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Information on EC 1.5.1.54 - methylenetetrahydrofolate reductase (NADH)
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1.5.1.54 methylenetetrahydrofolate reductase (NADH)IUBMB Comments
A flavoprotein (FAD). The enzyme, found in plants and some bacteria, catalyses the reversible conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate using NADH as the electron donor. It play an important role in folate metabolism by regulating the distribution of one-carbon moieties between cellular methylation reactions and nucleic acid synthesis. These proteins either contain a C-terminal domain that does not mediate allosteric regulation (as in plants) or lack this domain entirely (as in Escherichia coli). As a result, the plant enzymes are not inhibited by S-adenosyl-L-methionine, unlike other eukaryotic enzymes, and catalyse a reversible reaction. cf. EC 1.5.1.53, methylenetetrahydrofolate reductase (NADPH); EC 1.5.1.20, methylenetetrahydrofolate reductase [NAD(P)H]; and EC 1.5.7.1, methylenetetrahydrofolate reductase (ferredoxin).
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The expected taxonomic range for this enzyme is: Bacteria, Eukaryota
Synonyms
5,10-CH2-H4folate reductase, 5,10-methylenetetrahydrofolate reductase, 5,10-methylenetetrahydrofolate reductase (FADH2), 5,10-methylenetetrahydrofolic acid reductase, 5,10-methylenetetrahydropteroylglutamate reductase, 5-methyltetrahydrofolate:(acceptor) oxidoreductase, metF, methylenetetrahydrofolate (reduced riboflavin adenine dinucleotide) reductase, methylenetetrahydrofolate reductase, methylenetetrahydrofolic acid reductase, 
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SYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
5,10-CH2-H4folate reductase
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-
ambiguous
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5,10-methylenetetrahydrofolate reductase
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-
ambiguous
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5,10-methylenetetrahydrofolate reductase (FADH2)
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-
ambiguous
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5,10-methylenetetrahydrofolic acid reductase
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-
ambiguous
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5,10-methylenetetrahydropteroylglutamate reductase
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-
ambiguous
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5-methyltetrahydrofolate:(acceptor) oxidoreductase
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incorrect
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metF
methylenetetrahydrofolate (reduced riboflavin adenine dinucleotide) reductase
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methylenetetrahydrofolate reductase
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ambiguous
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methylenetetrahydrofolic acid reductase
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-
ambiguous
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MSMEG_6596
MSMEG_6649
MTHFR2
N5,10-methylenetetrahydrofolate reductase
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-
ambiguous
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N5,N10-methylenetetrahydrofolate reductase
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ambiguous
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metF
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-
-
-
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
5-methyltetrahydrofolate + NAD+ = 5,10-methylenetetrahydrofolate + NADH + H+
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-
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PATHWAY SOURCE
PATHWAYS
KEGG
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SYSTEMATIC NAME
IUBMB Comments
5-methyltetrahydrofolate:NAD+ oxidoreductase
A flavoprotein (FAD). The enzyme, found in plants and some bacteria, catalyses the reversible conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate using NADH as the electron donor. It play an important role in folate metabolism by regulating the distribution of one-carbon moieties between cellular methylation reactions and nucleic acid synthesis. These proteins either contain a C-terminal domain that does not mediate allosteric regulation (as in plants) or lack this domain entirely (as in Escherichia coli). As a result, the plant enzymes are not inhibited by S-adenosyl-L-methionine, unlike other eukaryotic enzymes, and catalyse a reversible reaction. cf. EC 1.5.1.53, methylenetetrahydrofolate reductase (NADPH); EC 1.5.1.20, methylenetetrahydrofolate reductase [NAD(P)H]; and EC 1.5.7.1, methylenetetrahydrofolate reductase (ferredoxin).
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
5,10-methylenetetrahydrofolate + NADPH + H+
5-methyltetrahydrofolate + NADP+
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-
-
?
