Any feedback?
Please rate this page
(literature.php)
(0/150)

BRENDA support

Literature summary 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 .
    View publication on PubMedView publication on EuropePMC

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.5.1.3 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
2.1.1.45 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

EC Number Protein Variants Comment Organism
1.5.1.3 additional information a helix deletion reduces the DHFR catalytic efficiency by 30fold Toxoplasma gondii
1.5.1.3 P292A site-directed mutagenesis, the mutation, reduces the DHFR catalytic efficiency by 7fold Toxoplasma gondii
1.5.1.3 W296A site-directed mutagenesis, reduces the DHFR catalytic efficiency by 100fold Toxoplasma gondii
2.1.1.45 S290G site-directed mutagenesis, the mutant shows 10fold reduced activity compared to the wild-type enzyme Toxoplasma gondii

Inhibitors

EC Number Inhibitors Comment Organism Structure
1.5.1.3 (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
1.5.1.3 N10-propargyl-5,8-dideazafolate PDDF, a TS folate inhibitor, binds at the TS active site Toxoplasma gondii
2.1.1.45 (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
2.1.1.45 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]

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

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+ Toxoplasma gondii
-
 5,6,7,8-tetrahydrofolate + NADP+
-
 r
2.1.1.45 5,10-methylenetetrahydrofolate + dUMP Toxoplasma gondii
-
 dihydrofolate + dTMP
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.5.1.3 Toxoplasma gondii Q07422
-
-
2.1.1.45 Toxoplasma gondii Q07422
-
-

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.5.1.3 7,8-dihydrofolate + NADPH + H+
-
 Toxoplasma gondii 5,6,7,8-tetrahydrofolate + NADP+
-
 r
1.5.1.3 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 ?
-
 ?
2.1.1.45 5,10-methylenetetrahydrofolate + dUMP
-
 Toxoplasma gondii dihydrofolate + dTMP
-
 ?
2.1.1.45 5,10-methylenetetrahydrofolate + dUMP dUMP substrate binding structure analysis Toxoplasma gondii dihydrofolate + dTMP
-
 ?
2.1.1.45 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

EC Number Subunits Comment Organism
1.5.1.3 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
2.1.1.45 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

EC Number Synonyms Comment Organism
1.5.1.3 bifunctional TS-DHFR
-
 Toxoplasma gondii
1.5.1.3 thymidylate synthase-dihydrofolate reductase
-
 Toxoplasma gondii
1.5.1.3 TS-DHFR
-
 Toxoplasma gondii
2.1.1.45 bifunctional TS-DHFR
-
 Toxoplasma gondii
2.1.1.45 thymidylate synthase-dihydrofolate reductase
-
 Toxoplasma gondii
2.1.1.45 TS-DHFR
-
 Toxoplasma gondii

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.5.1.3 7.8
-
 assay at Toxoplasma gondii
2.1.1.45 7.8
-
 assay at Toxoplasma gondii

Cofactor

EC Number Cofactor Comment Organism Structure
1.5.1.3 NADP+
-
 Toxoplasma gondii
1.5.1.3 NADPH binding structure analysis, residues interacting with the substrate NADPH include the conserved residues A10, I17, R81, T83, S103, and G153 and the nonconserved residue A154 Toxoplasma gondii

General Information

EC Number General Information Comment Organism
1.5.1.3 additional information DHFR domain structure analysis, the domain consists of 252 residues from the N-terminus to the start of the junctional region, overview Toxoplasma gondii
1.5.1.3 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
2.1.1.45 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
2.1.1.45 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