Literature summary for 2.1.1.45 extracted from

Arvizu-Flores, A.A.; Sugich-Miranda, R.; Arreola, R.; Garcia-Orozco, K.D.; Velazquez-Contreras, E.F.; Montfort, W.R.; Maley, F.; Sotelo-Mundo, R.R.
Role of an invariant lysine residue in folate binding on Escherichia coli thymidylate synthase: calorimetric and crystallographic analysis of the K48Q mutant (2008), Int. J. Biochem. Cell Biol., 40, 2206-2217.

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Cloned(Commentary)

Cloned (Comment)	Organism
expression in Escherichia coli	Escherichia coli

Crystallization (Commentary)

Crystallization (Comment)	Organism
hanging drop method at room temperature, the complexes of mutant TS with dUMP crystallize in the cubic form, binary and ternary complexes with 5NO2dUMP crystallize in the hexagonal form	Escherichia coli
mutant K48Q, in complex with dUMP or 5-nitro-dUMP, to 2.2 A and 2.7 A resolution, respectively. Binding of dUMP is not impaired in the mutant	Escherichia coli

Protein Variants

Protein Variants	Comment	Organism
K48Q	430fold decrease in kcat value, about 30fold increase in Km value for (R)-5,10-methylene-5,6,7,8-tetrahydrofolate. Decrease in binding affinity for folate-like inhibitors	Escherichia coli
K48Q	mutant of Escherichia coli thymidylate synthase	Escherichia coli

Inhibitors

Inhibitors	Comment	Organism	Structure
(S)-2-(5(((1,2-dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)methyl)amino)1-oxo-2-isoindolinyl)glutaric acid	BW1843U89, U89, distorts the thymidylate synthase active site	Escherichia coli
10-propargyl-5,8-dideazafolate	PDDF, folate-like TS inhibitor	Escherichia coli

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
0.05	-	dUMP	10fold higher than wild-type thymidylate synthase	Escherichia coli
0.05	-	dUMP	mutant K48Q	Escherichia coli
0.3	-	(R)-5,10-methylene-5,6,7,8-tetrahydrofolate	30fold higher than wild-type thymidylate synthase	Escherichia coli
0.3	-	(R)-5,10-methylenetetrahydrofolate	mutant K48Q	Escherichia coli

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
5,10-methylene-5,6,7,8-tetrahydrofolate + dUMP	Escherichia coli	thymidylate synthase is the third enzyme in the methylene tetrahydrofolate cycle besides dihydrofolate reductase and serine hydroxymethyltransferase, in many organisms, thymidylate synthase is the only de novo source for dTMP required for DNA synthesis	7,8-dihydrofolate + dTMP	-	?

Organism

Organism	UniProt	Comment	Textmining
Escherichia coli	P0A884	-	-

Purification (Commentary)

Purification (Comment)	Organism
after purification and precipitation with ammonium sulfate, wild-type and K48Q TS are frozen at -80°C	Escherichia coli

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
(R)-5,10-methylene-5,6,7,8-tetrahydrofolate + dUMP	-	Escherichia coli	7,8-dihydrofolate + dTMP	-	?
(R)-5,10-methylenetetrahydrofolate + dUMP	-	Escherichia coli	dihydrofolate + dTMP	-	?
5,10-methylene-5,6,7,8-tetrahydrofolate + dUMP	-	Escherichia coli	7,8-dihydrofolate + dTMP	-	?
5,10-methylene-5,6,7,8-tetrahydrofolate + dUMP	thymidylate synthase is the third enzyme in the methylene tetrahydrofolate cycle besides dihydrofolate reductase and serine hydroxymethyltransferase, in many organisms, thymidylate synthase is the only de novo source for dTMP required for DNA synthesis	Escherichia coli	7,8-dihydrofolate + dTMP	-	?

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism	Structure
0.016	-	dUMP	mutant K48Q	Escherichia coli

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Release 2023.1 (January 2023)