Cloned (Comment) | Organism |
---|---|
from genomic DNA, in pUC18 for complementation studies in Escherichia coli strain AB2829 (aroA auxotroph), in pTrc99A for inducible expression in Escherichia coli AB 2829 | Pseudomonas aeruginosa |
Protein Variants | Comment | Organism |
---|---|---|
G99A | similar substrate affinities and decreased turnover compared to wild-type, fails to complement aroA auxotrophy in transformed Escherichia coli | Pseudomonas aeruginosa |
L104P | lower affinity to phosphate and decreased turnover compared to wild-type, fails to complement aroA auxotrophy in transformed Escherichia coli | Pseudomonas aeruginosa |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
glyphosate | inhibits forward reaction only slightly at 0.2 mM but substantially at 20 mM | Pseudomonas aeruginosa |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
1.9 | - |
5-enolpyruvylshikimate 3-phosphate | +/-0.3 mM, mutant G99A | Pseudomonas aeruginosa | |
2.2 | - |
5-enolpyruvylshikimate 3-phosphate | +/-0.5 mM | Pseudomonas aeruginosa | |
2.5 | - |
5-enolpyruvylshikimate 3-phosphate | +/-0.4 mM, mutant L104P | Pseudomonas aeruginosa | |
5.6 | - |
phosphate | +/-1.6 | Pseudomonas aeruginosa | |
7.3 | - |
phosphate | +/-0.7 mM, mutant G99A | Pseudomonas aeruginosa | |
24.8 | - |
phosphate | +/-4.8 mM, mutant L104P | Pseudomonas aeruginosa |
Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
50000 | - |
wild-type and mutants, SDS-PAGE after induced overexpression in Escherichia coli | Pseudomonas aeruginosa |
54040 | - |
deduced from primary sequence | Pseudomonas aeruginosa |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Pseudomonas aeruginosa | - |
- |
- |
Purification (Comment) | Organism |
---|---|
from crude cell extracts by DEAE-sepharose anion-exchange chromatography (Pharmacia XK-16/40 column, elution with linear NaCl gradient), Superdex 200 gel filtration chromatography (Pharmacia Superdex 200 HR 10/30 column coupled to FPLC, pH 6.8), Resource Q anion-exchange chromatography (elution with linear NaCl gradient, pH 6.8) | Pseudomonas aeruginosa |
Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|
0.4 | - |
mutant G99A, after DEAE-Sepharose chromatography | Pseudomonas aeruginosa |
0.57 | - |
mutant G99A, in crude bacterial extracts | Pseudomonas aeruginosa |
0.62 | - |
mutant L104P, in crude bacterial extracts | Pseudomonas aeruginosa |
0.63 | - |
mutant L104P, after DEAE-Sepharose chromatography | Pseudomonas aeruginosa |
0.86 | - |
mutant G99A, after Superdex 200 gel filtration | Pseudomonas aeruginosa |
1.34 | - |
mutant G99A, after ResourceQ chromatography | Pseudomonas aeruginosa |
1.4 | - |
in crude bacterial extracts | Pseudomonas aeruginosa |
1.7 | - |
after DEAE-sepharose chromatography | Pseudomonas aeruginosa |
1.83 | - |
mutant L104P, after Superdex 200 gel filtration | Pseudomonas aeruginosa |
1.91 | - |
mutant L104P, after ResourceQ chromatography | Pseudomonas aeruginosa |
2 | - |
after Superdex 200 gel filtration | Pseudomonas aeruginosa |
8.5 | - |
after ResourceQ chromatography | Pseudomonas aeruginosa |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
phosphate + 5-enolpyruvylshikimate 3-phosphate | reverse reaction | Pseudomonas aeruginosa | shikimate 3-phosphate + phosphoenolpyruvate | phosphoenolpyruvate generation coupled to pyruvate kinase and lactate dehydrogenase and monitored spectrophotometrically at 340 nm, pH7.0, in presence of ADP, NADH, MgCl2 | r | |
shikimate 3-phosphate + phosphoenolpyruvate | forward reaction | Pseudomonas aeruginosa | phosphate + 5-enolpyruvylshikimate 3-phosphate | phosphate generation monitored by native gel analyses and subsequent calciumphosphate precipitation upon soaking in substrate solution in presence or absence of inhibitor glyphosate | r |
Synonyms | Comment | Organism |
---|---|---|
5-enol-pyruvylshikimate 3-phosphate synthase | - |
Pseudomonas aeruginosa |
AroA | - |
Pseudomonas aeruginosa |
EPSP synthase | - |
Pseudomonas aeruginosa |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
5-enolpyruvylshikimate 3-phosphate | kat/KM: 1100000 M(-1)s(-1), mutant L104P | Pseudomonas aeruginosa | |
additional information | - |
phosphate | kcat/KM: 110 M(-1)s(-1), mutant L104P | Pseudomonas aeruginosa | |
additional information | - |
phosphate | kcat/KM: 130 M(-1)s(-1), mutant G99A | Pseudomonas aeruginosa | |
additional information | - |
5-enolpyruvylshikimate 3-phosphate | kcat/KM: 3400000 M(-1)s(-1) | Pseudomonas aeruginosa | |
additional information | - |
5-enolpyruvylshikimate 3-phosphate | kcat/KM: 660000 M(-1)s(-1), mutant G99A | Pseudomonas aeruginosa | |
additional information | - |
phosphate | kcat/KM: 960 M(-1)s(-1) | Pseudomonas aeruginosa | |
1 | - |
phosphate | +/-0.03, mutant G99A | Pseudomonas aeruginosa | |
1.2 | - |
5-enolpyruvylshikimate 3-phosphate | +/-0.04, mutant G99A | Pseudomonas aeruginosa | |
2.7 | - |
5-enolpyruvylshikimate 3-phosphate | +/-0.1, mutant L104P | Pseudomonas aeruginosa | |
2.8 | - |
phosphate | +/-0.2, mutant L104P | Pseudomonas aeruginosa | |
5.4 | - |
phosphate | +/-0.4 | Pseudomonas aeruginosa | |
7.4 | - |
5-enolpyruvylshikimate 3-phosphate | +/-0.4 | Pseudomonas aeruginosa |