Information on EC 1.5.1.15 - methylenetetrahydrofolate dehydrogenase (NAD+)

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

EC NUMBER
COMMENTARY hide
1.5.1.15
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RECOMMENDED NAME
GeneOntology No.
methylenetetrahydrofolate dehydrogenase (NAD+)
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
5,10-methylenetetrahydrofolate + NAD+ = 5,10-methenyltetrahydrofolate + NADH + H+
show the reaction diagram
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-
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
-
-
-
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redox reaction
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-
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reduction
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-
-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
folate transformations I
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One carbon pool by folate
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Metabolic pathways
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SYSTEMATIC NAME
IUBMB Comments
5,10-methylenetetrahydrofolate:NAD+ oxidoreductase
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CAS REGISTRY NUMBER
COMMENTARY hide
82062-90-6
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
Antheraea eucalypti
moth, cell line (ATCC)CCL 80
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-
Manually annotated by BRENDA team
bovine
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-
Manually annotated by BRENDA team
spruce budworm, cell lines IPRI CF1, IPRI CF16T, IPRI CF124T, FPMI-CF-70, FPMI-CF-27 and FPMI-34
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-
Manually annotated by BRENDA team
black bellied hamster, cell line HA-Py(T), a polyoma virus-transformed hamster embryo fibroblast
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-
Manually annotated by BRENDA team
forest tent caterpillar, cell line IPRI-MD-66
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-
Manually annotated by BRENDA team
Syrian hamster
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-
Manually annotated by BRENDA team
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SwissProt
Manually annotated by BRENDA team
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SwissProt
Manually annotated by BRENDA team
Fischer
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-
Manually annotated by BRENDA team
strain KSY8
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-
Manually annotated by BRENDA team
fall armyworm, cell line Sf9
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Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
5,10-methylenetetrahydrofolate + NAD+
5,10-methenyltetrahydrofolate + NADH
show the reaction diagram
5,10-methylenetetrahydrofolate + NAD+
5,10-methenyltetrahydrofolate + NADH + H+
show the reaction diagram
5,10-methylenetetrahydrofolate + NADP+
5,10-methenyltetrahydrofolate + NADPH + H+
show the reaction diagram
about 15% of the rate with NAD+
-
-
?
L-5,10-methenyl-tetrahydrofolate + NADH
L-5,10-methylenetetrahydrofolate + NAD+
show the reaction diagram
L-5,10-methylenetetrahydrofolate + NAD+
5,10-methenyl-tetrahydrofolate + NADH
show the reaction diagram
additional information
?
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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
5,10-methylenetetrahydrofolate + NAD+
5,10-methenyltetrahydrofolate + NADH
show the reaction diagram
L-5,10-methenyl-tetrahydrofolate + NADH
L-5,10-methylenetetrahydrofolate + NAD+
show the reaction diagram
L-5,10-methylenetetrahydrofolate + NAD+
5,10-methenyl-tetrahydrofolate + NADH
show the reaction diagram
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
FAD
required for enzyme stability, binding site comprises amino acid residues 95-228
NADP+
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active with both NAD+ and NADP+
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ca2+
-
can substitute for Mg2+
phosphate
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required
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
EDTA
-
NAD+ dependent enzyme from Ehrlich ascites tumor cells
S-adenosyl-L-methionine
allosteric inhibitor, enzyme contains a binding site at the C-terminus
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
arsenate
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0.8 mM 104% activity
dithiothreitol
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phosphate
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.15 - 1
5,10-methenyltetrahydrofolate
0.793
arsenate
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-
0.0046 - 0.26
L-5,10-methylenetetrahydrofolate
0.03 - 0.096
methenyltetrahydrofolate
0.171 - 0.25
Mg2+
0.013 - 4
NAD+
0.255 - 2.12
NADP+
0.17 - 0.19
phosphate
additional information
5,10-methylenetetrahydrofolate
pH 6.0, 37C, the Km-value for 5,10-methylenetetrahydrofolate in the presence of 2.7 mM NAD+ is below 0.005 mM
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.58
L-5,10-methenyltetrahydrofolate
Acetobacterium woodii
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-
1.63
L-5,10-methylenetetrahydrofolate
Acetobacterium woodii
