Information on EC 3.5.4.9 - methenyltetrahydrofolate cyclohydrolase

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

EC NUMBER
COMMENTARY hide
3.5.4.9
-
RECOMMENDED NAME
GeneOntology No.
methenyltetrahydrofolate cyclohydrolase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
5,10-methenyltetrahydrofolate + H2O = 10-formyltetrahydrofolate
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
amidine hydrolysis
hydrolysis
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Carbon fixation pathways in prokaryotes
-
-
folate transformations I
-
-
folate transformations II
-
-
formate assimilation into 5,10-methylenetetrahydrofolate
-
-
L-histidine degradation III
-
-
Metabolic pathways
-
-
Microbial metabolism in diverse environments
-
-
N10-formyl-tetrahydrofolate biosynthesis
-
-
One carbon pool by folate
-
-
purine nucleobases degradation I (anaerobic)
-
-
purine nucleobases degradation II (anaerobic)
-
-
reductive acetyl coenzyme A pathway I (homoacetogenic bacteria)
-
-
tetrahydrofolate salvage from 5,10-methenyltetrahydrofolate
-
-
histidine metabolism
-
-
reductive acetyl coenzyme A pathway
-
-
tetrahydrofolate metabolism
-
-
SYSTEMATIC NAME
IUBMB Comments
5,10-methenyltetrahydrofolate 5-hydrolase (decyclizing)
In eukaryotes, the enzyme occurs as a trifunctional enzyme that also has methylenetetrahydrofolate dehydrogenase (NADP+) (EC 1.5.1.5) and formate---tetrahydrofolate ligase (EC 6.3.4.3) activity. In some prokaryotes, it occurs as a bifunctional enzyme that also has dehydrogenase (EC 1.5.1.5) activity or formimidoyltetrahydrofolate cyclodeaminase (EC 4.3.1.4) activity.
CAS REGISTRY NUMBER
COMMENTARY hide
9027-97-8
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
bifunctional enzyme
-
-
Manually annotated by BRENDA team
mosquito, Liverpool black-eye strain
-
-
Manually annotated by BRENDA team
mosquito, Liverpool black-eye strain
-
-
Manually annotated by BRENDA team
Allium sp.
onion
-
-
Manually annotated by BRENDA team
asparagus
-
-
Manually annotated by BRENDA team
cabbage
-
-
Manually annotated by BRENDA team
guinea-pig
-
-
Manually annotated by BRENDA team
pigeon
-
-
Manually annotated by BRENDA team
strain BE, bifunctional enzyme
-
-
Manually annotated by BRENDA team
cat
-
-
Manually annotated by BRENDA team
enokitake fungus
-
-
Manually annotated by BRENDA team
chicken, trifunctional enzyme
-
-
Manually annotated by BRENDA team
barley
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
shiitake fungus
-
-
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
AM1, aerobic methylotrophic proteobacterium, monofunctional enzyme
-
-
Manually annotated by BRENDA team
trifunctional enzyme
-
-
Manually annotated by BRENDA team
Petroselinum sp.
parsley
-
-
Manually annotated by BRENDA team
marine bioluminescent bacterium, bifunctional enzyme
-
-
Manually annotated by BRENDA team
ATCC 11299
-
-
Manually annotated by BRENDA team
spinach
-
-
Manually annotated by BRENDA team
strain Sf9
-
-
Manually annotated by BRENDA team
strain Sf9
-
-
Manually annotated by BRENDA team
Trifolium sp.
trefoil
-
-
Manually annotated by BRENDA team
wheat
-
-
Manually annotated by BRENDA team
bean
-
-
Manually annotated by BRENDA team
mappe bean
-
-
Manually annotated by BRENDA team
corn
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
metabolism
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(6R,S)-methenyltetrahydrofolate + H2O
?
show the reaction diagram
10-formyltetrahydrofolate
5-formyltetrahydrofolate + H2O
show the reaction diagram
-
low activity in the liver
-
-
?
