Literature summary for 1.5.1.5 extracted from

Shin, M.; Bryant, J.D.; Momb, J.; Appling, D.R.
Mitochondrial MTHFD2L is a dual redox cofactor-specific methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolate cyclohydrolase expressed in both adult and embryonic tissues (2014), J. Biol. Chem., 289, 15507-15517 .

Cloned(Commentary)

Cloned (Comment)	Organism
gene MTHFD2L, DNA and amino acid sequence determination and analysis, quantitative real-time PCR enzyme expression analysis, the truncated enzyme mutant rMTHFD2LDELTA1-40 is cloned into vector YEp24-based yeast expression vector containing the Saccharomyces cerevisiae MET6 promoter and expressed in Saccharomyces cerevisiae strain MWY4.4 as N-terminally His6-tagged protein, replacment of the mitochondrial targeting sequence of the rat MTHFD2L with an N-terminal His tag for localization in the yeast cytosol	Rattus norvegicus
gene Mthfd2l, MTHFD2L is alternatively spliced at exons 2 and 8, quantitative real-time PCR enzyme expression analysis, gene expression profiling of the MTHFD gene family in mouse embryos and adult organs	Mus musculus

Cloned (Comment)

Organism

gene MTHFD2L, DNA and amino acid sequence determination and analysis, quantitative real-time PCR enzyme expression analysis, the truncated enzyme mutant rMTHFD2LDELTA1-40 is cloned into vector YEp24-based yeast expression vector containing the Saccharomyces cerevisiae MET6 promoter and expressed in Saccharomyces cerevisiae strain MWY4.4 as N-terminally His6-tagged protein, replacment of the mitochondrial targeting sequence of the rat MTHFD2L with an N-terminal His tag for localization in the yeast cytosol

Rattus norvegicus

gene Mthfd2l, MTHFD2L is alternatively spliced at exons 2 and 8, quantitative real-time PCR enzyme expression analysis, gene expression profiling of the MTHFD gene family in mouse embryos and adult organs

Mus musculus

Inhibitors

Inhibitors	Comment	Organism	Structure
phosphate	the NADP+-dependent methylenetetrahydrofolate dehydrogenase activity is inhibited by phosphate, competitive inhibition	Mus musculus
phosphate	the NADP+-dependent methylenetetrahydrofolate dehydrogenase activity is inhibited by phosphate, competitive inhibition	Rattus norvegicus

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
additional information	-	additional information	Michaelis-Menten kinetics	Mus musculus
additional information	-	additional information	Michaelis-Menten kinetics	Rattus norvegicus
0.04	-	5,10-methylenetetrahydropteroylpoly-L-glutamate	pH 8.0, 30°C, monoglutamylated substrate, with NAD+	Mus musculus
0.042	-	5,10-methylenetetrahydropteroylpoly-L-glutamate	pH 8.0, 30°C, monoglutamylated substrate, with NADP+	Mus musculus
0.13	-	5,10-methylenetetrahydropteroylpoly-L-glutamate	pH 8.0, 30°C, polyglutamylated substrate, with NAD+	Mus musculus
0.147	-	NAD+	pH 8.0, 30°C	Mus musculus
0.153	-	5,10-methylenetetrahydropteroylpoly-L-glutamate	pH 8.0, 30°C, polyglutamylated substrate, with NADP+	Mus musculus
0.537	-	NADP+	pH 8.0, 30°C	Mus musculus

Localization

Localization	Comment	Organism	GeneOntology No.	Textmining
mitochondrion	-	Mus musculus	5739	-
mitochondrion	-	Rattus norvegicus	5739	-

Metals/Ions

Metals/Ions	Comment	Organism
Mg2+	required for methylenetetrahydrofolate dehydrogenase activity with NADH	Mus musculus
Mg2+	required for methylenetetrahydrofolate dehydrogenase activity with NADH	Rattus norvegicus
additional information	phosphate appears to counteract the Mg2+ effect with NADP+	Mus musculus
additional information	phosphate appears to counteract the Mg2+ effect with NADP+	Rattus norvegicus
phosphate	required for methylenetetrahydrofolate dehydrogenase activity with NADH	Mus musculus
phosphate	required for methylenetetrahydrofolate dehydrogenase activity with NADH	Rattus norvegicus

