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2.1.1.45: thymidylate synthase

This is an abbreviated version!
For detailed information about thymidylate synthase, go to the full flat file.

Word Map on EC 2.1.1.45

Reaction

5,10-methylenetetrahydrofolate
+
dUMP
=
dihydrofolate
 +
dTMP

Synonyms

BgDHFR-TS, bifunctional TS-DHFR, DFR-TS, DHFR-TS, DHFR–TS, dihydrofolate reductase-thymidylate synthase, dTMP synthase, HTS, human TS, LBRM_06_0830, methylenetetrahydrofolate:dUMP C-methyltransferase, More, Thy1, ThyA, thymidylate synthase, thymidylate synthase A, thymidylate synthase-dihydrofolate reductase, thymidylate synthetase, ThyX, TMP synthetase, TMPS, TS, TS-DHFR, TSase, TYMS, Y110A7A.4

ECTree

     2 Transferases
         2.1 Transferring one-carbon groups
             2.1.1 Methyltransferases
                2.1.1.45 thymidylate synthase

Crystallization

Crystallization on EC 2.1.1.45 - thymidylate synthase

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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
co-crystallization of the purified enzyme with inhibitor 2-amino-4-oxo-4,7-dihydro-pyrrolo[2,3-d]pyrimidine-methyl-phenyl-L-glutamic acid and FdUMP in the TS site and NADPH and methotrexate in the DHFR site, X-ray diffraction structure determination and analysis at 3.45 A resolution, PDB ID 4Q0D
in complex with inhibitor 2-amino-4-oxo-4,7-dihydro-pyrrolo[2,3-d]pyrimidine-methyl-phenyl-L-glutamic acid
-
structures in complex with inhibitors. Active site rigidity is a driving force in determining inhibitor selectivity
in complex with synthesized inhibitors and with methotrexate
crystallized in the I2(1)3 space group
crystals grown by the hanging drop vapor diffusion method
crystals of TS apoprotein grown using hanging drop method of vapor diffusion, cubic Laue group m3 with a = 133 A
-
determination of chemical shifts of the apoenzyme (lig0), the saturated holoenzyme (lig2), and the typically elusive singly bound (lig1) states. The two active sites in TS communicate with one another by using the intervening beta-sheet that also forms the dimer interface. The active sites have minimal communication in the lig0 state, but rather a network of correlated motions involving the two active sites is triggered by the first diligand binding event and amplified upon binding the second. Contacts between the diligand and a right-handed beta-bulge feature of this sheet are likely the triggering events
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
molecular modeling of complex with inhibitor N-[4-[(2-amino-6-methyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-5-yl)sulfanyl]benzoyl]-L-glutamic acid. The 6-methyl moiety of of the inhibitor makes important hydrophobic contacts with Trp109 and also serves to lock the 5-position side chain into favorable, low-energy conformations for thymidlyate synthase binding
-
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
in complex with co-factor FAD and substrate dUMP at 2.5 A resolution. Structure consists of a 1.5 tetramer of ThyX with a total of 1248 residues, six FAD and six dUMP molecules in an asymmetric unit
apo-enzyme crystallized in the active form with sulfate ions
crystals grown by vapor diffusion method in hanging drop setup
-
grown in low-salt conditions, 100 mM Tris, pH 9.0, 20 mM beta-mercaptoethanol, 3 mM KH2PO4, and 10-20% PEG 4K, by hanging drop diffusion at 4 °C
hanging drop vapor diffusion method, using 2.0 M ammonium sulfate, 0.1 M Tris, pH 8.5, and 20 mM beta-mercaptoethanol
hanging-drop vapour diffusion method
in complex with inhibitor 1,3-propanediphosphonic acid, to 2.5 A resolution
in-silico docking analysis of phytoconstituents from Crocus sativus, Curcuma longa, Cassia occidentalis and Moringa oleifera
ligand-free, binary and ternary complex structures with phosphate ions and dUMP
molecular docking of inhibitors
molecular modeling of complex with inhibitor N-[4-[(2-amino-6-methyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-5-yl)sulfanyl]benzoyl]-L-glutamic acid. The 6-methyl moiety of of the inhibitor makes important hydrophobic contacts with Trp109 and also serves to lock the 5-position side chain into favorable, low-energy conformations for thymidlyate synthase binding
mutant K47A in complex with inhibitor CQLYQRSG
mutant P254S
-
V3L, V3F and V3Y mutant enzymes, hanging drop vapor diffusion method, using 100 mM Tris-base, pH 8.5, 20 mM 2-mercaptoethanol, 32-40% (w/v) ammonium sulfate and 16-24% (w/v) polyethylene glycol 4000
-
wild-type and mutant R163K. Data show that all subunits of the mutant are in the active conformation, while wild-type crystallizes as the inactive conformer. Structure reveals differences in the environment of catalytic residue Cys195
in complex with substrate dUMP and antifolate inhibitor raltitrexed, to 1.74 A resolution.The structure reveals a well-ordered segment of 13 N-terminal amino acids, whose ordered conformation is stabilized due to specific crystal packing.The structure consists of two homodimers, differing in conformation, one being more closed (dimer AB) and thus supporting tighter binding of ligands, and the other being more open (dimer CD) and thus allowing weaker binding of ligands. Conformational changes lead to a ligand-induced closing of the active site cleft
in complexes with sulfate anion, 2'-deoxyuridine 50-monophosphate and 5-fluorodUMP and N5,10-methylenetetrahydrofolate. The loop 175-191, homologous to human TS loop 181-197, populates the active conformer, with catalytic Cys 189 buried in the active site and directed toward C(6) of the pyrimidine ring of dUMP/FdUMP
crystals belong to space group P2(1)2(1)2(1), a = 54.05 A, b = 66.16 A and c = 178.76 A
molecular modeling of complex with inhibitor N-[4-[(2-amino-6-methyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-5-yl)sulfanyl]benzoyl]-L-glutamic acid. The 6-methyl moiety of of the inhibitor makes important hydrophobic contacts with Trp109 and also serves to lock the 5-position side chain into favorable, low-energy conformations for thymidlyate synthase binding
-
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
in complexes with the dihydrotriazine-based or quinazoline-based antifolates C-448, cycloguanil, and Q-8, microbatch method, using 0.1-0.2 M ammonium acetate, 20-22% (w/v) PEG 4000 and 0.1 M sodium citrate buffer pH 5.6
-
the enzyme in folate-free state, in complex with trimetrexate, and in complex with methotrexate is crystallized by the hanging drop vapor diffusion method, using 16-20% (v/v) 5-methylpentane-2,3-diol, 8-12% (w/v) PEG 4000, 0.1 M potassium citrate pH 6.0, or 1.5-1.7 M ammonium sulfate, 0.01 M magnesium chloride, 5% (w/v) ethylene glycol, 0.05 M Tris pH 7.5, or 17% (v/v) 5-methylpentane-2,3-diol, 10% (w/v) PEG 4000, 0.1 M potassium citrate pH 6.0