10-formyltetrahydrofolate + H2O
5,10-methenyltetrahydrofolate + ?
show the reaction diagram
10-formyltetrahydrofolate + H2O
5,10-methenyltetrahydrofolate + H2O
show the reaction diagram
5,10-methenyl-tetrahydrofolate + H2O
10-formyltetrahydrofolate + H+
show the reaction diagram
trifunctional enzyme exhibits synthetase, dehydrogenase and cyclohydrolase activities
-
-
r
5,10-methenyltetrahydrofolate + H2O
10-formyltetrahydrofolate
show the reaction diagram
5,10-methenyltetrahydrofolate + H2O
10-formyltetrahydrofolate + ?
show the reaction diagram
-
cyclodehydrolase activity of MTHFD1
-
-
r
5,10-methenyltetrahydrofolate + H2O
10-formyltetrahydrofolate + H+
show the reaction diagram
5,10-methenyltetrahydrofolate + H2O
10-formyltetrahydrofolate + H+ + methanol
show the reaction diagram
5,10-methenyltetrahydrofolate + H2O
5-formyltetrahydrofolate + H+
show the reaction diagram
-
-
-
-
r
5,10-methylenetetrahydrofolate + NADP+
5,10-methenyltetrahydrofolate + NADPH + H+
show the reaction diagram
-
methylenetetrahydrofolate dehydrogenase (NADP+), EC 1.5.1.5, activity of the enzyme
-
-
r
N5,N10-methenyltetrahydrofolate + H2O
N10-formyltetrahydrofolate
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
10-formyltetrahydrofolate + H2O
5,10-methenyltetrahydrofolate + H2O
show the reaction diagram
5,10-methenyl-tetrahydrofolate + H2O
10-formyltetrahydrofolate + H+
show the reaction diagram
P07245, P09440
trifunctional enzyme exhibits synthetase, dehydrogenase and cyclohydrolase activities
-
-
r
5,10-methenyltetrahydrofolate + H2O
10-formyltetrahydrofolate
show the reaction diagram
5,10-methenyltetrahydrofolate + H2O
10-formyltetrahydrofolate + ?
show the reaction diagram
-
cyclodehydrolase activity of MTHFD1
-
-
r
5,10-methenyltetrahydrofolate + H2O
10-formyltetrahydrofolate + H+
show the reaction diagram
N5,N10-methenyltetrahydrofolate + H2O
N10-formyltetrahydrofolate
show the reaction diagram
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
NAD+
-
dependent on
NADP+
additional information
-
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2',5'-ADP
-
millimolar concentrations
2-[(2-chloro-6-fluorobenzyl)sulfanyl]-6-(trifluoromethyl)pyrimidin-4-ol
-
5,6,7,8-tetrahydro-N5,N10-carbonylfolic acid
i.e. LY354899
5-[(4-chlorophenyl)sulfonyl]-6-ethoxypyrimidine-2,4-diamine
-
6-benzyl-5-methyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-3-carbonitrile
-
aminopterin
-
slightly retards enzyme activity
di-2'-deoxyadenosine 5',5''-P1,P5,alpha,beta-methylene-delta,epsilon-methylene-pentaphosphate-gamma-borano
-
di-2'-deoxyadenosine 5',5''-P1,P5,alpha,beta-methylene-gamma,delta-methylene-tetraphosphate
-
diadenosine 5',5''-P1,P5,alpha,beta-methylene-delta,epsilon-methylene-pentaphosphate-gamma-borano
-
dihydrofolate
-
-
folate
-
competitive inhibition
folic acid
-
-
LY354899
i.e. 5,6,7,8-tetrahydro-N5,N10-carbonylfolic acid
LY374571
i.e. (2R)-2-[(4-{[(2,5-diamino-6-hydroxypyrimidin-4-yl)carbamoyl]amino}phenyl)formamido]pentanedioic acid
-
methotrexate
-
slightly retards enzyme activity
N5,N10-carbonyl tetrahydrofolate
NADP+
p-chloromercuribenzoate
-
-
