Cloned (Comment) | Organism |
---|---|
wild-type and mutant ThyX proteins expressed in either Escherichia coli DH5alpha (deltathyA) or BL21 | Paramecium bursaria Chlorella virus-1 |
Crystallization (Comment) | Organism |
---|---|
crystal structure of the ThyX protein complexed to its FAD cofactor and solved by molecular replacement, to 2.3 A resolution, crystals belong to the P21212 space group with predicted two molecules per asymmetric unit and a solvent content of 51% | Paramecium bursaria Chlorella virus-1 |
Protein Variants | Comment | Organism |
---|---|---|
E190G | does not confer thymidine-independent growth to an Escherichia coli strain lacking thymidylate synthase ThyA, is capable of binding FAD at a wild-type level, but lacks detectable oxidation and deprotonation | Paramecium bursaria Chlorella virus-1 |
H177K | does not confer thymidine-independent growth to an Escherichia coli strain lacking thymidylate synthase ThyA, 9.5% oxidation activity | Paramecium bursaria Chlorella virus-1 |
H177Q | confers thymidine-independent growth to an Escherichia coli strain lacking thymidylate synthase ThyA, 31% oxidation activity | Paramecium bursaria Chlorella virus-1 |
H53K | does not confer thymidine-independent growth to an Escherichia coli strain lacking thymidylate synthase ThyA, produces insoluble protein | Paramecium bursaria Chlorella virus-1 |
H53Q | does not confer thymidine-independent growth to an Escherichia coli strain lacking thymidylate synthase ThyA, produces insoluble protein | Paramecium bursaria Chlorella virus-1 |
H79K | does not confer thymidine-independent growth to an Escherichia coli strain lacking thymidylate synthase ThyA | Paramecium bursaria Chlorella virus-1 |
H79Q | confers thymidine-independent growth to an Escherichia coli strain lacking thymidylate synthase ThyA, 94% oxidation activity | Paramecium bursaria Chlorella virus-1 |
R182A | does not confer thymidine-independent growth to an Escherichia coli strain lacking thymidylate synthase ThyA, does not copurify with oxidized FAD, but is able to oxidize NADPH in the presence of 0.4 mM FAD | Paramecium bursaria Chlorella virus-1 |
R90A | does not confer thymidine-independent growth to an Escherichia coli strain lacking thymidylate synthase ThyA, looses 44% of its oxidation activity and shows no measurable deprotonation activity | Paramecium bursaria Chlorella virus-1 |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
5,10-methylenetetrahydrofolate | - |
Paramecium bursaria Chlorella virus-1 | |
diethyldicarbonate | decreases ThyX deprotonation activity at least 20fold, is partially reversible with hydroxylamine treatment | Paramecium bursaria Chlorella virus-1 |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Paramecium bursaria Chlorella virus-1 | - |
- |
- |
Purification (Comment) | Organism |
---|---|
by nickel chromatography and gel filtration, more than 95% pure | Paramecium bursaria Chlorella virus-1 |
Storage Stability | Organism |
---|---|
-80°C, 50 mM HEPES buffer, pH 7.0, 10% glycerol | Paramecium bursaria Chlorella virus-1 |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
5,10-methylenetetrahydrofolate + dUMP + FADH2 | - |
Paramecium bursaria Chlorella virus-1 | dTMP + tetrahydrofolate + FAD | - |
? |
Subunits | Comment | Organism |
---|---|---|
homotetramer | crystallographic studies | Paramecium bursaria Chlorella virus-1 |
Synonyms | Comment | Organism |
---|---|---|
flavin dependent thymidylate synthase | - |
Paramecium bursaria Chlorella virus-1 |
ThyX | - |
Paramecium bursaria Chlorella virus-1 |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
FAD | - |
Paramecium bursaria Chlorella virus-1 | |
NADPH | residue His53 is crucial for NADPH oxidation, is located in the vicinity of the redox active N-5 atom of the FAD ring system | Paramecium bursaria Chlorella virus-1 |
IC50 Value | IC50 Value Maximum | Comment | Organism | Inhibitor | Structure |
---|---|---|---|---|---|
0.016 | - |
wild-type | Paramecium bursaria Chlorella virus-1 | 5,10-methylenetetrahydrofolate | |
0.048 | - |
R182A mutant | Paramecium bursaria Chlorella virus-1 | 5,10-methylenetetrahydrofolate |