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.0001
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C-57/Bl, bone marrow and whole adrenal, CD-1, adrenal
0.0002
0.0003
0.0004
0.0005
0.0006
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RLE cells, virus 3611.MSV v-raf oncogene-transformed
0.0007
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thymus
0.00076
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fibroblasts, syrian hamster embryo
0.0011
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Swiss, bone marrow
0.0012
0.0014
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adrenal medulla
0.0015
0.0017
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cell line B-16 solid tumor
0.002
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RLE cells, virus pRNR16 v-Ha-ras oncogene-transformed
0.0021
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RLE cells, virus J2 v-raf/v-myc oncogene-transformed
0.0024
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cell line M4, human cutaneous melanoma
0.0027
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cell line MCF-7, 100% confluent
0.0028
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cell line KB
0.003
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cell line LR-73, chinese hamster ovary
0.0031
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cell line K562, human chronic myeolcytic leukemia
0.0033
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cell line TL3, EBV-transformed
0.0034
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bone marrow
0.0035
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cell line MCF-7, 50% confluent
0.0039
0.004
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cell line CCRF-CEM
0.0042
0.0045
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cell line CHO
0.0046
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cell line TL4, EBV-transformed
0.0047
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cell line MG-63, human osteosarcoma
0.0049
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cell line BeWo, human choriocarcinoma
0.0052
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adrenal cortex
0.0055
0.0058
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whole adrenal
0.0059
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cell line P815, ascites in vivo
0.0063
0.0066
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cell line EL4, ascites in vivo
0.0068
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L-cells, murine fibroblast
0.0069
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cell line MCF-7, human breast carcinoma
0.0071
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cell line P-388 leukemia ip
0.0078
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cell line CCRF-CEM, human leukemia
0.0089
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Ehrlich ascites, ascites in vivo
0.0094
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cell line EL4, murine T-cell lymphoma
0.0097
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cell line YAC, murine lymphoma (Moloney virus-induced)
0.0105
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cell line 3T3-SV40, SV40 transformed
0.02
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Ehrlich ascites, murine mastocytoma
0.024
mutant D190E, cosubstrate NAD+, pH 7.3
0.156
mutant R198A, cosubstrate NADP+, pH 7.3
0.16
mutant D133A, cosubstrate NADP+, pH 7.3
0.17
mutant R198A, cosubstrate NAD+, pH 7.3
0.207
mutant R198S, cosubstrate NAD+, pH 7.3
0.392
mutant D133A, cosubstrate NAD+, pH 7.3
1 - 2
pH 6.0, 37C, purified enzyme, recombinant
1.01
mutant D133S, cosubstrate NADP+, pH 7.3
1.24
mutant R198K, cosubstrate NADP+, pH 7.3
1.54
mutant R198S, cosubstrate NADP+, pH 7.3
2 - 2.5
wild-type, cosubstrate NAD+, pH 7.3
2.7
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fibroblast
2.92
wild-type, cosubstrate NADP+, pH 7.3
3.4
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embryo fibroblast
3.64
mutant D190N, cosubstrate NAD+, pH 7.3
3.96
mutant D190N, cosubstrate NADP+, pH 7.3
3.99
mutant D133S, cosubstrate NAD+, pH 7.3
4.6
mutant D133N, cosubstrate NAD+, pH 7.3
4.7
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osteosarcoma
5
pH 6.0, 37C, extract from Escherichia coli cells carrying the expression vector
5.5
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mastocytoma
6.9
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breast carcinoma
7.8
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leukemia
8.9
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adenocarcinoma
9.4
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T-cell lymphoma
9.7
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lymphoma
12.6
mutant R198K, cosubstrate NAD+, pH 7.3
12.9
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embryonal carcinoma
31.9
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40 - 44
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8
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enzyme activity decreases approximately 2fold at pH 7.0 or pH 9.0
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
45
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activity increases to the optimum, above 45C the enzyme begins to denature