P1,P4-bis(5'-adenosyl)-alpha,beta-gamma,delta-bismethylene-tetraphosphate
-
Pyridine nucleotide
-
-
-
suramin
-
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2',5'-ADP
5'-ADP
-
analog of NADP+ does not stimulate the reverse cyclohydrolase activity
phosphate
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0164 - 0.031
(6R,S)-methenyltetrahydrofolate
0.009 - 0.47
10-formyltetrahydrofolate
0.009 - 2.36
5,10-methenyltetrahydrofolate
0.019 - 0.23
methenyltetrahydrofolate
additional information
additional information
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
107 - 185
methenyltetrahydrofolate
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.018
5,6,7,8-tetrahydro-N5,N10-carbonylfolic acid
, pH 7.3, 37°C
0.009
di-2'-deoxyadenosine 5',5''-P1,P5,alpha,beta-methylene-delta,epsilon-methylene-pentaphosphate-gamma-borano
with NPP1, pH 7.3, 37°C
0.051
di-2'-deoxyadenosine 5',5''-P1,P5,alpha,beta-methylene-gamma,delta-methylene-tetraphosphate
with NPP1, pH 7.3, 37°C
0.02
diadenosine 5',5''-P1,P5,alpha,beta-methylene-delta,epsilon-methylene-pentaphosphate-gamma-borano
with NPP1, pH 7.3, 37°C
0.013
P1,P4-bis(5'-adenosyl)-alpha,beta-gamma,delta-bismethylene-tetraphosphate
with NPP1, pH 7.3, 37°C
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0025
2-[(2-chloro-6-fluorobenzyl)sulfanyl]-6-(trifluoromethyl)pyrimidin-4-ol
Pseudomonas aeruginosa
Q9I2U6
at pH 7.9 and 22°C
0.0009
5-[(4-chlorophenyl)sulfonyl]-6-ethoxypyrimidine-2,4-diamine
Pseudomonas aeruginosa
Q9I2U6
at pH 7.9 and 22°C
0.0038
6-benzyl-5-methyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-3-carbonitrile
Pseudomonas aeruginosa
Q9I2U6
at pH 7.9 and 22°C
0.013
di-2'-deoxyadenosine 5',5''-P1,P5,alpha,beta-methylene-delta,epsilon-methylene-pentaphosphate-gamma-borano
Leishmania major
Q4Q9F9
with NPP1, pH 7.3, 37°C
0.05
di-2'-deoxyadenosine 5',5''-P1,P5,alpha,beta-methylene-gamma,delta-methylene-tetraphosphate
Leishmania major
Q4Q9F9
with NPP1, pH 7.3, 37°C
0.063
diadenosine 5',5''-P1,P5,alpha,beta-methylene-delta,epsilon-methylene-pentaphosphate-gamma-borano
Leishmania major
Q4Q9F9
with NPP1, pH 7.3, 37°C
0.00003
LY374571
Pseudomonas aeruginosa
Q9I2U6
at pH 7.9 and 22°C
-
0.033
P1,P4-bis(5'-adenosyl)-alpha,beta-gamma,delta-bismethylene-tetraphosphate
Leishmania major
Q4Q9F9
with NPP1, pH 7.3, 37°C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.0002
-
cyclohydrolase activity, rat liver, formation of 5-formyltetrahydrofolate
0.09
-
source spleen
0.13
-
source kidney
0.16
-
source spleen
0.18
-
source liver
0.22
-
source liver
0.25
-
source kidney
0.27
-
source liver
0.28
-
0 day-old seedling
0.35
-
source kidney
0.36
-
source kidney
0.37
-
9 day-old seedling
0.4
-
source liver
0.41
-
source kidney
0.42
-
HCOOH/CO as growing substrate
0.434
-
-
0.44
-
3 day-old seedling
0.49
-
growth conditions H2 + CO2
0.63
-
growth conditions glucose + CO2
0.65
-
-
0.72
-
6 day-old sprout
0.82
-
source liver
0.87
-
-
0.94
-
growth conditions methanol + CO2
1.16
-
HCOOH as growing substrate
1.6
-
mutant K56R
2 - 3
pH 8.0, 37°C, extract from Escherichia coli cells carrying the expression vector
2.1
Petroselinum sp.
