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Literature summary for 4.1.3.1 extracted from
- Residues Asn214, Gln211, Glu219 and Gln221 contained in the subfamily 3 catalytic signature of the isocitrate lyase from Pseudomonas aeruginosa are involved in its catalytic and thermal properties (2013), World J. Microbiol. Biotechnol., 29, 991-999.
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| gene aceA, phylogenetic analysis, expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) | Pseudomonas aeruginosa |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| E219A | site-directed mutagenesis of the highly conserved residue in the ICL subfamily 3 catalytic pattern, the mutant enzyme shows reduced activity compared to the wild-type enzyme | Pseudomonas aeruginosa |
| N214D | site-directed mutagenesis of the highly conserved residue in the ICL subfamily 3 catalytic pattern, the mutant enzyme shows 10fold reduced activity compared to the wild-type enzyme | Pseudomonas aeruginosa |
| Q211H | site-directed mutagenesis of the highly conserved residue in the ICL subfamily 3 catalytic pattern, the mutant enzyme shows reduced activity and elevated temperature optimum compared to the wild-type enzyme | Pseudomonas aeruginosa |
| Q221K | site-directed mutagenesis of the highly conserved residue in the ICL subfamily 3 catalytic pattern, the mutant enzyme shows reduced activity and elevated temperature optimum compared to the wild-type enzyme | Pseudomonas aeruginosa |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| additional information | - |
additional information | Michaelis-Menten kinetics of wild-type and mutant enzyme's overview | Pseudomonas aeruginosa | |
| 0.25 | - |
isocitrate | recombinant His-tagged mutant N214D, pH 7.8, 30°C | Pseudomonas aeruginosa |  |
| 0.29 | - |
isocitrate | recombinant His-tagged mutant Q211H, pH 7.8, 30°C | Pseudomonas aeruginosa | |
| 0.44 | - |
isocitrate | recombinant His-tagged wild-type enzyme, pH 7.8, 30°C | Pseudomonas aeruginosa | |
| 0.47 | - |
isocitrate | recombinant His-tagged mutant Q221K, pH 7.8, 30°C | Pseudomonas aeruginosa | |
| 0.94 | - |
isocitrate | recombinant His-tagged mutant E219A, pH 7.8, 30°C | Pseudomonas aeruginosa | |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| isocitrate | Pseudomonas aeruginosa | - |
succinate + glyoxylate | - |
? | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Pseudomonas aeruginosa | Q9I0K4 | gene aceA | - |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| ecombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography | Pseudomonas aeruginosa |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| isocitrate | - |
Pseudomonas aeruginosa | succinate + glyoxylate | - |
? | |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 30 | - |
wild-type enzyme and mutant E219A | Pseudomonas aeruginosa |
| 36 | - |
mutant Q221K | Pseudomonas aeruginosa |
| 39 | - |
mutant Q211H | Pseudomonas aeruginosa |
Temperature Stability [°C]
| Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 45 | - |
purified enzyme, half-life of the wild-type enzyme is 40 min, half-lives of mutants E219A, Q211H, and Q221K are 25 min, 12 min, and 15 min | Pseudomonas aeruginosa |
Turnover Number [1/s]
| Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.56 | - |
isocitrate | recombinant His-tagged mutant N214D, pH 7.8, 30°C | Pseudomonas aeruginosa | |
| 3.25 | - |
isocitrate | recombinant His-tagged mutant Q221K, pH 7.8, 30°C | Pseudomonas aeruginosa | |
| 3.46 | - |
isocitrate | recombinant His-tagged mutant Q211H, pH 7.8, 30°C | Pseudomonas aeruginosa | |
| 4.63 | - |
isocitrate | recombinant His-tagged mutant E219A, pH 7.8, 30°C | Pseudomonas aeruginosa | |
| 9.81 | - |
isocitrate | recombinant His-tagged wild-type enzyme, pH 7.8, 30°C | Pseudomonas aeruginosa | |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 7.6 | - |
- |
Pseudomonas aeruginosa |
General Information
| General Information | Comment | Organism |
|---|---|---|
| evolution | the ICL family includes five subfamilies, and the 2-methylisocitrate lyase (MICL) family. The ICL from Pseudomonas aeruginosa (ICL-Pa) is identified in a different ICL node (subfamily 3) than other Pseudomonas ICL enzymes, phylogenetic analysis | Pseudomonas aeruginosa |
| additional information | conserved residues in the subfamily 3 signature of ICL-Pa play important roles in catalysis and thermostability and are likely associated with the catalytic loop structural conformation. Three-dimensional structural homology modeling of ICL-Pa wild-type and mutants, structure comparisons, active site modeling, overview. Residue N214 plays an essential role in ICL-Pa catalytic activity | Pseudomonas aeruginosa |
| physiological function | isocitrate lyase plays an important role in the ability of Pseudomonas aeruginosa to grow on fatty acids, acetate, acyclic terpenes, and amino acids | Pseudomonas aeruginosa |
kcat/KM [mM/s]
| kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 2.8 | - |
isocitrate | recombinant His-tagged mutant N214D, pH 7.8, 30°C | Pseudomonas aeruginosa | |
| 4.9 | - |
isocitrate | recombinant His-tagged mutant E219A, pH 7.8, 30°C | Pseudomonas aeruginosa | |
| 6.9 | - |
isocitrate | recombinant His-tagged mutant Q221K, pH 7.8, 30°C | Pseudomonas aeruginosa | |
| 11.9 | - |
isocitrate | recombinant His-tagged mutant Q211H, pH 7.8, 30°C | Pseudomonas aeruginosa | |
| 22.3 | - |
isocitrate | recombinant His-tagged wild-type enzyme, pH 7.8, 30°C | Pseudomonas aeruginosa | |
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