Crystallization (Comment) | Organism |
---|---|
the covalent adduct formed between irreversible mechanism based inhibitors, 3-methylene-4-penten-1-yl diphosphate or 3-oxiranyl-3-buten-1-yl diphosphate, and the flavin cofactor are investigated by X-ray crystallography and UV-visible spectroscopy. Both the crystal structures of enzyme binding the flavin-inhibitor adduct and the UV-visible spectra of the adducts indicate that the covalent bond is formed at C4a of flavin rather than at N5. The high-resolution crystal structures of enzyme-substrate complexes and the kinetic studies of new mutants confirm that only the flavin cofactor can catalyze protonation of the substrates and suggest that N5 of flavin is most likely to be involved in proton transfer | Saccharolobus shibatae |
Protein Variants | Comment | Organism |
---|---|---|
Q160E | 10-fold decrease in kcat/Km | Saccharolobus shibatae |
Q160E | the mutant shows a 10fold decrease in kcat/Km | Saccharolobus shibatae |
Q160H | 23-fold decrease in kcat/Km | Saccharolobus shibatae |
Q160H | the mutant shows a 23fold decrease in kcat/Km | Saccharolobus shibatae |
Q160K | 130-fold decrease in kcat/Km | Saccharolobus shibatae |
Q160K | the mutant shows a 130fold decrease in kcat/Km | Saccharolobus shibatae |
Q160L | 28-fold decrease in kcat/Km | Saccharolobus shibatae |
Q160L | the mutant shows a 28fold decrease in kcat/Km | Saccharolobus shibatae |
Q160N | 150-fold decrease in kcat, kcat/Km decreases 66-fold | Saccharolobus shibatae |
Q160N | the mutant shows a 150fold decrease in kcat, although kcat/Km only decreases 66fold | Saccharolobus shibatae |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
3-methylene-4-penten-1-yl diphosphate | irreversible inhibition; the covalent adduct formed between irreversible mechanism based inhibitors, 3-methylene-4-penten-1-yl diphosphate or 3-oxiranyl-3-buten-1-yl diphosphate, and the flavin cofactor are investigated by X-ray crystallography and UV-visible spectroscopy. Both the crystal structures of enzyme binding the flavin-inhibitor adduct and the UV-visible spectra of the adducts indicate that the covalent bond is formed at C4a of flavin rather than at N5 | Saccharolobus shibatae | |
3-oxiranyl-3-buten-1-yl diphosphate | irreversible inhibition; the covalent adduct formed between irreversible mechanism based inhibitors, 3-methylene-4-penten-1-yl diphosphate or 3-oxiranyl-3-buten-1-yl diphosphate, and the flavin cofactor are investigated by X-ray crystallography and UV-visible spectroscopy. Both the crystal structures of enzyme binding the flavin-inhibitor adduct and the UV-visible spectra of the adducts indicate that the covalent bond is formed at C4a of flavin rather than at N5 | Saccharolobus shibatae |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.0015 | - |
isopentenyl diphosphate | pH 6.0, 60°C, mutant enzyme Q160L | Saccharolobus shibatae | |
0.00152 | - |
isopentenyl diphosphate | mutant enzyme Q160L, in 50 mM MOPS-NaOH, pH 6.0, at 60°C | Saccharolobus shibatae | |
0.0032 | - |
isopentenyl diphosphate | pH 6.0, 60°C, mutant enzyme Q160N | Saccharolobus shibatae | |
0.00323 | - |
isopentenyl diphosphate | mutant enzyme Q160N, in 50 mM MOPS-NaOH, pH 6.0, at 60°C | Saccharolobus shibatae | |
0.0071 | - |
isopentenyl diphosphate | pH 6.0, 60°C, mutant enzyme Q160H | Saccharolobus shibatae | |
0.00713 | - |
isopentenyl diphosphate | mutant enzyme Q160H, in 50 mM MOPS-NaOH, pH 6.0, at 60°C | Saccharolobus shibatae | |
0.00739 | - |
isopentenyl diphosphate | wild type enzyme, in 50 mM MOPS-NaOH, pH 6.0, at 60°C | Saccharolobus shibatae | |