-
-
2.2
-
-
2.4
-
source leaf
2.7
-
source root
3.4
-
forward reaction, recombinant mutant S197R, substrate 5,10-methenyltetrahydrofolate
3.7
-
source petiole
6.3
-
cyclohydrolase activity is monitored by following the consumption of 5,10-methylenetetrahydrofolate yielding 10-formyltetrahydrofolate
6.4
-
reverse reaction, recombinant mutant S197A, substrate 10-formyltetrahydrofolate, in absence of 2',5'-ADP
6.7
-
mutants C147Q, S49Q
7.2
-
forward reaction, recombinant wild-type enzyme, substrate 5,10-methenyltetrahydrofolate
7.3
-
reverse reaction, recombinant mutant S197R, substrate 10-formyltetrahydrofolate, in absence of 2',5'-ADP
7.6
-
reverse reaction, recombinant mutant S197A, substrate 10-formyltetrahydrofolate, in presence of 2',5'-ADP
8
-
reverse reaction, recombinant mutant R173A, substrate 10-formyltetrahydrofolate, in presence of 2',5'-ADP
9
-
reverse reaction, recombinant mutant R173K, substrate 10-formyltetrahydrofolate, in absence of 2',5'-ADP
9.2
-
reverse reaction, recombinant mutant R173E, substrate 10-formyltetrahydrofolate, in presence of 2',5'-ADP; reverse reaction, recombinant mutant S197T, substrate 10-formyltetrahydrofolate, in absence of 2',5'-ADP
9.3
-
reverse reaction, recombinant mutant S197D, substrate 10-formyltetrahydrofolate, in absence of 2',5'-ADP
9.6
-
reverse reaction, recombinant wild-type enzyme, substrate 10-formyltetrahydrofolate, in absence of 2',5'-ADP
10.8
-
forward reaction, recombinant mutant S197T, substrate 5,10-methenyltetrahydrofolate; reverse reaction, recombinant mutant S197D, substrate 10-formyltetrahydrofolate, in presence of 2',5'-ADP
12.8
-
reverse reaction, recombinant mutant R173K, substrate 10-formyltetrahydrofolate, in presence of 2',5'-ADP
13.5
-
forward reaction, recombinant mutant S197A, substrate 5,10-methenyltetrahydrofolate
15.9
-
reverse reaction, recombinant mutant S197T, substrate 10-formyltetrahydrofolate, in presence of 2',5'-ADP
18
-
mutant C147Q
22.4
-
reverse reaction, recombinant wild-type enzyme, substrate 10-formyltetrahydrofolate, in presence of 2',5'-ADP
22.8
-
reverse reaction, recombinant mutant S197R, substrate 10-formyltetrahydrofolate, in presence of 2',5'-ADP
25
-
mutant S49Q
32.9
-
purified enzyme
39
-
mutant Y52S
47
-
mutant Y52A
51
-
recombinant loop3G mutant, cyclohydrolase forward reaction
61.6
-
forward reaction, recombinant mutant S197D, substrate 5,10-methenyltetrahydrofolate
66
pH 8.0, 37°C, purified enzyme, recombinant
66.7
-
recombinant loop5G mutant, cyclohydrolase forward reaction
76.5
-
recombinant mutant Y240A, cyclohydrolase forward reaction
97.4
-
recombinant enzyme
113
-
recombinant mutant R250A, cyclohydrolase forward reaction
128
-
mutantS49A
133
-
DC301, wild-type
146
-
mutants C147A, T148A
160
-
mutant Y52F
161
-
recombinant wild-type enzyme, cyclohydrolase forward reaction
329
-
forward reaction, recombinant mutant R173K, substrate 5,10-methenyltetrahydrofolate
470
-
purified enzyme, pH 7.2, 35°C
1173
-
forward reaction, recombinant mutant R173A, substrate 5,10-methenyltetrahydrofolate
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4 - 7
-
5-formyltetrahydrofolate formation
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
23
-
assay at
27
-
assay at
30
-
assay at
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
Allium sp.