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.
5,10-methylenetetrahydrofolate + NAD+	Mus musculus	-	5,10-methenyltetrahydrofolate + NADH + H+	-	r
5,10-methylenetetrahydrofolate + NAD+	Rattus norvegicus	-	5,10-methenyltetrahydrofolate + NADH + H+	-	r
5,10-methylenetetrahydrofolate + NAD+	Mus musculus C57BL/6	-	5,10-methenyltetrahydrofolate + NADH + H+	-	r
5,10-methylenetetrahydrofolate + NADP+	Mus musculus	-	5,10-methenyltetrahydrofolate + NADPH + H+	-	r
5,10-methylenetetrahydrofolate + NADP+	Rattus norvegicus	-	5,10-methenyltetrahydrofolate + NADPH + H+	-	r
5,10-methylenetetrahydrofolate + NADP+	Mus musculus C57BL/6	-	5,10-methenyltetrahydrofolate + NADPH + H+	-	r
additional information	Mus musculus	MTHFD2L catalyzes the oxidation of 5,10-methylenetetrahydrofolate (CH2-THF) in adult mammalian mitochondria. Enzyme MTHFD2L is bifunctional, possessing both methylenetetrahydrofolate dehydrogenase and methenyltetrahydrofolate cyclohydrolase activities, cf. EC 3.5.4.9	?	-	?
additional information	Rattus norvegicus	MTHFD2L catalyzes the oxidation of 5,10-methylenetetrahydrofolate in adult mammalian mitochondria. Enzyme MTHFD2L is bifunctional, possessing both methylenetetrahydrofolate dehydrogenase and methenyltetrahydrofolate cyclohydrolase activities, cf. EC 3.5.4.9	?	-	?
additional information	Mus musculus C57BL/6	MTHFD2L catalyzes the oxidation of 5,10-methylenetetrahydrofolate (CH2-THF) in adult mammalian mitochondria. Enzyme MTHFD2L is bifunctional, possessing both methylenetetrahydrofolate dehydrogenase and methenyltetrahydrofolate cyclohydrolase activities, cf. EC 3.5.4.9	?	-	?

Organism

Organism	UniProt	Comment	Textmining
Mus musculus	D3YZG8	-	-
Mus musculus C57BL/6	D3YZG8	-	-
Rattus norvegicus	D3ZUA0	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His-tagged truncated enzyme mutant rMTHFD2LDELTA1-40 from Saccharomyces cerevisiae strain MWY4.4 by nickel affinity chromatography, sodium carbonate in the extraction buffer is used to release MTHFD2L from membranes	Rattus norvegicus

Source Tissue

Source Tissue	Comment	Organism	Textmining
adipose tissue	-	Mus musculus	-
brain	high expression level	Mus musculus	-
embryo	enzyme MTHFD2L is expressed in embryonic tissues during neural tube closure. Expression of the MTHFD2L transcript is low in early mouse embryos but begins to increase at embryonic day 10.5 and remains elevated through birth, in situ hybridization of whole mouse embryos	Mus musculus	-
heart	-	Mus musculus	-
kidney	low expression level	Mus musculus	-
liver	low expression level	Mus musculus	-
lung	-	Mus musculus	-
additional information	in adults, MTHFD2L is expressed in all tissues examined, with the highest levels observed in brain and lung	Mus musculus	-
small intestine	-	Mus musculus	-
spleen	-	Mus musculus	-
testis	low expression level	Mus musculus	-

Storage Stability

Storage Stability	Organism
-20°C, purified MTHFD2L in 25 mM Tris-Cl, 10 mM 2-mercaptoethanol, 100mM KCl, and 50% v/v glycerol at pH 7.5. The purified enzyme retains about 75% of initial 5,10-CH2THF dehydrogenase activity after 1 week of storage	Rattus norvegicus

