Literature summary for 2.1.1.45 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 .

Application

Application	Comment	Organism
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)

Cloned (Comment)	Organism
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

Cloned (Comment)

Organism

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

Protein Variants	Comment	Organism
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

Inhibitors	Comment	Organism
5-fluorodeoxyuridine monophosphate	-	Trypanosoma brucei
5-fluorouracil	-	Trypanosoma brucei
methotrexate	-	Trypanosoma brucei
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
nolatrexed	-	Trypanosoma brucei
pemetrexed	-	Trypanosoma brucei
pyrimethamine	-	Trypanosoma brucei
raltitrexed	-	Trypanosoma brucei
trimethoprim	-	Trypanosoma brucei
trimetrexate	-	Trypanosoma brucei

KM Value [mM]

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

Metals/Ions

Metals/Ions	Comment	Organism	Structure
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
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)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.
5,10-methylenetetrahydrofolate + dUMP	Trypanosoma brucei	-	dihydrofolate + dTMP	-	?
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	?	-	?
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

Organism	UniProt	Comment	Textmining
Trypanosoma brucei	Q27783	-	-
Trypanosoma brucei 427	Q27783	-	-

Purification (Commentary)

Purification (Comment)	Organism
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]

Specific Activity Minimum [µmol/min/mg]	Specific Activity Maximum [µmol/min/mg]	Comment	Organism
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)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
5,10-methylenetetrahydrofolate + dUMP	-	Trypanosoma brucei	dihydrofolate + dTMP	-	?
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	?	-	?
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	?	-	?
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	?	-	?
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

Subunits	Comment	Organism
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

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

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
25	-	assay at	Trypanosoma brucei

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7	-	TS activity	Trypanosoma brucei

Cofactor

Cofactor	Comment	Organism	Structure
NADP+	-	Trypanosoma brucei
NADPH	-	Trypanosoma brucei

Ki Value [mM]

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

Expression

Organism	Comment	Expression
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

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