Cloned (Comment) | Organism |
---|---|
expressed in Escherichia coli BL21(lambdaDE3) | Pseudomonas putida |
Crystallization (Comment) | Organism |
---|---|
hanging drop method, PEG 4000 | Pseudomonas putida |
Protein Variants | Comment | Organism |
---|---|---|
R123A | no activity | Pseudomonas putida |
R123K | retains some activity | Pseudomonas putida |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
oxalate | competitive inhibitor | Pseudomonas putida |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.022 | - |
4-hydroxy-4-methyl-2-oxoglutarate | wild type protein, 1.0 mM Mn2+, pH 8.0, 25°C | Pseudomonas putida | |
0.036 | - |
oxaloacetate | wild type protein, 1.0 mM Mn2+, pH 8.0, 25°C; substrate inhibition constante 0.25 mM | Pseudomonas putida | |
0.071 | - |
2-hydroxy-4-oxopentanedioic acid | wild type protein, 1.0 mM Mn2+, pH 8.0, 25°C | Pseudomonas putida | |
0.15 | - |
2-hydroxy-4-oxopentanedioic acid | wild type protein, 1.0 mM Mg2+, pH 8.0, 25°C | Pseudomonas putida | |
0.26 | - |
4-hydroxy-4-methyl-2-oxoglutarate | wild type protein, 1.0 mM Mg2+, pH 8.0, 25°C | Pseudomonas putida | |
0.3 | - |
oxaloacetate | wild type protein, 1.0 mM Mg2+, pH 8.0, 25°C; substrate inhibition constante 0.37 mM | Pseudomonas putida | |
5.5 | - |
4-hydroxy-4-methyl-2-oxoglutarate | R123K mutant protein, pH 8.0, 25°C | Pseudomonas putida | |
8.8 | - |
4-hydroxy-2-oxopentanoate | wild type protein, 1.0 mM Mn2+, pH 8.0, 25°C | Pseudomonas putida | |
25 | - |
4-hydroxy-2-oxopentanoate | wild type protein, 1.0 mM Mg2+, pH 8.0, 25°C | Pseudomonas putida |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | Mn2+: 90.3% activity, Co2+: 64.7% activity, Zn2+: 49.6% activity | Pseudomonas putida |
Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
26000 | - |
6 * 26000, crystal structure, gel filtration and SDS-PAGE | Pseudomonas putida |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Pseudomonas putida | - |
- |
- |
Purification (Comment) | Organism |
---|---|
ion exchange chromatography, hydrophobic interaction chromatography, gel filtration | Pseudomonas putida |
Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|
37.7 | - |
wild type protein, pH 8.0, 25°C | Pseudomonas putida |
Storage Stability | Organism |
---|---|
-80°C, 20 mM sodium HEPES buffer, pH7.5, stable for 2 months | Pseudomonas putida |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
2-hydroxy-4-oxopentanedioic acid | - |
Pseudomonas putida | ? | - |
? | |
4-hydroxy-2-oxopentanoate | - |
Pseudomonas putida | ? | - |
? | |
4-hydroxy-4-methyl-2-oxoglutarate | - |
Pseudomonas putida | 2 pyruvate | - |
r | |
oxaloacetate | - |
Pseudomonas putida | ? | - |
? |
Subunits | Comment | Organism |
---|---|---|
homohexamer | 6 * 26000, crystal structure, gel filtration and SDS-PAGE | Pseudomonas putida |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.048 | - |
4-hydroxy-2-oxopentanoate | wild type protein, 1.0 mM Mg2+, pH 8.0, 25°C | Pseudomonas putida | |
0.058 | - |
2-hydroxy-4-oxopentanedioic acid | wild type protein, 1.0 mM Mg2+, pH 8.0, 25°C | Pseudomonas putida | |
0.099 | - |
4-hydroxy-4-methyl-2-oxoglutarate | R123K mutant protein, pH 8.0, 25°C | Pseudomonas putida | |
0.19 | - |
4-hydroxy-2-oxopentanoate | wild type protein, 1.0 mM Mn2+, pH 8.0, 25°C | Pseudomonas putida | |
0.25 | - |
2-hydroxy-4-oxopentanedioic acid | wild type protein, 1.0 mM Mn2+, pH 8.0, 25°C | Pseudomonas putida | |
1.52 | - |
oxaloacetate | wild type protein, 1.0 mM Mn2+, pH 8.0, 25°C | Pseudomonas putida | |
1.85 | - |
oxaloacetate | wild type protein, 1.0 mM Mg2+, pH 8.0, 25°C | Pseudomonas putida | |
13.7 | - |
4-hydroxy-4-methyl-2-oxoglutarate | wild type protein, 1.0 mM Mn2+, pH 8.0, 25°C | Pseudomonas putida | |
19.3 | - |
4-hydroxy-4-methyl-2-oxoglutarate | wild type protein, 1.0 mM Mg2+, pH 8.0, 25°C | Pseudomonas putida |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
additional information | - |
broad optimum (log KCAT) pH 8 - pH 10.5 | Pseudomonas putida |
pH Minimum | pH Maximum | Comment | Organism |
---|---|---|---|
6 | 10.5 | - |
Pseudomonas putida |
Ki Value [mM] | Ki Value maximum [mM] | Inhibitor | Comment | Organism | Structure |
---|---|---|---|---|---|
0.0017 | - |
oxalate | 1 mM MnCl, pH 8.0, 25°C | Pseudomonas putida | |
0.005 | - |
oxalate | 1 mM MgCl, pH 8.0, 25°C | Pseudomonas putida |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.0019 | - |
4-hydroxy-2-oxopentanoate | wild type protein, 1.0 mM Mg2+, pH 8.0, 25°C | Pseudomonas putida | |
0.022 | - |
4-hydroxy-2-oxopentanoate | wild type protein, 1.0 mM Mn2+, pH 8.0, 25°C | Pseudomonas putida | |
0.38 | - |
2-hydroxy-4-oxopentanedioic acid | wild type protein, 1.0 mM Mg2+, pH 8.0, 25°C | Pseudomonas putida | |
3.5 | - |
2-hydroxy-4-oxopentanedioic acid | wild type protein, 1.0 mM Mn2+, pH 8.0, 25°C | Pseudomonas putida | |
6.2 | - |
oxaloacetate | wild type protein, 1.0 mM Mg2+, pH 8.0, 25°C | Pseudomonas putida | |
74 | - |
4-hydroxy-4-methyl-2-oxoglutarate | wild type protein, 1.0 mM Mg2+, pH 8.0, 25°C | Pseudomonas putida | |
420 | - |
oxaloacetate | wild type protein, 1.0 mM Mn2+, pH 8.0, 25°C | Pseudomonas putida | |
620 | - |
4-hydroxy-4-methyl-2-oxoglutarate | wild type protein, 1.0 mM Mn2+, pH 8.0, 25°C | Pseudomonas putida |