BRENDA team
-
low activity
Manually annotated by BRENDA team
-
low content
Manually annotated by BRENDA team
-
a neuroblastoma cell line
Manually annotated by BRENDA team
-
moderate enzyme expression
Manually annotated by BRENDA team
additional information
-
tissue specific expression
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
22000
-
monomeric recombinant enzyme, gel filtration
23000
-
gel filtration
25210
-
calculated from amino acid sequence
25220
-
MALDI-TOF
26500 - 29000
-
gel filtration, HPLC gel filtration, equilibrium ultracentrifugation
44000
-
dimeric recombinant enzyme, gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
additional information
structure modelling, overview
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cocrystallization with folinate, ATP and Mg2+, sitting drop vapour diffusion method using 25% polyethylene glycol 3350, 100 mM bis-Tris pH 5.5
purified recombinant wild-type MTHFS in a binary complex with ADP, with the N5-iminium phosphate reaction intermediate, or as an enzyme-product complex, hanging drop method at 16C, 600 nl protein solution, with or without 5 mM 5-formyltetrahydrofolate and 5 mM ATP, are equilibrated against 0.05 ml of reservoir solution containing 100 mM HEPES, pH 6.6, 20 mM MgCl2, 20 mM NiCl2, and 20% w/v PEG 3350, 2 days, X-ray diffraction structure determination and analysis
purified recombinant enzyme complexed with ADP, phosphate, and 5-formyltetrahydrofolate, hanging drop vapour diffusion method, 20C, 30 mg/ml protein with 20% PEG 4000, 5 mM ATP, and 10 mM 5-formylTHF, flash freezing of all crystals in 20% PEG 4000 and 30% ethylene glycol, X-ray diffraction structure determination and analysis at 2.5 A resolution, crystallization of the enzyme with ATP analogues ATP-gamma-S, AMP-PNP, or AMP-PCP is not successful
-
purified recombinant His-tagged wild-type and selenomethionine-labeled enzyme, hanging drop vapour diffusion method, 30 mg/ml protein in 20 mM Tris-HCl, pH 8.0, 0.1 M NaCl, 1 mM EDTA, 1 mM DTT, at room temperature, 30 mg/ml selenomethionine-labeled enzyme in 0.2 M ammonium sulfate, 0.1 M sodium acetate, 21.25% PEG 4000, 15% PEG 400, pH 4.6, at 20C, flash freezing of all crystals in a solution containing 0.17 M ammonium sulfate, 0.085 M sodium acetate, 21.25% PEG 4000, 15% PEG 400, pH 4.6, X-ray diffraction structure determination and analysis at 2.2 A resolution
-
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.5 - 7.5
-
below pH 6.5 and above pH 7.5, 4C, in presence of 2-mercaptoethanol, 24 h, 90% loss of activity
1321
7
-
4C, 2-mercaptoethanol, 24 h, 10% loss of activity
1321
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4
-
extremely labile at
45
-
5-10 min, stable
50
-
5 min, 23% loss of activity, 10 min, 50% loss of activity
55
-
5 min, 82% loss of activity, 10 min, 91% loss of activity
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
enzyme loses activity when subjected to dialysis or concentration by ultrafiltration
-
non-ionic detergent, 0.1% v/v Tween 20, and 10% v/v glycerol stabilize the enzyme which is extremely labile at 4 C
-
stabilized by non-ionic detergent Tween 20
-
the enzyme is stabilized by Tween 20
OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
rapid loss of activity unless kept in the presence of reducing agents, e.g. DTT
-
1320
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-70C, 0.1% Tween 20, stable for a long time
-
-80C, pH 7.0, 0.1% v/v Tween 20, 10% v/v glycerol, stable for several months
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Ni-NTA affinity chromatography and gel filtration
recombinant His-tagged enzyme 171fold to near homogeneity from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, the His-tag is removed by thrombin cleavage and dialysis
recombinant His-tagged enzyme from Escherichia coli
-
recombinant His-tagged enzyme from Escherichia coli strain BL21 by nickel affinity chromatography
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by metal affinity chromatography to homogeneity
-
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by metal affinity chromatography to over 95% homogeneity
-
recombinant truncation mutant comprising residues 6-202 from Escherichia coli strain BL21(DE3) by affinity chromatography and substrate competition elution
-
Talon metal affinity column chromatography
-
Talon metal affinity resin chromatography and Sephadex G-25 gel filtration
to near homogeneity, recombinant enzyme from Escherichia coli
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cloning from brain cDNA library, expression of the His-tagged enzyme in Escherichia coli strain BL21
cytsolic enzyme
-
