Literature summary for 2.5.1.19 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.

Cloned(Commentary)

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

Protein Variants

Protein Variants	Comment	Organism
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
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
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

General Stability	Organism
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

Organism

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

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

KM Value [mM]

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

Molecular Weight [Da]

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

Organism

Organism	UniProt	Comment	Textmining
Escherichia coli	P0A6D3	-	-

Purification (Commentary)

Purification (Comment)	Organism
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]

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

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
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

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

Turnover Number [1/s]

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

IC50 Value

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