5,10-methylenetetrahydrofolate + reduced methylene blue
5-methyltetrahydrofolate + methylene blue
5-methyltetrahydrofolate + methylene blue
5,10-methylenetetrahydrofolate + reduced methylene blue
5,10-methylenetetrahydrofolate + menadiol
5-methyltetrahydrofolate + menadione
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-
-
r
5,10-methylenetetrahydrofolate + menadiol
5-methyltetrahydrofolate + menadione
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-
-
r
5,10-methylenetetrahydrofolate + menadiol
5-methyltetrahydrofolate + menadione
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-
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r
5,10-methylenetetrahydrofolate + NADH + H+
5-methyltetrahydrofolate + NAD+
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-
-
ir
5,10-methylenetetrahydrofolate + NADH + H+
5-methyltetrahydrofolate + NAD+
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-
-
ir
5,10-methylenetetrahydrofolate + NADH + H+
5-methyltetrahydrofolate + NAD+
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-
-
?
5,10-methylenetetrahydrofolate + NADH + H+
5-methyltetrahydrofolate + NAD+
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-
-
?
5,10-methylenetetrahydrofolate + NADH + H+
5-methyltetrahydrofolate + NAD+
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-
-
-
?
5,10-methylenetetrahydrofolate + NADH + H+
5-methyltetrahydrofolate + NAD+
Blautia producta Marburg
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-
-
-
?
5,10-methylenetetrahydrofolate + NADH + H+
5-methyltetrahydrofolate + NAD+
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-
-
?
5,10-methylenetetrahydrofolate + NADH + H+
5-methyltetrahydrofolate + NAD+
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-
-
?
5,10-methylenetetrahydrofolate + NADH + H+
5-methyltetrahydrofolate + NAD+
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-
-
?
5,10-methylenetetrahydrofolate + NADH + H+
5-methyltetrahydrofolate + NAD+
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-
-
?
5,10-methylenetetrahydrofolate + reduced methylene blue
5-methyltetrahydrofolate + methylene blue
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-
-
-
r
5,10-methylenetetrahydrofolate + reduced methylene blue
5-methyltetrahydrofolate + methylene blue
Blautia producta Marburg
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-
-
-
r
5-methyltetrahydrofolate + menadione
5,10-methylenetetrahydrofolate + menadiol
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-
-
r
5-methyltetrahydrofolate + menadione
5,10-methylenetetrahydrofolate + menadiol
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-
-
?
5-methyltetrahydrofolate + menadione
5,10-methylenetetrahydrofolate + menadiol
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-
-
r
5-methyltetrahydrofolate + menadione
5,10-methylenetetrahydrofolate + menadiol
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-
r
5-methyltetrahydrofolate + methylene blue
5,10-methylenetetrahydrofolate + reduced methylene blue
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r
5-methyltetrahydrofolate + methylene blue
5,10-methylenetetrahydrofolate + reduced methylene blue
Blautia producta Marburg
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r
NADH + H+ + methylviologen
NAD+ + reduced methylviologen
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r
NADH + H+ + methylviologen
NAD+ + reduced methylviologen
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r
additional information
?
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no substrate: ferredoxin
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-
additional information
?
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the enzyme catalyzes the methylenetetrahydrofolate reduction with methylene blue as an artificial electron donor. The oxidation of methyltetrahydrofolate is mediated with methylene blue as the electron acceptor. Neither NAD+ nor viologen dyes can replace methylene blue in this reaction
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additional information
?
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Blautia producta Marburg
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the enzyme catalyzes the methylenetetrahydrofolate reduction with methylene blue as an artificial electron donor. The oxidation of methyltetrahydrofolate is mediated with methylene blue as the electron acceptor. Neither NAD+ nor viologen dyes can replace methylene blue in this reaction
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NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
5,10-methylenetetrahydrofolate + NADH + H+
5-methyltetrahydrofolate + NAD+
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-
-
-
?
