Crystallization (Comment) | Organism |
---|---|
to 2.35 A resolution, in complex with thiamine diphosphate and Mn2+. basic residues Arg32, Arg106, Arg409, Arg428, and Lys299 interact with carboxylate and hydroxyl groups to align substrates for catalysis in combination with a cluster of the non-polar residues Ile489, Phe490, and Leu493 on one side of the active site. Arg409 plays a significant role in binding both substrates while Arg428 contributes mainly to binding of 2-oxoglutarate. Arg32 and in particular Arg106 are critical for recognition of isochorismate | Bacillus subtilis |
Protein Variants | Comment | Organism |
---|---|---|
F490A | 0.6% of wild-type activity | Bacillus subtilis |
I489A | 1% of wild-type activity | Bacillus subtilis |
K299A | 150% of wild-type activity | Bacillus subtilis |
L493A | 80% of wild-type activity | Bacillus subtilis |
R106A | mutation in the second subunit forming the active site, 10% of wild-type activity | Bacillus subtilis |
R32A | mutation in the second subunit forming the active site, 30% of wild-type activity | Bacillus subtilis |
R409A | 3% of wild-type activity | Bacillus subtilis |
R428A | 1% of wild-type activity | Bacillus subtilis |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.0077 | - |
2-oxoglutarate | mutant R106A, pH 7.8, 23°C | Bacillus subtilis | |
0.018 | - |
2-oxoglutarate | mutant L493A, pH 7.8, 23°C | Bacillus subtilis | |
0.022 | - |
2-oxoglutarate | mutant K299A, pH 7.8, 23°C | Bacillus subtilis | |
0.022 | - |
2-oxoglutarate | mutant R32A, pH 7.8, 23°C | Bacillus subtilis | |
0.022 | - |
2-oxoglutarate | wild-type, pH 7.8, 23°C | Bacillus subtilis | |
0.159 | - |
2-oxoglutarate | mutant R409A, pH 7.8, 23°C | Bacillus subtilis | |
0.274 | - |
2-oxoglutarate | mutant F490A, pH 7.8, 23°C | Bacillus subtilis | |
0.346 | - |
2-oxoglutarate | mutant I489A, pH 7.8, 23°C | Bacillus subtilis | |
0.514 | - |
2-oxoglutarate | mutant R428A, pH 7.8, 23°C | Bacillus subtilis |
Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
290000 | - |
gel filtration | Bacillus subtilis |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Bacillus subtilis | P23970 | - |
- |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
isochorismate + 2-oxoglutarate = 5-enolpyruvoyl-6-hydroxy-2-succinyl-cyclohex-3-ene-1-carboxylate + CO2 | two-stage mechanism that is primarily driven by the chemical properties of the cofactor thiamine diphosphate | Bacillus subtilis |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
isochorismate + 2-oxoglutarate | - |
Bacillus subtilis | 5-enolpyruvoyl-6-hydroxy-2-succinyl-cyclohex-3-ene-1-carboxylate + CO2 | - |
? |
Subunits | Comment | Organism |
---|---|---|
tetramer | dimer of dimer, crystallization data, 4 x 64100, calculated | Bacillus subtilis |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.01 | - |
2-oxoglutarate | mutant R106A, pH 7.8, 23°C | Bacillus subtilis | |
0.02 | - |
2-oxoglutarate | mutant F490A, pH 7.8, 23°C | Bacillus subtilis | |
0.05 | - |
2-oxoglutarate | mutant R409A, pH 7.8, 23°C | Bacillus subtilis | |
0.06 | - |
2-oxoglutarate | mutant R428A, pH 7.8, 23°C | Bacillus subtilis | |
0.08 | - |
2-oxoglutarate | mutant R32A, pH 7.8, 23°C | Bacillus subtilis | |
0.1 | - |
2-oxoglutarate | mutant I489A, pH 7.8, 23°C | Bacillus subtilis | |
0.17 | - |
2-oxoglutarate | mutant L493A, pH 7.8, 23°C | Bacillus subtilis | |
0.23 | - |
2-oxoglutarate | wild-type, pH 7.8, 23°C | Bacillus subtilis | |
0.37 | - |
2-oxoglutarate | mutant K299A, pH 7.8, 23°C | Bacillus subtilis |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
thiamine diphosphate | in absence of any residue that can act as a general acid/base the cofactor N4' atom is a critical component of the mechanism | Bacillus subtilis |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.07 | - |
2-oxoglutarate | mutant F490A, pH 7.8, 23°C | Bacillus subtilis | |
0.12 | - |
2-oxoglutarate | mutant R428A, pH 7.8, 23°C | Bacillus subtilis | |
0.27 | - |
2-oxoglutarate | mutant I489A, pH 7.8, 23°C | Bacillus subtilis | |
1.5 | - |
2-oxoglutarate | mutant R106A, pH 7.8, 23°C | Bacillus subtilis | |
2.7 | - |
2-oxoglutarate | mutant R409A, pH 7.8, 23°C | Bacillus subtilis | |
3.7 | - |
2-oxoglutarate | mutant R32A, pH 7.8, 23°C | Bacillus subtilis | |
8.3 | - |
2-oxoglutarate | mutant L493A, pH 7.8, 23°C | Bacillus subtilis | |
10.7 | - |
2-oxoglutarate | wild-type, pH 7.8, 23°C | Bacillus subtilis | |
16.7 | - |
2-oxoglutarate | mutant K299A, pH 7.8, 23°C | Bacillus subtilis |