-
-
Manually annotated by BRENDA team
additional information
-
the cytoplasmic MTHFD1 is ubiquitously expressed in all mammalian cells; the mitochondrial MTHFD2 is detected only in transformed cell lines and embryogenic tissues
Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
22000
-
2 * 22000, SDS-PAGE
25500
-
2 * 25500, SDS-PAGE
30000
2 * 30000, SDS-PAGE
30600
-
estimated from amino acid sequence
31060
-
calculated from amino acid sequence
35000
-
SDS-PAGE
38500
-
SDS-PAGE
41000
-
sedimentation equilibrium centrifugation
45000
-
gel filtration
57000
-
SDS-PAGE
58000
-
gel filtration
65000
recombinant detagged enzyme, gel filtration
70000
-
gel filtration
100000
150000
-
gel filtration
200000
-
gel filtration
218000
-
isoenzyme
318700
x * 318700, calculated from sequence
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homodimer
monomer
additional information
structure of the TaMTHFDC-NADP+ complex dimer, overview
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
small platelike crystals belong to body centered orthorhombic space group I222
-
crystal structure of DC301, the cytosolic DC domain of the trifunctional enzyme
-
purified recombinant apo-DHCH, hanging drop vapor diffusion, mixing of 0.002 ml 9 mg/ml protein in 50 mM Tris–HCl, 100 mM NaCl, pH 7.5, with 0.002 ml reservoir solution containing 20% PEG 3350 and 0.2 M ammonium acetate, equilibration against 0.07 ml of reservoir solution at 22°C, 3 days, X-ray diffraction structure determination and analysis at 2.7 A resolution, modelling
hanging drop vapor diffusion method, using 25% (w/v) PEG 3350 and 0.2 M magnesium formate
purified enzyme free and in complex with NADP+, sitting drop vapour diffusion method, mixing of 1.8 mg/ml protein in 10 mM Tris-HCl, 50 mM KCl, and 2 mM DTT, with 18% PEG 4000, 400 mM NaCl, and 100 mM Tris-HCl, pH 8.0 at 22°C, 5 mM NADP+ is added during crystallization, X-ray diffraction structure determination and analysis, molecular replacement
in the presence of NADP+, at 22°C, using polyethylene glycol 4000 as a precipitant, sitting drop vapour diffusion method
-
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
45
-
loses activity rapidly above, after 2 min at 55°C 33% activity remains, after 2 min at 65°C 4% activity remains
50 - 60
-
67% activity lost at 50°C for 5 min, 80% activity lost at 50°C for 10 min, 91% activity lost at 60°C for 5 min
55
-
70% activity lost within 2 min, rapidly inactivated at temperatures above
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
purified enzyme stored in 20% glycerol, 0.1 M potassium phosphate pH 7.3 stable for about 1 week
-
quite unstable, loses all activity after 96 h of storage
-
unstable, stabilized by addition of 25% glycerol
-
ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Glycerol
-
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20°C purified enzyme in 50% glycerol, 95% active after 6 months
-
-20°C, 25% glycerol, 20% cyclohydrolase activity remains after 1 month
-
-20°C, loses 15% activity in 1 day, 50% in 5 days and 70% in 30 days
-
-80°C, 50 mM potassium phosphate, 30% glycerol buffer, stable for over 1 month
-
0°C, relatively unstable, losing 40% activity in 1 day and 75% in 4 days
-
4°C, 25% glycerol, 60% cyclohydrolase activity remains after 1 month
-
5°C, 50 mM phosphate buffer, pH 7.0, stable for at least 1 week
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
by anion-exchange chromatography
-
His-Trap affinity column chromatography and Superdex-S200 gel filtration
-
Ni2+-charged HisTrap column chromatography and Superdex 200 gel filtration
recombinant DHCH1 protein is purified by metal-affinity chromatography
-
recombinant His-tagged enzyme by nickel affinity chromatography, cleavage of the His-tag by TEV protease, His affinity chromatography, and gel filtration, to over 95% purity
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli by Ni2+-chelate affinity chromatography
-
recombinant N-terminally 6-His-tagged MTHFDC from Escherichia coli strain BL21(DE3) by heat-treatment, nickel affinity chromatography, and gel filtration
recombinant protein expressed in Escherichia coli
-
recombinant protein expressed in Escherichia coli K38
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
amplified genomic library transformed into yest YA3-1, yeast-DNA transformed into Escherichia coli K38 pBKe, gene folD encoding PPDC expressed
-
