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Literature summary for 5.4.99.5 extracted from

  • Westfall, C.S.; Xu, A.; Jez, J.M.
    Structural evolution of differential amino acid effector regulation in plant chorismate mutases (2014), J. Biol. Chem., 289, 28619-28628 .
    View publication on PubMedView publication on EuropePMC

Activating Compound

Activating Compound Comment Organism Structure
cysteine
-
Arabidopsis thaliana
histidine
-
Arabidopsis thaliana
additional information although AtCM2 contains the putative regulatory effector binding domain, phenylalanine, tyrosine, and tryptophan do not affect its activity Arabidopsis thaliana
tryptophan
-
Arabidopsis thaliana
tryptophan activates about 3fold Arabidopsis thaliana

Cloned(Commentary)

Cloned (Comment) Organism
AtCM1, sequence comparisons, recombinant expression of N-terminally His6-tagged wild-type and mutant enzymes in Escherichia coli strain Rosetta II (DE3) Arabidopsis thaliana
AtCM3, sequence comparisons, recombinant expression of N-terminally His6-tagged wild-type and mutant enzymes in Escherichia coli strain Rosetta II (DE3) Arabidopsis thaliana
recombinant His6-tagged wild-type enzyme from Escherichia coli strain Rosetta II (DE3) by nickel affinity chromatography, tag cleavage with thrombin, dialysis, benzamidine/nickel affinity chromatography, and gel filtration Arabidopsis thaliana

Crystallization (Commentary)

Crystallization (Comment) Organism
purified recombinant detagged wild-type enzyme in complex with phenylalanine or tyrosine, hanging drop vapordiffusion method, mixing 0f 0.001 ml of 9 mg/ml protein in 25 mM HEPES, pH 7.5, and 100 mM NaCl, with 0.001 ml of reservoir solution containing 30% PEG 400, 0.1 M HEPES, pH 7.5, 0.2 M MgCl2, and 1 mM of either phenylalanine or tyrosine, X-ray diffraction structure determination and analysis at 2.30-2.45 A resolution, molecular replacement using yeast chorismate mutase structure, PDB ID 4CSM, as a search model Arabidopsis thaliana

Protein Variants

Protein Variants Comment Organism
D132G site-directed mutagenesis, the mutant enzyme kinetically resembles isozyme AtCM1, it retains activation by tryptophan, although to a lesser extent than observed with wild-type AtCM3 Arabidopsis thaliana
G149A site-directed mutagenesis, the mutation eliminates the effector action of both phenylalanine and tyrosine Arabidopsis thaliana
G149D site-directed mutagenesis, the mutation eliminates the effector action of both phenylalanine and tyrosine Arabidopsis thaliana
G213A site-directed mutagenesis, mutation in effector binding site, the mutation eliminates the effect of aromatic amino acids on enzymatic activity Arabidopsis thaliana
G213P site-directed mutagenesis, mutation in effector binding site, the mutation eliminates the effect of aromatic amino acids on enzymatic activity Arabidopsis thaliana
H145Q site-directed mutagenesis, mutation in effector binding site, the mutation has varyring effects on the EC50 values for the aromatic amino acid effectors but does not change either positive or negative effects on enzymatic activity Arabidopsis thaliana
additional information generation of the expression construct N-terminally hexahistidine-tagged AtCM1 lacking the plastid localization peptide, AtCM1DELTA66 Arabidopsis thaliana
R79k site-directed mutagenesis, mutation in effector binding site, the mutation has varyring effects on the EC50 values for the aromatic amino acid effectors but does not change either positive or negative effects on enzymatic activity Arabidopsis thaliana
V217T site-directed mutagenesis, mutation in effector binding site Arabidopsis thaliana

Inhibitors

Inhibitors Comment Organism Structure
additional information although AtCM2 contains the putative regulatory effector binding domain, phenylalanine, tyrosine, and tryptophan do not affect its activity Arabidopsis thaliana
phenylalanine model of the AtCM1x02phenylalanine complex including residues Arg79-Val290 and Val307-Asp340, the phenylalanine ligand, and 83 waters, inhibits about 20fold Arabidopsis thaliana
tyrosine inhibits about 20fold Arabidopsis thaliana

