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Literature summary for 2.5.1.19 extracted from

  • Healy-Fried, M.L.; Funke, T.; Priestman, M.A.; Han, H.; Schoenbrunn, E.
    Structural basis of glyphosate tolerance resulting from mutations of Pro101 in Escherichia coli 5-enolpyruvylshikimate 3-phosphate synthase (2007), J. Biol. Chem., 282, 32949-32955.
    View publication on PubMed
Search for more literature for 2.5.1.19

Cloned(Commentary)

Cloned (Comment) Organism
wild-type and mutants in pET-24d for expression in Escherichia coli BL21(DE3) Escherichia coli

Crystallization (Commentary)

Crystallization (Comment) Organism
mutants P101S and P101L in complex with shikimate 3-phosphate and with or without inhibitor glyphosate, long-range structural change in glyphosate binding site compared to wild-type, Gly96 and Thr97 are shifted towards the glyphosate-binding site, which gets slightly narrowed, crystals: space group: P2(1)2(1)2(1), unit cell parameters: a: 57.6-57.8, b: 85.1-85.6, c: 87.9-88.3, beta: 90°, hanging-drop vapour-diffusion, protein solution (37.5 mg/ml, 5 mM inhibitor and/or 5 mM shikimate 3-phosphate), 19°C, molecular replacement using PDB: 1G6S as model Escherichia coli

Protein Variants

Protein Variants Comment Organism
P101A slight decrease in catalytic efficiency, decreased inhibitory potency of glyphosate Escherichia coli
P101G slight decrease in catalytic efficiency, decreased inhibitory potency of glyphosate Escherichia coli
P101L lowest catalytic efficiency, decreased inhibitory potency of glyphosate due to long-range conformational changes Escherichia coli
P101S slight decrease in catalytic efficiency, decreased inhibitory potency of glyphosate due to long-range conformational changes, flexibility of Ser101 side chain disables hydrophobic bonding with carbonyl oxygen of Thr97 Escherichia coli

Inhibitors

Inhibitors Comment Organism Structure
glyphosate
-
 Escherichia coli

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
 phosphoenolpyruvate KM/KI: 13, mutant P101S Escherichia coli
additional information
-
 phosphoenolpyruvate KM/KI: 150 Escherichia coli
additional information
-
 phosphoenolpyruvate KM/KI: 2.3, mutant P101L Escherichia coli
additional information
-
 phosphoenolpyruvate KM/KI: 4.2, mutant P101A Escherichia coli
additional information
-
 phosphoenolpyruvate KM/KI: 7.5, mutant P101G Escherichia coli
additional information
-
 additional information small residue substitutions for Pro101 do not alter the KM for phosphoenolpyruvate and shikimate 3-phosphate Escherichia coli
0.06
-
 shikimate 3-phosphate +/-0.004 mM, mutant P101A Escherichia coli
0.06
-
 phosphoenolpyruvate +/-0.006 mM Escherichia coli
0.06
-
 shikimate 3-phosphate +/-0.006 mM Escherichia coli
0.06
-
 shikimate 3-phosphate +/-0.010 mM, mutant P101G Escherichia coli
0.07
-
 phosphoenolpyruvate +/-0.005 mM, mutant P101S Escherichia coli
0.08
-
 phosphoenolpyruvate +/-0.005 mM, mutant P101A Escherichia coli
0.08
-
 shikimate 3-phosphate +/-0.006 mM, mutant P101S Escherichia coli
0.09
-
 phosphoenolpyruvate +/-0.010 mM, mutant P101G Escherichia coli
0.1
-
 shikimate 3-phosphate +/-0.007 mM, mutant P101L Escherichia coli
0.15
-
 phosphoenolpyruvate +/-0.020 mM, mutant P101L Escherichia coli

Organism

Organism UniProt Comment Textmining
Escherichia coli P0A6D3
-
-

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
8
-
 +/-0.2 micromol/min/mg, mutant P101L Escherichia coli
22
-
 +/-0.4 micromol/min/mg, mutant P101S Escherichia coli
28
-
 +/-1 micromol/min/mg, mutant P101G Escherichia coli
31
-
 +/-0.5 micromol/min/mg, mutant P101A Escherichia coli
50
-
 +/-1 micromol/min/mg Escherichia coli

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
shikimate 3-phosphate + phosphoenolpyruvate 30 min, 25°C, pH 7.5, in presence of 2 mM dithiothreitol Escherichia coli phosphate + 5-enolpyruvylshikimate 3-phosphate detection of phosphate generation with Lanzetta reagent and change in absorbance at 650 nm ?

Synonyms

Synonyms Comment Organism
5-enolpyruvylshikimate 3-phosphate synthase
-
 Escherichia coli
E. coli EPSPS
-
 Escherichia coli
EPSPS
-
 Escherichia coli

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
additional information
-
 shikimate 3-phosphate kcat/KM: 200000 1/M*s, mutant P101S Escherichia coli
additional information
-
 phosphoenolpyruvate kcat/KM: 240000 1/M*s, mutant P101G Escherichia coli
additional information
-
 phosphoenolpyruvate kcat/KM: 280000 1/M*s, mutant P101A Escherichia coli
additional information
-
 phosphoenolpyruvate kcat/KM: 290000 1/M*s, mutant P101S Escherichia coli
additional information
-
 shikimate 3-phosphate kcat/KM: 390000 1/M*s, mutant P101A Escherichia coli
additional information
-
 shikimate 3-phosphate kcat/KM: 390000 1/M*s, mutant P101G Escherichia coli
additional information
-
 phosphoenolpyruvate kcat/KM: 41000 1/M*s, mutant P101L Escherichia coli
additional information
-
 shikimate 3-phosphate kcat/KM: 62000 1/M*s, mutant P101L Escherichia coli
additional information
-
 phosphoenolpyruvate kcat/KM: 640000 1/M*s Escherichia coli
additional information
-
 shikimate 3-phosphate kcat/KM: 640000 1/M*s Escherichia coli

Ki Value [mM]

Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
additional information
-
 glyphosate any substitutions for residue Pro101 decrease glyphosate binding affinity and inhibitor potency Escherichia coli
0.0004
-
 glyphosate +/-0.00006 mM Escherichia coli
0.0055
-
 glyphosate +/-0.0003 mM, mutant P101S Escherichia coli
0.012
-
 glyphosate +/-0.004 mM, mutant P101G Escherichia coli
0.019
-
 glyphosate +/-0.003 mM, mutant P101A Escherichia coli
0.066
-
 glyphosate +/-0.002 mM, mutant P101L Escherichia coli