bifunctional dehydrogenase/cyclohydrolase portion of the cytoplasmic trifunctional enzyme cloned and heterologous expressed in the yeast strain YA3-1
-
cDNA encoding bifunctional enzyme cloned and expressed in Escherichia coli K38
-
cDNA encoding trifunctional enzyme used to complete the purine auxotrophic yeast strain YA3-1
-
deletion of the MIS1 gene has little effect
DNA fragment containing the bifunctional enzyme from Escherichia coli BE cloned and transformed into Escherichia coli BL21 for protein expression
-
expressed in Escherichia coli BL21(DE3) cells
expressed in Escherichia coli strain BL21 (DE3)
-
expression of the His-tagged enzyme in Escherichia coli
expression of the N-terminally 6-His-tagged MTHFDC in Escherichia coli strain BL21(DE3)
expression of wild-type and mutant enzymes in Escherichia coli BL21 as C-terminally His-tagged enzymes
-
expression of wild-type and mutant enzymes in Escherichia coli JM109 as C-terminally His-tagged enzymes
-
gene folD for bifunctional dehydrogenase/cyclohydrolase cloned and sequenced
-
heterologously overproduced in Escherichia coli with a C-terminal His6-tag
hexa-His tagged DHCH1 is expressed in Escherichia coli, production of a genetic knock-out
-
null mutation is embryonic lethal at about 12 days gestation, knockout of enzyme establishes the important role of formate production during embryogenesis
-
the wild type and R653Q mutant pBKeDCS constructs are transformed into Escherichia coli BL21 DE3 to express the full-length protein
-
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
MTHFD2 expression is high during early development but decreases as birth approaches
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D125A
-
site-directed mutagenesis, inactive mutant
D125C
-
site-directed mutagenesis, inactive mutant
D125E
-
site-directed mutagenesis, 95% reduced activity in the forward reaction compared to the wild-type enzyme
D125M
-
site-directed mutagenesis, inactive mutant
D125N
-
site-directed mutagenesis, inactive mutant
D125Q
-
site-directed mutagenesis, inactive mutant
K56Q/Q100K
Q100K
-
site-directed mutagenesis, inactive mutant
Q100M
-
site-directed mutagenesis, inactive mutant
Q100N
-
site-directed mutagenesis, inactive mutant
R173A
-
site-directed mutagenesis, at least 500fold increased Km for NADP+ compared to the wild-type, 163fold increased activity in the forward and reduced activity in the reverse reaction of the cyclohydrolase, unaltered channeling efficiency
R173E
-
site-directed mutagenesis, no dehydrogenase forward reaction activity, no forward channeling
R173K
-
site-directed mutagenesis, at least 500fold increased Km for NADP+ compared to the wild-type, 46fold increased activity in the forward and reduced activity in the reverse reaction of the cyclohydrolase, unaltered channeling efficiency
R250A
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site-directed mutagenesis, 29.7% reduced activity in forward and reduced activity in the reverse reaction compared to the wild-type enzyme
S197A
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site-directed mutagenesis, 20fold increased Km for NADP+ compared to the wild-type, reduced activity in the reverse reaction of the cyclohydrolase, no stimulation by 2',5'-ADP, unaltered channeling efficiency
S197D
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site-directed mutagenesis, 8.5fold increased activity in the forward reaction of the cyclohydrolase, unaltered channeling efficiency
S197R
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site-directed mutagenesis, 10fold increased Km for methenyltetrahydrofolate, reduced activity in the forward and reverse reaction of the cyclohydrolase, unaltered channeling efficiency
S197T
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site-directed mutagenesis, slightly increased activity in the forward reaction of the cyclohydrolase, unaltered channeling efficiency
Y240A
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site-directed mutagenesis, 52,5% reduced activity in forward and reduced activity in the reverse reaction compared to the wild-type enzyme
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
drug development
medicine
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enzyme represents a potential pharmaceutical target for manipulation of cell growth and development and hence cancer prevention or treatment
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