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Literature summary extracted from

  • Haghani, K.; Salmanian, A.H.; Ranjbar, B.; Zakikhan-Alang, K.; Khajeh, K.
    Comparative studies of wild type Escherichia coli 5-enolpyruvylshikimate 3-phosphate synthase with three glyphosate-insensitive mutated forms: activity, stability and structural characterization (2008), Biochim. Biophys. Acta, 1784, 1167-1175.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.5.1.19 in pET24d for expression in Escherichia coli DE3 Escherichia coli

Protein Variants

EC Number Protein Variants Comment Organism
2.5.1.19 A183T 38fold decrease in phosphoenolpyruvate-binding affinity, more solvent-exposed tryptophan residues and lower stability against guanidine hydrochloride compared to wild-type and mutant G96A, midpoint guanidine hydrochloride concentration of unfolding: 0.7 M, higher structural flexibility and decrease of secondary structure (28% alpha helix, 35% beta sheet) compared to wild-type (40% alpha helix, 31% beta sheet) and lowest resistance against proteolysis, residue A183 located on the exterior in the N-terminal domain Escherichia coli
2.5.1.19 G96A glyphosate-insensitive, 31fold decrease in phosphoenolpyruvate-binding affinity, little change with respect to structural flexibility and stability compared to wild-type, lower structural flexibility than wild-type and mutants A183T and G96A/A183T, midpoint guanidine hydrochloride concentration of unfolding: 1.1 M, residue Gly96 located at interphase between two domains Escherichia coli
2.5.1.19 G96A/A183T glyphosate-insensitive, 8fold decrease in phosphoenolpyruvate-binding affinity, more solvent-exposed tryptophan residues and lower stability against guanidine hydrochloride compared to wild-type and mutant G96A, midpoint guanidine hydrochloride concentration of unfolding: 0.65 M, higher structural flexibility and decrease of secondary structure (36% alpha helix, 38% beta sheet) compared to wild-type (40% alpha helix, 31% beta sheet) Escherichia coli

General Stability

EC Number General Stability Organism
2.5.1.19 complete unfolding of the protein at 2.5 M guanidine hydrochloride at pH 7.8 (midpoint guanidine hydrochloride concentration of unfolding: 0.8 +/-0.1 M), enhanced flexibility, lower stability and increased proteolysis of mutants A183T and G96A/A183T compared to wild-type and mutant G96A as revealed by susceptibility to proteolytic digestion with trypsin, thermolysin, pronase (25°C for various time intervals), unfolding profiles (intrinsic tryptophan fluorescence measurements at 340 nm) after treatment with guanidine hydrochloride (30 min at 25°C) and dynamic quenching with acrylamide or not, and secondary structure determination by circular dichroism spectroscopy Escherichia coli

Inhibitors

EC Number Inhibitors Comment Organism Structure
2.5.1.19 glyphosate N-(phosphonomethyl)glycine, broad-spectrum herbicide Escherichia coli

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
2.5.1.19 0.1
-
 phosphoenolpyruvate in presence of 1 mM shikimate 3-phosphate Escherichia coli
2.5.1.19 0.8
-
 phosphoenolpyruvate mutant G96A/A183T, in presence of 1 mM shikimate 3-phosphate Escherichia coli
2.5.1.19 3.1
-
 phosphoenolpyruvate mutant G96A, in presence of 1 mM shikimate 3-phosphate Escherichia coli
2.5.1.19 3.8
-
 phosphoenolpyruvate mutant A183T, in presence of 1 mM shikimate 3-phosphate Escherichia coli

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
2.5.1.19 44000
-
 SDS-PAGE Escherichia coli

Organism

EC Number Organism UniProt Comment Textmining
2.5.1.19 Escherichia coli P0A6D3
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.5.1.19 from supernatant from 25% (w/v) ammonium sulfate precipitation of crude bacterial extracts, Q-sepharose anion exchange chromatography (NaCl gradient elution) and FPLC (Mono-Q column, NaCl gradient elution) Escherichia coli

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
2.5.1.19 18.9
-
 mutant A183T, with respect to phosphoenolpyruvate Escherichia coli
2.5.1.19 26.5
-
 mutant G96A/A183T, with respect to phosphoenolpyruvate Escherichia coli
2.5.1.19 34
-
 with respect to phosphoenolpyruvate Escherichia coli
2.5.1.19 37.4
-
 mutant G96A, with respect to phosphoenolpyruvate Escherichia coli

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.5.1.19 shikimate 3-phosphate + phosphoenolpyruvate
-
 Escherichia coli phosphate + 5-enolpyruvylshikimate 3-phosphate detection of inorganic phosphate by reaction with Lanzetta reagent and change in optical density at 660 nm ?

Synonyms

EC Number Synonyms Comment Organism
2.5.1.19 5-enolpyruvylshikimate 3-phosphate synthase
-
 Escherichia coli
2.5.1.19 AroA
-
 Escherichia coli
2.5.1.19 EPSP synthase
-
 Escherichia coli

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
2.5.1.19 15
-
 phosphoenolpyruvate mutant A183T, in presence of 1 mM shikimate 3-phosphate Escherichia coli
2.5.1.19 21
-
 phosphoenolpyruvate mutant G96A/A183T, in presence of 1 mM shikimate 3-phosphate Escherichia coli
2.5.1.19 26.4
-
 phosphoenolpyruvate in presence of 1 mM shikimate 3-phosphate Escherichia coli
2.5.1.19 29.7
-
 phosphoenolpyruvate mutant G96A, in presence of 1 mM shikimate 3-phosphate Escherichia coli

IC50 Value

EC Number IC50 Value IC50 Value Maximum Comment Organism Inhibitor Structure
2.5.1.19 additional information
-
 more than 10 mM for mutant G96A Escherichia coli glyphosate
2.5.1.19 additional information
-
 more than 10 mM for mutant G96A/A183T Escherichia coli glyphosate
2.5.1.19 0.05
-
-
 Escherichia coli glyphosate
2.5.1.19 8.2
-
 mutant A183T Escherichia coli glyphosate