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
5,10-methylenetetrahydrofolate + NAD+	-	Mus musculus	5,10-methenyltetrahydrofolate + NADH + H+	-	r
5,10-methylenetetrahydrofolate + NAD+	-	Rattus norvegicus	5,10-methenyltetrahydrofolate + NADH + H+	-	r
5,10-methylenetetrahydrofolate + NAD+	-	Mus musculus C57BL/6	5,10-methenyltetrahydrofolate + NADH + H+	-	r
5,10-methylenetetrahydrofolate + NADP+	-	Mus musculus	5,10-methenyltetrahydrofolate + NADPH + H+	-	r
5,10-methylenetetrahydrofolate + NADP+	-	Rattus norvegicus	5,10-methenyltetrahydrofolate + NADPH + H+	-	r
5,10-methylenetetrahydrofolate + NADP+	-	Mus musculus C57BL/6	5,10-methenyltetrahydrofolate + NADPH + H+	-	r
5,10-methylenetetrahydropteroylpoly-L-glutamate + NADP+	-	Mus musculus	5,10-methenyltetrahydropteroylpoly-L-glutamate + NADPH + H+	-	?
5,10-methylenetetrahydropteroylpoly-L-glutamate + NADP+	-	Mus musculus C57BL/6	5,10-methenyltetrahydropteroylpoly-L-glutamate + NADPH + H+	-	?
additional information	MTHFD2L catalyzes the oxidation of 5,10-methylenetetrahydrofolate (CH2-THF) in adult mammalian mitochondria. Enzyme MTHFD2L is bifunctional, possessing both methylenetetrahydrofolate dehydrogenase and methenyltetrahydrofolate cyclohydrolase activities, cf. EC 3.5.4.9	Mus musculus	?	-	?
additional information	MTHFD2L catalyzes the oxidation of 5,10-methylenetetrahydrofolate in adult mammalian mitochondria. Enzyme MTHFD2L is bifunctional, possessing both methylenetetrahydrofolate dehydrogenase and methenyltetrahydrofolate cyclohydrolase activities, cf. EC 3.5.4.9	Rattus norvegicus	?	-	?
additional information	enzyme MTHFD2L uses the mono- and polyglutamylated forms of 5,10-methylenetetrahydrofolate with similar catalytic efficiencies	Rattus norvegicus	?	-	?
additional information	enzyme MTHFD2Luses the mono- and polyglutamylated forms of 5,10-methylenetetrahydrofolate with similar catalytic efficiencies	Mus musculus	?	-	?
additional information	MTHFD2L catalyzes the oxidation of 5,10-methylenetetrahydrofolate (CH2-THF) in adult mammalian mitochondria. Enzyme MTHFD2L is bifunctional, possessing both methylenetetrahydrofolate dehydrogenase and methenyltetrahydrofolate cyclohydrolase activities, cf. EC 3.5.4.9	Mus musculus C57BL/6	?	-	?
additional information	enzyme MTHFD2Luses the mono- and polyglutamylated forms of 5,10-methylenetetrahydrofolate with similar catalytic efficiencies	Mus musculus C57BL/6	?	-	?

Subunits

Subunits	Comment	Organism
?	x * 33000, recombinant His6-tagged truncated enzyme mutant rMTHFD2LDELTA1-40, SDS-PAGE	Rattus norvegicus

Synonyms

Synonyms	Comment	Organism
CH2-THF dehydrogenase	-	Mus musculus
CH2-THF dehydrogenase	-	Rattus norvegicus
methylenetetrahydrofolate dehydrogenase	-	Mus musculus
methylenetetrahydrofolate dehydrogenase	-	Rattus norvegicus
methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolate cyclohydrolase	-	Mus musculus
methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolate cyclohydrolase	-	Rattus norvegicus
MTHFD2L	-	Mus musculus
MTHFD2L	-	Rattus norvegicus

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
30	-	assay at	Mus musculus
30	-	assay at	Rattus norvegicus

