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Literature summary for 2.5.1.19 extracted from

  • Vaithanomsat, P.; Brown, K.A.
    Isolation and mutation of recombinant EPSP synthase from pathogenic bacteria Pseudomonas aeruginosa (2007), Process Biochem., 42, 592-598.
No PubMed abstract available

Cloned(Commentary)

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

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

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 [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 [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

Organism UniProt Comment Textmining
Pseudomonas aeruginosa
-
-
-

Purification (Commentary)

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 [micromol/min/mg]

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 and Products (Substrate)

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

Synonyms Comment Organism
5-enol-pyruvylshikimate 3-phosphate synthase
-
Pseudomonas aeruginosa
AroA
-
Pseudomonas aeruginosa
EPSP synthase
-
Pseudomonas aeruginosa

Turnover Number [1/s]

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