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
additional information steady-state kinetics, isozyme AtCM1 shows Michaelis-Menten kinetics. Effect of aromatic amino acids on wild-type and mutant AtCM1, overview Arabidopsis thaliana
additional information
-
additional information steady-state kinetics, isozyme AtCM2, shows Michaelis-Menten kinetics Arabidopsis thaliana
additional information
-
additional information steady-state kinetics, isozyme AtCM3 displays positive cooperativity with a Hill coefficient of 2.1, indicating that substrate binding at one active site of the homodimer enhanced interaction at the second active site Arabidopsis thaliana
0.15
-
chorismate recombinant enzyme, pH 8.0, temperature not specified in the publication Arabidopsis thaliana
0.55
-
chorismate recombinant enzyme, pH 8.0, temperature not specified in the publication Arabidopsis thaliana
1.1
-
chorismate recombinant enzyme, pH 8.0, temperature not specified in the publication Arabidopsis thaliana

Localization

Localization Comment Organism GeneOntology No. Textmining
chloroplast
-
Arabidopsis thaliana 9507
-
cytosol
-
Arabidopsis thaliana 5829
-

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
60000
-
about, recombinant enzyme, gel filtration Arabidopsis thaliana

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
Chorismate Arabidopsis thaliana
-
Prephenate
-
?

Organism

Organism UniProt Comment Textmining
Arabidopsis thaliana P42738
-
-
Arabidopsis thaliana Q9C544
-
-
Arabidopsis thaliana Q9S7H4
-
-

Purification (Commentary)

Purification (Comment) Organism
AtCM1, sequence comparisons, recombinant expression of N-terminally His6-tagged wild-type and mutant enzymes in Escherichia coli strain Rosetta II (DE3) Arabidopsis thaliana
recombinant His6-tagged wild-type and mutant enzymes from Escherichia coli strain Rosetta II (DE3) by nickel affinity chromatography, tag cleavage with thrombin, dialysis, benzamidine/nickel affinity chromatography, and gel filtration Arabidopsis thaliana

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
Chorismate
-
Arabidopsis thaliana Prephenate
-
?

Subunits

Subunits Comment Organism
homodimer 2 * 32600, recombinant enzyme, SDS-PAGE Arabidopsis thaliana

Synonyms

Synonyms Comment Organism
AtCM1
-
Arabidopsis thaliana
AtCM2
-
Arabidopsis thaliana
AtCM3
-
Arabidopsis thaliana

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
13
-
chorismate recombinant enzyme, pH 8.0, temperature not specified in the publication Arabidopsis thaliana
16.1
-
chorismate recombinant enzyme, pH 8.0, temperature not specified in the publication Arabidopsis thaliana
38.7
-
chorismate recombinant enzyme, pH 8.0, temperature not specified in the publication Arabidopsis thaliana

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8
-
assay at Arabidopsis thaliana

General Information

General Information Comment Organism
additional information the crystal structure of AtCM1 in complex with tyrosine and phenylalanine identifies differences in the effector sites of the allosterically regulated yeast enzyme and the other two Arabidopsis isoforms. The catalytic efficiency (kcat/Km) of AtCM2 is 11 and 22fold higher than that of AtCM1 and AtCM3, respectively. This results from a combination of a more rapid turnover rate and a lower Km value for chorismate displayed by AtCM2 compared with the other two isoforms. Two catalytic residues (Arg229 and Lys240 in AtCM1) are invariant across the AtCM isoforms. These two basic residues are essential for substrate binding, orient the two negatively charged carboxylic acids of chorismate, and provide transition state stabilization during catalysis Arabidopsis thaliana
physiological function AtCM2 is a nonallosteric form Arabidopsis thaliana
physiological function isozyme AtCM1 is alosterically regulated Arabidopsis thaliana
physiological function isozyme AtCM3 is alosterically regulated Arabidopsis thaliana

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
11.8
-
chorismate recombinant enzyme, pH 8.0, temperature not specified in the publication Arabidopsis thaliana
29.27
-
chorismate recombinant enzyme, pH 8.0, temperature not specified in the publication Arabidopsis thaliana
258
-
chorismate recombinant enzyme, pH 8.0, temperature not specified in the publication Arabidopsis thaliana