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Literature summary for 1.5.1.5 extracted from

  • Murta, S.M.; Vickers, T.J.; Scott, D.A.; Beverley, S.M.
    Methylene tetrahydrofolate dehydrogenase/cyclohydrolase and the synthesis of 10-CHO-THF are essential in Leishmania major (2009), Mol. Microbiol., 71, 1386-1401.
    View publication on PubMedView publication on EuropePMC

Application

Application Comment Organism
medicine Leishmania major is shown to require 10-formyl tetrahydrofolate metabolism, thus this is a possible target for chemotherapy in Leishmania major and potential other protozoan parasites Leishmania major

Cloned(Commentary)

Cloned (Comment) Organism
hexa-His tagged DHCH1 is expressed in Escherichia coli, production of a genetic knock-out Leishmania major
PCR-amplification, expressed in Escherichia coli, overexpression and null-mutant production Leishmania major

Protein Variants

Protein Variants Comment Organism
additional information successful double replacement of the DHCH1 gene is a result of double electroporation with two replacement fragments but an intact gene copy is retained (probably aneuploidy), additional metabolic complementation to bypass the requirement for DHCH1 is not successful either, ectopic addition of DHCH1 or formate-tetrahydrofolate ligase before the second replacement step finally results in a null mutant without chromosomal DHCH1 gene (dhch1-/pXNG4-DHCH1 or dhch1-/pXNG4-FTL) or addition to wild-type in overexpressing transfectants (WT/pXNG4-DHCH1 or WT/pXNG4-FTL) Leishmania major

Inhibitors

Inhibitors Comment Organism Structure
(2S)-2-[[4-[(6aR)-3-amino-1,9-dioxo-5,6,6a,7-tetrahydro-4H-imidazol[3,4-f]pteridin-8-yl]benzoyl]amino]pentanedioate
-
Leishmania major
(2S)-2-[[4-[(6aR)-3-amino-1,9-dioxo-5,6,6a,7-tetrahydro-4H-imidazol[3,4-f]pteridin-8-yl]benzoyl]amino]pentanedioic acid 5,10-formyltetrahydrofolate substrate analogue inhibits dehydrogenase activity Leishmania major
(2S)-2-[[4-[(6aR)-3-amino-1,9-dioxo-5,6,6a,7-tetrahydro-4H-imidazo[3,4-f]pteridin-8-yl]benzoyl]amino]pentanedioic acid competitive substrate analogue Leishmania major
2,4-diamino-6-(3,4-dichlorophenoxy)-quinazoline
-
Leishmania major
2,4-diamino-6-benzyl-5-(3-phenylpropyl)-pyrimidine
-
Leishmania major
methotrexate
-
Leishmania major
additional information no inhibition by pteridine analogues, such as methotrexate, or by hydrophobic diamino-quinazoline, -pyrimidine or -pteridine analogues, such as 2,4-diamino-6-(3,4-dichlorophenoxy)-quinazoline, or 2,4-diamino-6-benzyl-5-(3-phenylpropyl)-pyrimidine, or 2,4-diamino-6,7-diisopropylpteridine Leishmania major

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.038
-
NADP+ 25 mM MOPS pH 7.3, 2.5 mM formaldehyde, 30 mM 2-mercaptoethanol, 27°C, with 250 microM 5,10-methylene-tetrahydrofolate Leishmania major
0.038
-
NADP+ recombinant DHCH1 Leishmania major
0.12
-
5,10-methylene-tetrahydrofolate 25 mM MOPS pH 7.3, 2.5 mM formaldehyde, 1 mM NADP+, 30 mM 2-mercaptoethanol, 27°C Leishmania major
0.12
-
5,10-methylene-tetrahydrofolate recombinant DHCH1 Leishmania major

Localization

Localization Comment Organism GeneOntology No. Textmining
cytosol
-
Leishmania major 5829
-
cytosol not in mitochondria as in other organisms Leishmania major 5829
-

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
5,10-methylene tetrahydrofolate + NADP+ Leishmania major DH activity 5,10-methenyl tetrahydrofolate + NADPH + H+
-
r
5,10-methylene-tetrahydrofolate + NADP+ Leishmania major
-
5,10-methenyl-tetrahydrofolate + NADPH + H+ 10-formyl tetrahydrofolate (10-CHO-THF)is the product of subsequently active methenyltetrahydrofolate cyclohydrolase, ATP-dependent formate-tetrahydrofolate ligase catalyzes an alternative one step reaction from tetrahydrofolate + format to 10-formyl tetrahydrofolate ?
additional information Leishmania major dehydrogenase and cyclohydrogenase activities are contained in the single bifunctional protein DHCH1 ?
-
?