5,10-methylenetetrahydrofolate + NADH + H+
5-methyltetrahydrofolate + NAD+
Blautia producta Marburg
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-
-
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?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
[4Fe-4S]-center
complex contains 23.5 Fe and 24.5 S, corresponding to predicted six [4Fe4S] clusters in MetV and RnfC2
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Fe
complex contains 23.5 Fe and 24.5 S, corresponding to predicted six [4Fe4S] clusters in MetV and RnfC2
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INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
genistein
0.5 mM, almost complete inhibition. The inhibition is increased when genistein is preincubated with the enzyme and NADH
additional information
not inhibitory: S-Methyl-L-methionine; not inhibitory: S-Methyl-L-methionine
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additional information
not inhibitory: S-Methyl-L-methionine; not inhibitory: S-Methyl-L-methionine
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
the NADH:methylenetetrahydrofolate reductase activity is not stimulated by ferredoxin
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.0005
5,10-methylenetetrahydrofolate
wild-type, cosubstrate NADH, pH 7.2, 25°C
0.027
5,10-methylenetetrahydrofolate
mutant D120N, cosubstrate NADH, pH 7.2, 25°C
0.106
5,10-methylenetetrahydrofolate
Chimera-1, cosubstrate NADPH, pH 7.2, temperature not specified in the publication
0.152
5,10-methylenetetrahydrofolate
Chimera-1, cosubstrate NADH, pH 7.2, temperature not specified in the publication
0.287
5,10-methylenetetrahydrofolate
wild-type, cosubstrate NADH, pH 7.2, temperature not specified in the publication
0.085
5-methyltetrahydrofolate
wild-type, cosubstrate menadione, pH 7.2, 25°C
0.106
5-methyltetrahydrofolate
mutant D120N, cosubstrate menadione, pH 7.2, 25°C
0.873
NADH
wild-type, pH 7.2, temperature not specified in the publication
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
cf. EC 1.5.1.20
Uniprot
brenda
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LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
metabolism
the half-reactions proceed at rates sufficiently fast to account for overall turnover, the enzyme is kinetically competent to catalyze these oxidoreductions by a ping-pong Bi-Bi mechanism. Reoxidation of the reduced flavin by CH2-H4folate is substantially rate limiting in the physiological NADH-CH2-H4folate oxidoreductase reaction. In the NADH-menadione oxidoreductase reaction, the reduction of the flavin by NADH is rate limiting as is the reduction of flavin by CH3-H4folate in the CH3-H4folate-menadione oxidoreductase reaction
physiological function
physiological function
enzyme is able to complement a Saccharomyces cerevisiae met12 met13 double disruptant
physiological function
enzyme is able to complement a Saccharomyces cerevisiae met12 met13 double disruptant
physiological function
expression of MTHFR1 rescues an Escherichia coli MTHFR deletion mutant. The Mycolicibacterium smegmatis MTHFR1 mutant strain is partially auxotrophic for methionine and grows only poorly without methionine. The mutant strain is more sensitive to folate pathway inhibitors such as sulfachloropyridazine, 4-aminosalicylic acid, sulfamethoxazole, and trimethoprim
physiological function
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expression of MTHFR1 rescues an Escherichia coli MTHFR deletion mutant. The Mycolicibacterium smegmatis MTHFR1 mutant strain is partially auxotrophic for methionine and grows only poorly without methionine. The mutant strain is more sensitive to folate pathway inhibitors such as sulfachloropyridazine, 4-aminosalicylic acid, sulfamethoxazole, and trimethoprim
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UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). A0A0B8QP67_LACLL
283
0
31864
TrEMBL
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A0R6M0_MYCS2
Mycolicibacterium smegmatis (strain ATCC 700084 / mc(2)155)
302
0
33311
TrEMBL
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A0R6S0_MYCS2
Mycolicibacterium smegmatis (strain ATCC 700084 / mc(2)155)
296
0
32941
TrEMBL
-
H6LBX9_ACEWD
Acetobacterium woodii (strain ATCC 29683 / DSM 1030 / JCM 2381 / KCTC 1655 / WB1)
298
0
32752
TrEMBL
-
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
119000
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SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
heterotrimer
monomer
octamer
heterotrimer
1 * 71000, i.e.subunit RnfC2, 1 * 32800, i.e. subunit MetF, 1 * 22700, i.e. subunit MetV, respectively