0.0074 | - |
isopentenyl diphosphate | pH 6.0, 60°C, wild-type enzyme | Saccharolobus shibatae | |
0.00826 | - |
isopentenyl diphosphate | mutant enzyme Q160E, in 50 mM MOPS-NaOH, pH 6.0, at 60°C | Saccharolobus shibatae | |
0.0083 | - |
isopentenyl diphosphate | pH 6.0, 60°C, mutant enzyme Q160E | Saccharolobus shibatae |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
isopentenyl diphosphate | Saccharolobus shibatae | - |
dimethylallyl diphosphate | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Saccharolobus shibatae | - |
- |
- |
Saccharolobus shibatae | P61615 | - |
- |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
isopentenyl diphosphate | - |
Saccharolobus shibatae | dimethylallyl diphosphate | - |
? | |
isopentenyl diphosphate | a mechanism is proposed where the reduced flavin cofactor acts as a general acid/base catalyst and helps stabilize the carbocationic intermediate formed by protonation | Saccharolobus shibatae | dimethylallyl diphosphate | - |
? |
Synonyms | Comment | Organism |
---|---|---|
Idi-2 | - |
Saccharolobus shibatae |
isopentenyl diphosphate isomerase | - |
Saccharolobus shibatae |
type-2 isopentenyl diphosphate isomerase | - |
Saccharolobus shibatae |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
60 | - |
assay at | Saccharolobus shibatae |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.000197 | - |
isopentenyl diphosphate | pH 6.0, 60°C, mutant enzyme Q160N | Saccharolobus shibatae | |
0.00022 | - |
isopentenyl diphosphate | pH 6.0, 60°C, mutant enzyme Q160L | Saccharolobus shibatae | |
0.001 | - |
isopentenyl diphosphate | pH 6.0, 60°C, mutant enzyme Q160H | Saccharolobus shibatae | |
0.003 | - |
isopentenyl diphosphate | pH 6.0, 60°C, mutant enzyme Q160E | Saccharolobus shibatae | |
0.03 | - |
isopentenyl diphosphate | pH 6.0, 60°C, wild-type enzyme | Saccharolobus shibatae | |
197 | - |
isopentenyl diphosphate | mutant enzyme Q160N, in 50 mM MOPS-NaOH, pH 6.0, at 60°C | Saccharolobus shibatae | |
221 | - |
isopentenyl diphosphate | mutant enzyme Q160L, in 50 mM MOPS-NaOH, pH 6.0, at 60°C | Saccharolobus shibatae | |
1210 | - |
isopentenyl diphosphate | mutant enzyme Q160H, in 50 mM MOPS-NaOH, pH 6.0, at 60°C | Saccharolobus shibatae | |
3280 | - |
isopentenyl diphosphate | mutant enzyme Q160E, in 50 mM MOPS-NaOH, pH 6.0, at 60°C | Saccharolobus shibatae | |
29900 | - |
isopentenyl diphosphate | wild type enzyme, in 50 mM MOPS-NaOH, pH 6.0, at 60°C | Saccharolobus shibatae |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
6 | - |
assay at | Saccharolobus shibatae |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
FMN | a mechanism is proposed where the reduced flavin cofactor acts as a general acid/base catalyst and helps stabilize the carbocationic intermediate formed by protonation | Saccharolobus shibatae |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.031 | - |
isopentenyl diphosphate | mutant enzyme Q160K, in 50 mM MOPS-NaOH, pH 6.0, at 60°C | Saccharolobus shibatae | |
0.061 | - |
isopentenyl diphosphate | mutant enzyme Q160N, in 50 mM MOPS-NaOH, pH 6.0, at 60°C | Saccharolobus shibatae | |
0.145 | - |
isopentenyl diphosphate | mutant enzyme Q160L, in 50 mM MOPS-NaOH, pH 6.0, at 60°C | Saccharolobus shibatae | |
0.17 | - |
isopentenyl diphosphate | mutant enzyme Q160H, in 50 mM MOPS-NaOH, pH 6.0, at 60°C | Saccharolobus shibatae | |
0.397 | - |
isopentenyl diphosphate | mutant enzyme Q160E, in 50 mM MOPS-NaOH, pH 6.0, at 60°C | Saccharolobus shibatae | |
4.05 | - |
isopentenyl diphosphate | wild type enzyme, in 50 mM MOPS-NaOH, pH 6.0, at 60°C | Saccharolobus shibatae |