Cloned (Comment) | Organism |
---|---|
expressed in Escherichia coli BL21(DE3) cells | Escherichia coli |
Crystallization (Comment) | Organism |
---|---|
mutant enzyme R395K bound to thiamine diphosphate, hanging drop vapor diffusion method, using 0.2 M ammonium acetate, 0.06 M magnesium formate, 3% (w/v) polyethylene glycol 3350, and 12% (w/v) polyethylene glycol 10000 in 0.1 M Tris-HCl (pH 7.5). Mutant enzyme R395A bound to thiamine diphosphate, hanging drop vapor diffusion method, using 30% (v/v) Jeffamine M-600 (pH 7.0), 11.2% (w/v) polyethylene glycol 3350 and 0.16 M magnesium formate in 0.1 M HEPES (pH 7.5). Mutant enzyme R413A bound to thiamine diphosphate, hanging drop vapor diffusion method, using 0.16 M magnesium formate, 1% (w/v) tacsimate (pH 7.0), 14% (w/v) polyethylene glycol 3350, and 2% (w/v) polyethylene glycol MME 5000 in 0.02 M HEPES (pH 7.0) | Escherichia coli |
Protein Variants | Comment | Organism |
---|---|---|
Q118A | the mutant is completely inactive | Escherichia coli |
R395A | the mutant shows a 160 and 1000fold decrease in kcat/Km value with respect to 2-oxoglutarate and isochorismate, respectively, compared to the wild type enzyme | Escherichia coli |
R395K | the mutant shows a 45 and 66fold decrease in kcat/Km value with respect to 2-oxoglutarate and isochorismate, respectively, compared to the wild type enzyme | Escherichia coli |
R395K/R413K | the mutant shows a 6300 and 1300fold decrease in kcat/Km value with respect to 2-oxoglutarate and isochorismate, respectively, compared to the wild type enzyme | Escherichia coli |
R413A | the mutant is completely inactive | Escherichia coli |
R413K | the mutant shows a 270 and 37fold decrease in kcat/Km value with respect to 2-oxoglutarate and isochorismate, respectively, compared to the wild type enzyme | Escherichia coli |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.00008 | - |
isochorismate | wild type enzyme, at pH 7.0 and 21°C | Escherichia coli | |
0.00035 | - |
isochorismate | mutant enzyme R413K, at pH 7.0 and 21°C | Escherichia coli | |
0.00082 | - |
isochorismate | mutant enzyme R395K/R413K, at pH 7.0 and 21°C | Escherichia coli | |
0.0022 | - |
isochorismate | mutant enzyme R395K, at pH 7.0 and 21°C | Escherichia coli | |
0.0029 | - |
2-oxoglutarate | wild type enzyme, at pH 7.0 and 21°C | Escherichia coli | |
0.016 | - |
isochorismate | mutant enzyme R395A, at pH 7.0 and 21°C | Escherichia coli | |
0.029 | - |
2-oxoglutarate | mutant enzyme R395A, at pH 7.0 and 21°C | Escherichia coli | |
0.067 | - |
2-oxoglutarate | mutant enzyme R395K, at pH 7.0 and 21°C | Escherichia coli | |
0.115 | - |
2-oxoglutarate | mutant enzyme R413K, at pH 7.0 and 21°C | Escherichia coli | |
0.172 | - |
2-oxoglutarate | mutant enzyme R395K/R413K, at pH 7.0 and 21°C | Escherichia coli |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | 5 mM used in assay conditions | Escherichia coli |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
isochorismate + 2-oxoglutarate | Escherichia coli | - |
5-enolpyruvoyl-6-hydroxy-2-succinyl-cyclohex-3-ene-1-carboxylate + CO2 | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Escherichia coli | P17109 | - |
- |
Purification (Comment) | Organism |
---|---|
DEAE column chromatography and Sephacryl S-200 gel filtration | Escherichia coli |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
isochorismate + 2-oxoglutarate | - |
Escherichia coli | 5-enolpyruvoyl-6-hydroxy-2-succinyl-cyclohex-3-ene-1-carboxylate + CO2 | - |
? |
Synonyms | Comment | Organism |
---|---|---|
2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate synthase | - |
Escherichia coli |
MenD | - |
Escherichia coli |
SEPHCHC synthase | - |
Escherichia coli |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.0025 | - |
isochorismate | mutant enzyme R395K/R413K, at pH 7.0 and 21°C | Escherichia coli | |
0.0027 | - |
2-oxoglutarate | mutant enzyme R395K/R413K, at pH 7.0 and 21°C | Escherichia coli | |
0.018 | - |
2-oxoglutarate | mutant enzyme R395A, at pH 7.0 and 21°C | Escherichia coli | |
0.038 | - |
isochorismate | mutant enzyme R413K, at pH 7.0 and 21°C | Escherichia coli | |
0.043 | - |
2-oxoglutarate | mutant enzyme R413K, at pH 7.0 and 21°C | Escherichia coli | |
0.065 | - |
isochorismate | mutant enzyme R395A, at pH 7.0 and 21°C | Escherichia coli | |
0.135 | - |
isochorismate | mutant enzyme R395K, at pH 7.0 and 21°C | Escherichia coli | |
0.143 | - |
2-oxoglutarate | mutant enzyme R395K, at pH 7.0 and 21°C | Escherichia coli | |
0.287 | - |
2-oxoglutarate | wild type enzyme, at pH 7.0 and 21°C | Escherichia coli | |
0.337 | - |
isochorismate | wild type enzyme, at pH 7.0 and 21°C | Escherichia coli |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
thiamine diphosphate | - |
Escherichia coli |
General Information | Comment | Organism |
---|---|---|
metabolism | the enzyme catalyzes an essential Stetter reaction in menaquinone (vitamin K2) biosynthesis via thiamine diphosphate (ThDP)-bound tetrahedral postdecarboxylation intermediates | Escherichia coli |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.016 | - |
2-oxoglutarate | mutant enzyme R395K/R413K, at pH 7.0 and 21°C | Escherichia coli | |
0.3 | - |
isochorismate | mutant enzyme R395K/R413K, at pH 7.0 and 21°C | Escherichia coli | |
0.37 | - |
2-oxoglutarate | mutant enzyme R413K, at pH 7.0 and 21°C | Escherichia coli | |
0.62 | - |
2-oxoglutarate | mutant enzyme R395A, at pH 7.0 and 21°C | Escherichia coli | |
2.2 | - |
2-oxoglutarate | mutant enzyme R395K, at pH 7.0 and 21°C | Escherichia coli | |
4 | - |
isochorismate | mutant enzyme R395A, at pH 7.0 and 21°C | Escherichia coli | |
6.2 | - |
isochorismate | mutant enzyme R395K, at pH 7.0 and 21°C | Escherichia coli | |
98.3 | - |
2-oxoglutarate | wild type enzyme, at pH 7.0 and 21°C | Escherichia coli | |
110 | - |
isochorismate | mutant enzyme R413K, at pH 7.0 and 21°C | Escherichia coli | |
400 | - |
isochorismate | wild type enzyme, at pH 7.0 and 21°C | Escherichia coli |