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
1.1	-	NADP+	pH 8.0, 30°C	Mus musculus
1.3	-	5,10-methylenetetrahydropteroylpoly-L-glutamate	pH 8.0, 30°C, monoglutamylated substrate, with NADP+	Mus musculus
2.7	-	5,10-methylenetetrahydropteroylpoly-L-glutamate	pH 8.0, 30°C, monoglutamylated substrate, with NAD+	Mus musculus
3.6	-	NAD+	pH 8.0, 30°C	Mus musculus
7.2	-	5,10-methylenetetrahydropteroylpoly-L-glutamate	pH 8.0, 30°C, polyglutamylated substrate, with NADP+	Mus musculus
8.8	-	5,10-methylenetetrahydropteroylpoly-L-glutamate	pH 8.0, 30°C, polyglutamylated substrate, with NAD+	Mus musculus

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
8	-	assay at	Mus musculus
8	-	assay at	Rattus norvegicus

Cofactor

Cofactor	Comment	Organism
additional information	enzyme MTHFD2L uses both NAD+ and NADP+ for methylenetetrahydrofolate dehydrogenase activity but requires both phosphate and Mg2+ when using NAD+	Rattus norvegicus
additional information	the enzyme MTHFD2L uses both NAD+ and NADP+ for methylenetetrahydrofolate dehydrogenase activity but requires both phosphate and Mg2+ when using NAD+	Mus musculus
NAD+	-	Mus musculus
NAD+	-	Rattus norvegicus
NADH	-	Mus musculus
NADH	-	Rattus norvegicus
NADP+	-	Mus musculus
NADP+	-	Rattus norvegicus
NADPH	-	Mus musculus
NADPH	-	Rattus norvegicus

General Information

General Information	Comment	Organism
evolution	the redox cofactor requirements of the isozymes from Mus musculus and Rattus norvegicus are quite similar, both exhibit lower Km values for NAD+ than for NADP+, their NAD+-dependent activities require phosphate and Mg2+, and their NADP+-dependent activities are inhibited by phosphate	Mus musculus
evolution	the redox cofactor requirements of the isozymes from Mus musculus and Rattus norvegicus are quite similar, both exhibit lower Km values for NAD+ than for NADP+, their NAD+-dependent activities require phosphate and Mg2+, and their NADP+-dependent activities are inhibited by phosphate	Rattus norvegicus
additional information	MTFHD2 isozyme has been named NAD+-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase but in fact exhibits dehydrogenase activity with NADP+, albeit with a much higher Km and lower Vmax	Mus musculus
additional information	MTFHD2 isozyme has been named NAD+-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase but in fact exhibits dehydrogenase activity with NADP+, albeit with a much higher Km and lower Vmax	Rattus norvegicus
physiological function	MTHFD2L uses both NAD+ and NADP+ and is expressed in embryonic tissues during neural tube closure. The cofactor specificity allows for rapid response to changing metabolic conditions. MTHFD2 uses Mg2+ and phosphate to convert an NADP+ binding site into an NAD+ binding site. Phosphate binds in close proximity to the 2'-hydroxyl of NAD+ and competes with NADP+ binding. Mg2 plays a role in positioning phosphatei and NAD+	Mus musculus
physiological function	MTHFD2L uses both NAD+ and NADP+ and is expressed in embryonic tissues during neural tube closure. The cofactor specificity allows for rapid response to changing metabolic conditions. MTHFD2 uses Mg2+ and phosphate to convert an NADP+ binding site into an NAD+ binding site. Phosphate binds in close proximity to the 2'-hydroxyl of NAD+ and competes with NADP+ binding. Mg2 plays a role in positioning phosphatei and NAD+	Rattus norvegicus

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
2.05	-	NADP+	pH 8.0, 30°C	Mus musculus
24.49	-	NAD+	pH 8.0, 30°C	Mus musculus
30.95	-	5,10-methylenetetrahydropteroylpoly-L-glutamate	pH 8.0, 30°C, monoglutamylated substrate, with NADP+	Mus musculus
47.06	-	5,10-methylenetetrahydropteroylpoly-L-glutamate	pH 8.0, 30°C, polyglutamylated substrate, with NADP+	Mus musculus
67.5	-	5,10-methylenetetrahydropteroylpoly-L-glutamate	pH 8.0, 30°C, monoglutamylated substrate, with NAD+	Mus musculus
67.69	-	5,10-methylenetetrahydropteroylpoly-L-glutamate	pH 8.0, 30°C, polyglutamylated substrate, with NAD+	Mus musculus