Organism

Organism UniProt Comment Textmining
Leishmania major
-
-
-

Purification (Commentary)

Purification (Comment) Organism
metal affinity chromatography Leishmania major
recombinant DHCH1 protein is purified by metal-affinity chromatography Leishmania major

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
22
-
25 mM MOPS pH 7.3, 2.5 mM formaldehyde, 1 mM NADP+, 30 mM 2-mercaptoethanol, 27°C, 6.3 microM/min/mg activity of the methenyltetrahydrofolate cyclohydrolase Leishmania major
22
-
dehydrogenase activity is measured by following the conversion of 5,10-methylenetetrahydrofolate to 5,10-methenyltetrahydrofolate Leishmania major

Storage Stability

Storage Stability Organism
10% glycerol required to prevent loss of activity during storage Leishmania major

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
5,10-methylene tetrahydrofolate + NADP+ DH activity Leishmania major 5,10-methenyl tetrahydrofolate + NADPH + H+
-
r
5,10-methylene-tetrahydrofolate + NADP+
-
Leishmania major 5,10-methenyl-tetrahydrofolate + NADPH + H+ 10-formyl tetrahydrofolate (10-CHO-THF)is the product of subsequently active methenyltetrahydrofolate cyclohydrolase, ATP-dependent formate-tetrahydrofolate ligase catalyzes an alternative one step reaction from tetrahydrofolate + format to 10-formyl tetrahydrofolate ?
additional information dehydrogenase and cyclohydrogenase activities are contained in the single bifunctional protein DHCH1 Leishmania major ?
-
?

Synonyms

Synonyms Comment Organism
5,10-mehtylene tetrahydrofolate dehydrogenase
-
Leishmania major
5,10-methenyltetrahydrofolate cyclohydrolase/5,10-methylene tetrahydrofolate dehydrogenase
-
Leishmania major
DHCH1
-
Leishmania major
DHCH1 bifuntional enzyme with 5,10-mehtylene tetrahydrofolate dehydrogenase and methenyltetrahydrofolate cyclohydrolase activity in Leishmania major Leishmania major
methylene tetrahydrofolate dehydrogenase/cyclohydrolase
-
Leishmania major

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
27
-
assay at Leishmania major

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
10
-
NADP+ 25 mM MOPS pH 7.3, 2.5 mM formaldehyde, 30 mM 2-mercaptoethanol, 27°C Leishmania major
10
-
NADP+ recombinant DHCH1, 1 mM NADP+ Leishmania major
14
-
5,10-methylene-tetrahydrofolate 25 mM MOPS pH 7.3, 2.5 mM formaldehyde, 1 mM NADP+, 30 mM 2-mercaptoethanol, 27°C Leishmania major
14
-
5,10-methylene tetrahydrofolate recombinant DHCH1, 0.25 mM substrate Leishmania major

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.3
-
assay at Leishmania major

Cofactor

Cofactor Comment Organism Structure
NADP+
-
Leishmania major
NADP+ no dependence on NAD+ in presence of K3PO4 and MgCl2 (required for human mitochondrial NAD+ dependent activity of bifunctional dehydrogenase cyclase) Leishmania major

Ki Value [mM]

Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
0.000105
-
(2S)-2-[[4-[(6aR)-3-amino-1,9-dioxo-5,6,6a,7-tetrahydro-4H-imidazol[3,4-f]pteridin-8-yl]benzoyl]amino]pentanedioate
-
Leishmania major
0.000105
-
(2S)-2-[[4-[(6aR)-3-amino-1,9-dioxo-5,6,6a,7-tetrahydro-4H-imidazo[3,4-f]pteridin-8-yl]benzoyl]amino]pentanedioic acid pH 7.3, 27°C, Leishmania wild-type growth is inhibited with an EC50 of 1.1 microM, DHCH1 or formate-tetrahydrofolate ligase overexpressing (FTL) Leishmania transfectants show 4-5fold or 3fold resistance, respectively Leishmania major