heterotrimer
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1 * 71000, i.e.subunit RnfC2, 1 * 32800, i.e. subunit MetF, 1 * 22700, i.e. subunit MetV, respectively
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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
modeling of the methyltetrahydrofolate product into the enzyme active site and shows 150fold decreased activity
molecular docking of genistein. Gensitein may interfere with the binding site of NADH
structure provides a model for the catalytic domain shared by all MTHFRs. This domain is a beta8alpha8 barrel that binds FAD in a special fashion. Ala 177, corresponding to Ala 222 in human MTHFR, is near the bottom of the barrel and distant from the FAD. The mutation A177V does not affect Km or kcat but instead increases the propensity for bacterial MTHFR to lose its essential flavin cofactor
structures of ligand-free mutants F223L and F223L/E28Q in complex with 5,10-methylentetrahydrofolate, at 1.65 and 1.70 A resolution, respectively. The folate is bound in a catalytically competent conformation, and residue Leu223 undergoes a conformational change similar to that observed for Phe223 in the E28Q-5,10-methylentetrahydrofolate structure
structures of the enzyme complexes of wild-type with NADH and mutant E28Q with methytetrahydrofolate. Residue Gln183 makes key hydrogen-bonding interactions with both NADH and folate in their respective halfreactions
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
A177V
mutation mimicks human polymorphism A222V. Mutation does not affect kcat or the Km values but alters FAD binding
D120A
midpoint potential of the mutant increases, the mutant exhibits a 1.2- to 1.5fold faster reduction rate than the wild-type enzyme. Catalytic efficiency (kcat/Km) in the 5-10-methylentetrahydrofolate oxidative half-reaction is significantly decreased
D120K
midpoint potential of the mutant increases, the mutant exhibits a 1.2- to 1.5fold faster reduction rate than the wild-type enzyme. Catalytic efficiency (kcat/Km) in the 5-10-methylentetrahydrofolate oxidative half-reaction is significantly decreased
D120N
D120S
midpoint potential of the mutant increases, the mutant exhibits a 1.2- to 1.5fold faster reduction rate than the wild-type enzyme. Catalytic efficiency (kcat/Km) in the 5-10-methylentetrahydrofolate oxidative half-reaction is significantly decreased
D120V
midpoint potential of the mutant increases, the mutant exhibits a 1.2- to 1.5fold faster reduction rate than the wild-type enzyme. Catalytic efficiency (kcat/Km) in the 5-10-methylentetrahydrofolate oxidative half-reaction is significantly decreased
E28Q
mutant is unable to catalyze the reduction of 5,10-methylentetrahydrofolate and is inactive in the physiological oxidoreductase reaction. The mutant is able to bind methyltetrahydrofolate, but reduction of the FAD cofactor is not observed
F223A
mutation impairs NADH and 5,10-methylentetrahydrofolate binding each 40fold yet slows catalysis of both half-reactions less than 2fold
F223L
affinity for 5,10-methylentetrahydrofolate is unaffected by the mutation, the variant catalyzes the oxidative half-reaction 3fold faster than the wild-type enzyme
Q183A
in the reductive half-reaction, NADH binding affinity and the rate of flavin reduction are not hindered by the mutation. Q183A exhibits a 6-10fold lower rate of folate reduction and binds methylentetrahydrofolate with 7-fold lower affinity
Q183E
in the reductive half-reaction, NADH binding affinity and the rate of flavin reduction are not hindered by the mutation and Gln183 plays a minor in the oxidative half-reaction. The mutant displays catalytic constants within 3fold of the wild-type enzyme
additional information
chromosomal copy of MET13 is replaced by an Arabidopsis thaliana MTHFR cDNA (AtMTHFR-1) or by a chimeric sequence (Chimera-1) comprising the yeast N-terminal domain and the AtMTHFR-1 C-terminal domain. Chimera-1 uses both NADH and NADPH and is insensitive to S-adenosyl-L-methionine. Engineered yeast expressing Chimera-1 accumulates 140fold more S-adenosyl-L-methionine and 7fold more methionine than does the wild-type and grows normally. Yeast expressing AtMTHFR-1 accumulates 8fold more S-adenosyl-L-methionine
D120N
midpoint potential of the mutant increases, the mutant exhibits a 1.2- to 1.5fold faster reduction rate than the wild-type enzyme. Catalytic efficiency (kcat/Km) in the 5-10-methylentetrahydrofolate oxidative half-reaction is significantly decreased
D120N
mutant is reduced by NADH 30% more rapidly than the wild-type enzyme
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE