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Literature summary extracted from

  • Gibson, M.W.; Dewar, S.; Ong, H.B.; Sienkiewicz, N.; Fairlamb, A.H.
    Trypanosoma brucei DHFR-TS revisited characterisation of a bifunctional and highly unstable recombinant dihydrofolate reductase-thymidylate synthase (2016), PLoS Negl. Trop. Dis., 10, e0004714 .
    View publication on PubMedView publication on EuropePMC

Application

EC Number Application Comment Organism
1.5.1.3 drug development the enzyme is a target for drug development in the treatment of Human African trypanosomiasis (HAT), an infectious disease caused by two distinct subspecies of the protozoan parasite Trypanosoma brucei subsp. gambiense and Trypanosoma brucei subsp. rhodesiense Trypanosoma brucei
2.1.1.45 drug development the enzyme is a target for drug development in the treatement of Human African trypanosomiasis (HAT), an infectious disease caused by two distinct subspecies of the protozoan parasite Trypanosoma brucei subsp. gambiense and Trypanosoma brucei subsp. rhodesiense Trypanosoma brucei

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.5.1.3 recombinant expression of bifunctional Trypanosoma brucei DHFR-TS in form of a fusion protein incorporating Escherichia coli elongation factor Ts (Tsf), in Escherichia coli strain BL21 Star (DE3). The Tsf open reading frame is amplified by PCR from the genomic DNA of Eschrichia coli strain K12, cloning procedure overview. Expression constructs carrying a thymidylate synthase (TS) domain from Trypanosoma brucei, Leishmania major and Homo sapiens are expressed in a TS-deficient Escherichia coli strain (thyA-), while TbDHFR without thymidylate synthase is expressed in the parental thyA+ strain Trypanosoma brucei
2.1.1.45 recombinant expression of bifunctional Trypanosoma brucei DHFR-TS in form of a fusion protein incorporating Escherichia coli elongation factor Ts (Tsf), in Escherichia coli strain BL21 Star (DE3). The Tsf open reading frame is amplified by PCR from the genomic DNA of Eschrichia coli strain K12, cloning procedure overview. Expression constructs carrying a thymidylate synthase (TS) domain from Trypanosoma brucei, Leishmania major and Homo sapiens are expressed in a TS-deficient Escherichia coli strain (thyA-), while TbDHFR without thymidylate synthase is expressed in the parental thyA+ strain Trypanosoma brucei

Protein Variants

EC Number Protein Variants Comment Organism
1.5.1.3 additional information DHFR activity in lysates of Escherichia coli expressing Tsf-TbDHFR-TS is about 6fold more active than those expressing His6-TbDHFR-TS. In addition, thymidylate synthase activity, which has proved elusive with the His6-protein, is detectable. Stabilization of the TS activity with dUMP, without affect on DHFR stability. No stabilisation observed with CH2THF and other pyrimidine nucleotides, including the uracil-containing ribonucleotides and deoxyribonucleotides, and the thymidine-containing deoxyribonucleotides, overview Trypanosoma brucei
2.1.1.45 additional information DHFR activity in lysates of Escherichia coli expressing Tsf-TbDHFR-TS is about 6fold more active than those expressing His6-TbDHFR-TS. In addition, thymidylate synthase activity, which has proved elusive with the His6-protein, is detectable. Stabilization of the TS activity with dUMP, without affect on DHFR stability. No stabilisation observed with CH2THF and other pyrimidine nucleotides, including the uracil-containing ribonucleotides and deoxyribonucleotides, and the thymidine-containing deoxyribonucleotides, overview Trypanosoma brucei

Inhibitors

EC Number Inhibitors Comment Organism Structure
1.5.1.3 5-fluorodeoxyuridine monophosphate
-
Trypanosoma brucei
1.5.1.3 5-fluorouracil
-
Trypanosoma brucei
1.5.1.3 methotrexate
-
Trypanosoma brucei
1.5.1.3 additional information analysis of enzyme susceptibility to a range of classical inhibitors normally used in the treatment of cancer, bacterial or protozoal infections, in vivo effects in presence of absence of thymidine and/or folate, overview. Modulating certain medium components can affect drug sensitivity, presumably by either competition for uptake and competition for the active site of DHFR-TS. In the case of one human thymidylate synthase inhibitor raltitrexed, the inhibitor is more potent against the intact parasite. Addition of extra glutamic acid residues not only improves retention in the cell, but also increases potency against thymidylate synthase, as it does in human cells. No inhibition by FdUMP Trypanosoma brucei
1.5.1.3 nolatrexed
-
Trypanosoma brucei
1.5.1.3 pemetrexed
-
Trypanosoma brucei
1.5.1.3 pyrimethamine
-
Trypanosoma brucei
1.5.1.3 raltitrexed
-
Trypanosoma brucei
1.5.1.3 trimethoprim
-
Trypanosoma brucei
1.5.1.3 trimetrexate
-
Trypanosoma brucei
2.1.1.45 5-fluorodeoxyuridine monophosphate
-
Trypanosoma brucei
2.1.1.45 5-fluorouracil
-
Trypanosoma brucei
2.1.1.45 methotrexate
-
Trypanosoma brucei
2.1.1.45 additional information analysis of enzyme susceptibility to a range of classical inhibitors normally used in the treatment of cancer, bacterial or protozoal infections, in vivo effects in presence of absence of thymidine and/or folate, overview. Modulating certain medium components can affect drug sensitivity, presumably by either competition for uptake and competition for the active site of DHFR-TS. In the case of one human thymidylate synthase inhibitor raltitrexed, the inhibitor is more potent against the intact parasite. Addition of extra glutamic acid residues not only improves retention in the cell, but also increases potency against thymidylate synthase, as it does in human cells. No inhibition by FdUMP Trypanosoma brucei
2.1.1.45 nolatrexed
-
Trypanosoma brucei
2.1.1.45 pemetrexed
-
Trypanosoma brucei
2.1.1.45 pyrimethamine
-
Trypanosoma brucei
2.1.1.45 raltitrexed
-
Trypanosoma brucei
2.1.1.45 trimethoprim
-
Trypanosoma brucei
2.1.1.45 trimetrexate
-
Trypanosoma brucei

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.5.1.3 additional information
-
additional information Michaelis-Menten kinetics Trypanosoma brucei
2.1.1.45 additional information
-
additional information Michaelis-Menten kinetics Trypanosoma brucei

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
1.5.1.3 KCl the optimal ionic strength for both enzymes requires 100 mM KCl, with DHFR displaying 2.5fold activation and TS 4.5fold activation Trypanosoma brucei
2.1.1.45 KCl the optimal ionic strength for both enzymes requires 100 mM KCl, with DHFR displaying 2.5fold activation and TS 4.5fold activation Trypanosoma brucei
2.1.1.45 Mg2+ TS is activated 4.5fold at 10 mM MgCl2, but inhibited at higher concentrations of MgCl2. The activating effect is not additive with activation by KCl Trypanosoma brucei

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.5.1.3 7,8-dihydrofolate + NADPH + H+ Trypanosoma brucei
-
5,6,7,8-tetrahydrofolate + NADP+
-
r
1.5.1.3 7,8-dihydrofolate + NADPH + H+ Trypanosoma brucei 427
-
5,6,7,8-tetrahydrofolate + NADP+
-
r
1.5.1.3 additional information Trypanosoma brucei the bifunctional folate and pyrimidine-metabolising enzyme dihydrofolate reductase-thymidylate synthase dihydrofolate reductase-thymidylate synthase is expressed from a single gene as a homodimer comprising of an N-terminal DHFR domain fused via a linker peptide to a thymidylate synthase domain at the C-terminus ?
-
?
1.5.1.3 additional information Trypanosoma brucei 427 the bifunctional folate and pyrimidine-metabolising enzyme dihydrofolate reductase-thymidylate synthase dihydrofolate reductase-thymidylate synthase is expressed from a single gene as a homodimer comprising of an N-terminal DHFR domain fused via a linker peptide to a thymidylate synthase domain at the C-terminus ?
-
?
2.1.1.45 5,10-methylenetetrahydrofolate + dUMP Trypanosoma brucei
-
dihydrofolate + dTMP
-
?
2.1.1.45 additional information Trypanosoma brucei the bifunctional folate and pyrimidine-metabolising enzyme dihydrofolate reductase-thymidylate synthase dihydrofolate reductase-thymidylate synthase is expressed from a single gene as a homodimer comprising of an N-terminal DHFR domain fused via a linker peptide to a thymidylate synthase domain at the C-terminus ?
-
?
2.1.1.45 additional information Trypanosoma brucei 427 the bifunctional folate and pyrimidine-metabolising enzyme dihydrofolate reductase-thymidylate synthase dihydrofolate reductase-thymidylate synthase is expressed from a single gene as a homodimer comprising of an N-terminal DHFR domain fused via a linker peptide to a thymidylate synthase domain at the C-terminus ?
-
?

Organism

EC Number Organism UniProt Comment Textmining
1.5.1.3 Trypanosoma brucei Q27783
-
-
1.5.1.3 Trypanosoma brucei 427 Q27783
-
-
2.1.1.45 Trypanosoma brucei Q27783
-
-
2.1.1.45 Trypanosoma brucei 427 Q27783
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.5.1.3 recombinant Trypanosoma brucei Tsf-DHFR-TS from Escherichia coli strain BL21 Star (DE3) by methotrexate affinity chromatography and gel filtration Trypanosoma brucei
2.1.1.45 recombinant Trypanosoma brucei Tsf-DHFR-TS from Escherichia coli strain BL21 Star (DE3) by methotrexate affinity chromatography and gel filtration Trypanosoma brucei

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
1.5.1.3 1.2
-
purified recombinant bifunctional enzyme DHFR-TS with Tsf tag, pH 7.4, 25°C from a TS-deficient Escherichia coli strain (thyA-) Trypanosoma brucei
2.1.1.45 0.0088
-
purified recombinant bifunctional enzyme DHFR-TS with Tsf tag, pH 7.4, 25°C from a TS-deficient Escherichia coli strain (thyA-) Trypanosoma brucei

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.5.1.3 7,8-dihydrofolate + NADPH + H+
-
Trypanosoma brucei 5,6,7,8-tetrahydrofolate + NADP+
-
r
1.5.1.3 7,8-dihydrofolate + NADPH + H+
-
Trypanosoma brucei 427 5,6,7,8-tetrahydrofolate + NADP+
-
r
1.5.1.3 additional information the bifunctional folate and pyrimidine-metabolising enzyme dihydrofolate reductase-thymidylate synthase dihydrofolate reductase-thymidylate synthase is expressed from a single gene as a homodimer comprising of an N-terminal DHFR domain fused via a linker peptide to a thymidylate synthase domain at the C-terminus Trypanosoma brucei ?
-
?
1.5.1.3 additional information no activity with folic acid and the structurally related pterins (biopterin, dihydrobiopterin, sepiapterin and neopterin) as substrates by enzyme DHFR-TS Trypanosoma brucei ?
-
?
1.5.1.3 additional information the bifunctional folate and pyrimidine-metabolising enzyme dihydrofolate reductase-thymidylate synthase dihydrofolate reductase-thymidylate synthase is expressed from a single gene as a homodimer comprising of an N-terminal DHFR domain fused via a linker peptide to a thymidylate synthase domain at the C-terminus Trypanosoma brucei 427 ?
-
?
1.5.1.3 additional information no activity with folic acid and the structurally related pterins (biopterin, dihydrobiopterin, sepiapterin and neopterin) as substrates by enzyme DHFR-TS Trypanosoma brucei 427 ?
-
?
2.1.1.45 5,10-methylenetetrahydrofolate + dUMP
-
Trypanosoma brucei dihydrofolate + dTMP
-
?
2.1.1.45 additional information the bifunctional folate and pyrimidine-metabolising enzyme dihydrofolate reductase-thymidylate synthase dihydrofolate reductase-thymidylate synthase is expressed from a single gene as a homodimer comprising of an N-terminal DHFR domain fused via a linker peptide to a thymidylate synthase domain at the C-terminus Trypanosoma brucei ?
-
?
2.1.1.45 additional information no activity with folic acid and the structurally related pterins (biopterin, dihydrobiopterin, sepiapterin and neopterin) as substrates by enzyme DHFR-TS Trypanosoma brucei ?
-
?
2.1.1.45 additional information the bifunctional folate and pyrimidine-metabolising enzyme dihydrofolate reductase-thymidylate synthase dihydrofolate reductase-thymidylate synthase is expressed from a single gene as a homodimer comprising of an N-terminal DHFR domain fused via a linker peptide to a thymidylate synthase domain at the C-terminus Trypanosoma brucei 427 ?
-
?
2.1.1.45 additional information no activity with folic acid and the structurally related pterins (biopterin, dihydrobiopterin, sepiapterin and neopterin) as substrates by enzyme DHFR-TS Trypanosoma brucei 427 ?
-
?

