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 (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 | 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 | 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 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 | Comment | Organism | GeneOntology No. | Textmining |
---|---|---|---|---|
cytosol | - |
Leishmania major | 5829 | - |
cytosol | not in mitochondria as in other organisms | Leishmania major | 5829 | - |
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 | UniProt | Comment | Textmining |
---|---|---|---|
Leishmania major | - |
- |
- |
Purification (Comment) | Organism |
---|---|
metal affinity chromatography | Leishmania major |
recombinant DHCH1 protein is purified by metal-affinity chromatography | Leishmania major |
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 | Organism |
---|---|
10% glycerol required to prevent loss of activity during storage | Leishmania major |
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 | 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 Maximum [°C] | Comment | Organism |
---|---|---|---|
27 | - |
assay at | Leishmania major |
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 Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.3 | - |
assay at | Leishmania major |
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 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 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 | 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 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 |