DNA and amino acid sequence determination and analysis, expression in Escherichia coli strain BL21(DE3) as N-terminally His-tagged enzyme, yield of purified recombinant enzyme depends on the growth conditions, M9 media is referred, overview
DNA sequence determination and analysis, expression in Escherichia coli BL21 as full length or truncated protein, the latter lacking the mitochondrial targeting sequence, functional complementation of enzyme deficient yeast fau1 mutant, yeast strain WHY1.3.1
DNA sequence determination and analysis, expression in Escherichia coli BL21(DE3) as full length or truncated protein, the latter lacking the mitochondrial targeting sequence
DNA sequence determination and analysis, expression of His-tagged enzyme in Escherichia coli strain BL21(DE3)
-
expressed in Escherichia coli (strain Rosetta pLysS)
expressed in Escherichia coli BL21 Star cells and MCF-7 cells
expressed in Escherichia coli BL21(DE3)/pSJS1244 cells
-
expression in Escherichia coli
expression in MCF-7 mammary adenocarcinoma cell line and in SH-SY5Y neuroblastoma cell line
-
expression of truncation mutant comprising residues 6-202 in Escherichia coli strain BL21(DE3)
-
gene FAU1, orf YER183c, functional expression as His-tagged enzyme in Escherichia coliBL21(DE3)
-
insertional mutant DNA sequence determination and analysis
-
overexpression of His-tagged enzyme in Escherichia coli strain BL21(DE3)
-
wild type and mutant enzymes are expressed in Escherichia coli strain BL21(DE3)/pSJS1244
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
5,10-methenyltetrahydrofolate synthetase expression is upregulated in tumors
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D154A
site-directed mutagenesis, the mutant shows 0.9% of the wild-type catalytic activity
E61A
site-directed mutagenesis, the mutant shows 8.6% of the wild-type catalytic activity
F55A
site-directed mutagenesis, the mutant shows 53.6% of the wild-type catalytic activity
F85A
site-directed mutagenesis, the mutant shows 72.3% of the wild-type catalytic activity
K10A
site-directed mutagenesis, the mutant shows 7.5% of the wild-type catalytic activity
K150A
site-directed mutagenesis, the mutant shows wild-type catalytic activity
M58A
site-directed mutagenesis, the mutant shows 121.6% of the wild-type catalytic activity
M90A
site-directed mutagenesis, the mutant shows 49.2% of the wild-type catalytic activity
R145A
site-directed mutagenesis, the mutant shows 2.3% of the wild-type catalytic activity
R148A
site-directed mutagenesis, the mutant shows 30.7% of the wild-type catalytic activity
R14A
site-directed mutagenesis, the mutant shows 12.9% of the wild-type catalytic activity
W109A
site-directed mutagenesis, the mutant shows 39.7% of the wild-type catalytic activity
Y152A
site-directed mutagenesis, the mutant shows 24.9% of the wild-type catalytic activity
Y153A
site-directed mutagenesis, the mutant shows 3.2% of the wild-type catalytic activity
Y83A
site-directed mutagenesis, the mutant shows wild-type catalytic activity
Y151
-
able to use 5-formyltetrahydrohomofolate as a substrate
Y152A
-
increased turnover rate compared to the wild type enzyme
Y152F
-
decreased turnover rate compared to the wild type enzyme
D124A
-
inactive
D151A
-
the mutant shows increased turnover number compared to the wild type enzyme
D154A
-
inactive
D81A
-
the mutant shows reduced turnover number compared to the wild type enzyme
K3A
-
the mutant shows strongly reduced turnover number compared to the wild type enzyme
K3D/D151K
-
the mutant shows strongly reduced turnover number compared to the wild type enzyme
K3D/D154 K
-
inactive
R115A
the mutant shows no activity
R125A
-
the mutant shows reduced turnover number compared to the wild type enzyme
R7A
-
the mutant shows severely reduced turnover number compared to the wild type enzyme
W153A
-
the mutant shows reduced turnover number compared to the wild type enzyme
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
E0IWA2
development of a functional complementation assay for 5-CHO-THF metabolism in Escherichia coli, based on deleting the gene encoding 5-FCL. The deletion mutant accumulates 5-formyltetrahydrofolate and,with glycine as sole nitrogen source, shows a growth defect, both phenotypes are complemented by bacterial or archaeal genes encoding glutamate formiminotransferase. Glutamate formiminotransferases functionally replace 5-formyltetrahydrofolate cyclo-ligases in certain prokaryotes
medicine
-
the enzyme is important in cancer treatment to rescue cells from high dose levels of the anti-folate methotrexate, and to potentiate the antitumor activity of 5-fluorouracil
pharmacology
-
the enzyme could be a potentially important enzyme as a target in chemotherapy
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