Subunits

EC Number Subunits Comment Organism
1.5.1.3 More the bifunctional folate and pyrimidine-metabolising enzyme dihydrofolate reductase-thymidylate synthase dihydrofolate reductase-thymidylate synthase is expressed from a single gene as a homodimer comprising of an N-terminal DHFR domain fused via a linker peptide to a TS domain at the C-terminus Trypanosoma brucei
2.1.1.45 More the bifunctional folate and pyrimidine-metabolising enzyme dihydrofolate reductase-thymidylate synthase dihydrofolate reductase-thymidylate synthase is expressed from a single gene as a homodimer comprising of an N-terminal DHFR domain fused via a linker peptide to a TS domain at the C-terminus Trypanosoma brucei

Synonyms

EC Number Synonyms Comment Organism
1.5.1.3 DHFR-TS
-
Trypanosoma brucei
1.5.1.3 dihydrofolate reductase-thymidylate synthase
-
Trypanosoma brucei
1.5.1.3 More cf. EC 2.1.1.45 Trypanosoma brucei
2.1.1.45 DHFR-TS
-
Trypanosoma brucei
2.1.1.45 dihydrofolate reductase-thymidylate synthase
-
Trypanosoma brucei
2.1.1.45 More cf. EC 1.5.1.3 Trypanosoma brucei

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.5.1.3 25
-
assay at Trypanosoma brucei
2.1.1.45 25
-
assay at Trypanosoma brucei

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.5.1.3 5.5
-
DHFR activity Trypanosoma brucei
2.1.1.45 7
-
TS activity Trypanosoma brucei

Cofactor

EC Number Cofactor Comment Organism Structure
1.5.1.3 NADP+
-
Trypanosoma brucei
1.5.1.3 NADPH
-
Trypanosoma brucei
2.1.1.45 NADP+
-
Trypanosoma brucei
2.1.1.45 NADPH
-
Trypanosoma brucei

Ki Value [mM]

EC Number Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
1.5.1.3 0.000000095
-
methotrexate pH 7.4, 25°C, recombinant enzyme Trypanosoma brucei
1.5.1.3 0.000000597
-
trimetrexate pH 7.4, 25°C, recombinant enzyme Trypanosoma brucei
1.5.1.3 0.00000635
-
pyrimethamine pH 7.4, 25°C, recombinant enzyme Trypanosoma brucei
1.5.1.3 0.0000176
-
trimethoprim pH 7.4, 25°C, recombinant enzyme Trypanosoma brucei
1.5.1.3 0.0000931
-
raltitrexed pH 7.4, 25°C, recombinant enzyme Trypanosoma brucei
1.5.1.3 0.00029
-
pemetrexed pH 7.4, 25°C, recombinant enzyme Trypanosoma brucei
1.5.1.3 0.000348
-
nolatrexed pH 7.4, 25°C, recombinant enzyme Trypanosoma brucei
2.1.1.45 0.000000095
-
methotrexate pH 7.4, 25°C, recombinant enzyme Trypanosoma brucei
2.1.1.45 0.000000597
-
trimetrexate pH 7.4, 25°C, recombinant enzyme Trypanosoma brucei
2.1.1.45 0.00000635
-
pyrimethamine pH 7.4, 25°C, recombinant enzyme Trypanosoma brucei
2.1.1.45 0.0000176
-
trimethoprim pH 7.4, 25°C, recombinant enzyme Trypanosoma brucei
2.1.1.45 0.0000931
-
raltitrexed pH 7.4, 25°C, recombinant enzyme Trypanosoma brucei
2.1.1.45 0.00029
-
pemetrexed pH 7.4, 25°C, recombinant enzyme Trypanosoma brucei
2.1.1.45 0.000348
-
nolatrexed pH 7.4, 25°C, recombinant enzyme Trypanosoma brucei

Expression

EC Number Organism Comment Expression
2.1.1.45 Trypanosoma brucei DHFR activity in lysates of Escherichia coli expressing Tsf-TbDHFR-TS is about 6fold more active than those expressing His6-TbDHFR-TS. In addition, thymidylate synthase activity, which has proved elusive with the His6-protein, is detectable. Stabilization of the TS activity with dUMP, without affect on DHFR stability. No stabilisation observed with CH2THF and other pyrimidine nucleotides, including the uracil-containing ribonucleotides and deoxyribonucleotides, and the thymidine-containing deoxyribonucleotides, overview additional information

General Information

EC Number General Information Comment Organism
1.5.1.3 physiological function DHFR-TS is essential for cell survival of Trypanosoma brucei Trypanosoma brucei
2.1.1.45 physiological function DHFR-TS is essential for cell survival of Trypanosoma brucei Trypanosoma brucei