Literature summary for 2.1.1.45 extracted from

Sharma, H.; Landau, M.; Vargo, M.; Spasov, K.; Anderson, K.
First three-dimensional structure of Toxoplasma gondii thymidylate synthase-dihydrofolate reductase Insights for catalysis, interdomain interactions, and substrate channeling (2013), Biochemistry, 52, 7305-7317 .

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant wild-type TS-DHFR enzyme and truncated TS-DHFR mutant lacking the surface loops, complexed with dUMP and NADPH, as well as with inhibitors methotrexate and N10-propargyl-5,8-dideazafolate, 10 mg/ml protein with 10 mM of each ligand is mixed with mixed with 18% PEG 3350, 0.1 M potassium formate in a 1:1 ratio, 4-6 days, X-ray diffraction structure determination and analysis at 3.7 A and 2.2 A resolution, respectively	Toxoplasma gondii

Crystallization (Comment)

Organism

purified recombinant wild-type TS-DHFR enzyme and truncated TS-DHFR mutant lacking the surface loops, complexed with dUMP and NADPH, as well as with inhibitors methotrexate and N10-propargyl-5,8-dideazafolate, 10 mg/ml protein with 10 mM of each ligand is mixed with mixed with 18% PEG 3350, 0.1 M potassium formate in a 1:1 ratio, 4-6 days, X-ray diffraction structure determination and analysis at 3.7 A and 2.2 A resolution, respectively

Toxoplasma gondii

Protein Variants

Protein Variants	Comment	Organism
S290G	site-directed mutagenesis, the mutant shows 10fold reduced activity compared to the wild-type enzyme	Toxoplasma gondii

Inhibitors

Inhibitors	Comment	Organism	Structure
(2S)-2-[(4-([(2,4-diaminopteridin-6-yl)methyl](methyl)amino)phenyl)formamido]pentanedioic acid	i.e, methotrexate, A DHFR inhibitor, binds at the DHFR active site	Toxoplasma gondii
N10-propargyl-5,8-dideazafolate	PDDF, a TS folate inhibitor, binds at the TS active site. Residues that are involved in binding the folate analogue PDDF include I402, D513, L516, F520, as well as the nonconserved R603 and conserved M608 located on the C-terminal tail of the TS-domain	Toxoplasma gondii

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
additional information	-	additional information	single turnover, stopped-flow, and steady-state kinetics	Toxoplasma gondii

Natural Substrates/ Products (Substrates)

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

Organism

Organism	UniProt	Comment	Textmining
Toxoplasma gondii	Q07422	-	-

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
5,10-methylenetetrahydrofolate + dUMP	-	Toxoplasma gondii	dihydrofolate + dTMP	-	?
5,10-methylenetetrahydrofolate + dUMP	dUMP substrate binding structure analysis	Toxoplasma gondii	dihydrofolate + dTMP	-	?
additional information	molecular mechanism of the distinct differences in substrate channeling behavior between Toxoplasma gondii TS-DHFR and Cryptosporidium hominis TS-DHFR, overview	Toxoplasma gondii	?	-	?

Subunits

Subunits	Comment	Organism
More	three-dimensional structures of Toxoplasma gondii enzyme TS-DHFR at 3.7 A and of a loop truncated TS-DHFR enzyme, removing several flexible surface loops in the DHFR domain, improving resolution to 2.2 A. The TS-DHFR homodimer includes a junctional region containing a linked crossover helix between the DHFR domains of the two adjacent monomers, a long linker connecting the TS and DHFR domains, and a DHFR domain that is positively charged, importance of this region not only in DHFR catalysis but also in modulating the distal TS activity suggests a role for TS-DHFR interdomain interactions. The interactions between the TS and the DHFR domains within the same monomer are comprised of both electrostatic and hydrophobic interactions that are predominately hydrophobic, these include hydrophobic contacts between F231, F319, and M297 as well as P242, I573, and V596	Toxoplasma gondii

Synonyms

Synonyms	Comment	Organism
bifunctional TS-DHFR	-	Toxoplasma gondii
thymidylate synthase-dihydrofolate reductase	-	Toxoplasma gondii
TS-DHFR	-	Toxoplasma gondii

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.8	-	assay at	Toxoplasma gondii

General Information

General Information	Comment	Organism
additional information	TS domain structure analysis, overview. The TS domain of the enzyme forms the largest portion of the dimerization interface mainly through a five-stranded beta-sheet from each monomer. The catalytic cysteine 489 is responsible for catalysis, te active site is comprised of residues from both monomers. Several conserved arginines bind dUMP, i.e. R344, R510 as well as R469 and R470 from the adjacent monomer	Toxoplasma gondii
physiological function	several parasitic protozoa, including Toxoplasma gondii, contain a unique bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) having the catalytic activities contained on a single polypeptide chain in contrast to the human enzyme. Three-dimensional structures of Toxoplasma gondii enzyme TS-DHFR and of a loop truncated TS-DHFR enzyme, removing several flexible surface loops in the DHFR domain, shows that the TS-DHFR homodimer includes a junctional region containing a linked crossover helix between the DHFR domains of the two adjacent monomers, a long linker connecting the TS and DHFR domains, and a DHFR domain that is positively charged. The crystal structure suggests that the positively charged DHFR domain governs this electrostatically mediated movement of dihydrofolate, preventing release from the enzyme. Importance of this region not only in DHFR catalysis but also in modulating the distal TS activity suggests a role for TS-DHFR interdomain interactions	Toxoplasma gondii