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
additional information
PDB
SCOP
CATH
ORGANISM
UNIPROT
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
33000 - 38000
37000
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55000
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purified enzyme, gel filtration
58000
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gel filtration
60000
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sedimentation equilibrium centrifugation
64000
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gel filtration
68000
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cross-linking with bis(sulfosuccinimidyl)suberate
69000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
construction of homology model
protein crystallized from 7% PEG 8000 in 50 mM Tris-HCl, pH 8.2 at 4C in both hanging and sitting drop conditions, space group P4212 with cell constants a/b/c 75.9/75,9/160.0 A, crystals diffract to 2.9 A resolution, crystals form as tetragonal bipyramids and may grow as large as 0.45 mm on a side
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8
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enzyme activity decreases approximately 2fold at pH 7.0 or pH 9.0
392083
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
50
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stable for 30 min at temperatures below, loses 25% of its activity during incubation
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
requires phosphate anions for stability
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OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
oxygen-labile
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392076
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-80C, enzyme is stable in 20-30% glycerol buffer and can be frozen for months without loss of activity
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4C, can be stored dissolved in 0.1 M potassium maleate, pH 7.0 for at least 1 month without significant loss of activity
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4C, enzyme activity is stable for several weeks after storage in the final elution buffer
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
from ascites tumor cells
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native enzyme from yeast as well as recombinant yest enzyme expressed in Escherichia coli
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recombinant enzyme, expressed in Escherichia coli
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cDNA clones of gene DNMDMC encoding the bifunctional protein
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cloned cDNA for the human enzyme expressed in Escherichia coli K-38
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DNA sequencing; isolation of cDNA and the gene encoding the murine cytoplasmic methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase (DCS)
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full-length cDNA clone encoding NAD-dependent dehydrogenase-cyclohydrolase
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heterologously overproduced in Escherichia coli with a C-terminal His6-tag
isolation of cDNA and the gene encoding the murine cytoplasmic methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase (DCS)
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recombinant enzyme, expressed in Escherichia coli
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yeast gene MTD1 cloned and sequenced
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C677T
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one of several polymorphisms, leads to increased risk of hepatocellular carcinoma in patients with alcoholic cirrhosis, determination of genotypes in liver transplant patients with cirrhosis with and without hepatocellular carcinoma and in healthy persons
D133A
cosubstrate NAD+, 1.7% of wild-type activity, cosubstrate NADP+, 5.5% of wild-type activity
D133E
no enzymic activity
D133N
cosubstrate NAD+, 20.4% of wild-type activity, cosubstrate NADP+, 82.2% of wild-type activity
D133S
cosubstrate NAD+, 17.8% of wild-type activity, cosubstrate NADP+, 34.5% of wild-type activity
D190A
no enzymic activity
D190E
cosubstrate NAD+, 1% of wild-type activity
D190N
cosubstrate NAD+, 16% of wild-type activity
D190S
no enzymic activity
R166A
no enzymic activity
R166K
no enzymic activity
R166S
no enzymic activity
R198A
cosubstrate NAD+, 0.7% of wild-type activity, cosubstrate NADP+, 5.3% of wild-type activity
R198K
cosubstrate NAD+, 56% of wild-type activity, cosubstrate NADP+, 42% of wild-type activity
R198S
cosubstrate NAD+, 0.9% of wild-type activity, cosubstrate NADP+, 53% of wild-type activity
K56Q
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inactivation of methenyltetrahydrofolate cyclohydrolase activity of bifunctional enzyme. Transfection with mutant enzyme rescues auxotrophy of enzyme-deficient fibroblasts, but only poorly. Rescued cells demonstrate a decrease in the ratio of incorporation of exogenous formate to serine
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine
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