IC50 Value

IC50 Value IC50 Value Maximum Comment Organism Inhibitor Structure
0.000036
-
wild type enzyme Leishmania major methotrexate
0.000041
-
dhch1-/pXNG4-DHCH1 mutant, FV1 line Leishmania major methotrexate
0.0004
-
dhch1-/pXNG4-DHCH1 mutant, FV1 line Leishmania major 2,4-diamino-6-benzyl-5-(3-phenylpropyl)-pyrimidine
0.00044
-
wild type enzyme Leishmania major 2,4-diamino-6-benzyl-5-(3-phenylpropyl)-pyrimidine
0.00068
-
wild type enzyme Leishmania major 2,4-diamino-6-(3,4-dichlorophenoxy)-quinazoline
0.00076
-
dhch1-/pXNG4-DHCH1 mutant, FV1 line Leishmania major 2,4-diamino-6-(3,4-dichlorophenoxy)-quinazoline
0.0011
-
wild type enzyme Leishmania major (2S)-2-[[4-[(6aR)-3-amino-1,9-dioxo-5,6,6a,7-tetrahydro-4H-imidazol[3,4-f]pteridin-8-yl]benzoyl]amino]pentanedioic acid
0.0011
-
50% growth inhibition of wild-type cell line Leishmania major (2S)-2-[[4-[(6aR)-3-amino-1,9-dioxo-5,6,6a,7-tetrahydro-4H-imidazo[3,4-f]pteridin-8-yl]benzoyl]amino]pentanedioic acid
0.0033
-
50% growth inhibition of null mutant with plasmid expressing formate-tetrahydrofolate ligase Leishmania major (2S)-2-[[4-[(6aR)-3-amino-1,9-dioxo-5,6,6a,7-tetrahydro-4H-imidazo[3,4-f]pteridin-8-yl]benzoyl]amino]pentanedioic acid
0.0037
-
50% growth inhibition of wild-type with plasmid expressing formate-tetrahydrofolate ligase Leishmania major (2S)-2-[[4-[(6aR)-3-amino-1,9-dioxo-5,6,6a,7-tetrahydro-4H-imidazo[3,4-f]pteridin-8-yl]benzoyl]amino]pentanedioic acid
0.0045
-
50% growth inhibition of null mutant with plasmid expressing the enzyme Leishmania major (2S)-2-[[4-[(6aR)-3-amino-1,9-dioxo-5,6,6a,7-tetrahydro-4H-imidazo[3,4-f]pteridin-8-yl]benzoyl]amino]pentanedioic acid
0.0045
-
dhch1-/pXNG4-DHCH1 mutant, FV1 line Leishmania major (2S)-2-[[4-[(6aR)-3-amino-1,9-dioxo-5,6,6a,7-tetrahydro-4H-imidazol[3,4-f]pteridin-8-yl]benzoyl]amino]pentanedioic acid
0.0051
-
50% growth inhibition of wild-type with plasmid expressing the enzyme (overexpressing it as a result) Leishmania major (2S)-2-[[4-[(6aR)-3-amino-1,9-dioxo-5,6,6a,7-tetrahydro-4H-imidazo[3,4-f]pteridin-8-yl]benzoyl]amino]pentanedioic acid
0.0051
-
WT/pXNG4-DHCH mutant, FV1 line Leishmania major (2S)-2-[[4-[(6aR)-3-amino-1,9-dioxo-5,6,6a,7-tetrahydro-4H-imidazol[3,4-f]pteridin-8-yl]benzoyl]amino]pentanedioic acid

General Information

General Information Comment Organism
metabolism product of the bifuncional enzyme, 10-formyl tetrahydrofolate, is not essential for de novo purine synthesis in Leishmania and other parasitic protozoans contrary to other organisms, it is utilized for methionyl-tRNA formulation in mitochondria, the bifunctional enzyme product is essential for Leishmania major and may even reveal novel pathways Leishmania major

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
120
-
5,10-methylene-tetrahydrofolate 25 mM MOPS pH 7.3, 2.5 mM formaldehyde, 1 mM NADP+, 30 mM 2-mercaptoethanol, 27°C Leishmania major
260
-
NADP+ 25 mM MOPS pH 7.3, 2.5 mM formaldehyde, 30 mM 2-mercaptoethanol, 27°C, with 250 microM 5,10-methylene-tetrahydrofolate Leishmania major