Crystallization (Comment) | Organism |
---|---|
wild-type dimeric enzyme, wild-type enzyme in complex with products ADP and shikimate 3-phosphate, enzyme mutant R57A, and enzyme mutant E114A in complex with selective inhibitor NSC162535, hanging drop vapour ddiffusion metho, 50 mg/ml protein in 40 mM Tris-HCl, pH 7.0, containing 100 mM NaCl mixed with an equal volume of reservoir solution and equilibrated against 0.06 ml of reservoir solution, containing 0.2 M Li2SO4, 30% w/v PEG 8000, and 0.1 M sodium acetate, pH 6.5 for the apo-enzyme, or containing 0.1 M HEPES sodium salt, pH 7.5, 0.1 M sodium acetate, 18% w/v PEG 8000, 2% w/v 2-propanol, and 5 mM shikimate and 5 mM MgATP for the product complex enzyme, or containing 0.1 M HEPES sodium salt, pH 8.0, 8% w/v 2-propanol and 18% w/v PEG 4000 for enzyme mutant R57A, or containing 0.1 M HEPES sodium salt, pH 6.7, and 1.2 M potassium sodium tartrate tetrahydrate for the enzyme mutant E114A with inhibitor, X-ray diffraction structure determination and analysis at 1.8 A, 2.3 A, 2.4 A, and 2.53 A resolution, respectively, molecular replacement | Helicobacter pylori |
Protein Variants | Comment | Organism |
---|---|---|
D33A | site-directed mutagensis, inactive mutant | Helicobacter pylori |
D33E | site-directed mutagensis, inactive mutant | Helicobacter pylori |
E114A | site-directed mutagensis, the mutant shows 82% of wlld-type activity | Helicobacter pylori |
F48A | site-directed mutagensis, inactive mutant | Helicobacter pylori |
F48Y | site-directed mutagensis, the mutant shows 40% of wlld-type activity | Helicobacter pylori |
M10A | site-directed mutagensis, the mutant shows 38% of wlld-type activity | Helicobacter pylori |
R116A | site-directed mutagensis, inactive mutant | Helicobacter pylori |
R116K | site-directed mutagensis, inactive mutant | Helicobacter pylori |
R132A | site-directed mutagensis, the mutant shows 5% of wlld-type activity | Helicobacter pylori |
R132K | site-directed mutagensis, inactive mutant | Helicobacter pylori |
R57A | site-directed mutagensis, the mutant shows 2% of wlld-type activity | Helicobacter pylori |
R57K | site-directed mutagensis, the mutant shows 2% of wlld-type activity | Helicobacter pylori |
General Stability | Organism |
---|---|
differential scanning calorimetry experiments for evaluaton of the stability and unfolding of each of the enzyme mutants, overview | Helicobacter pylori |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
NSC162535 | selective inhibitor, identification and binding analysis with enzyme mutant E144A by virtual docking analysis, isothermal titration calorimetry, and crystals structure analysis revealing an induced-fit mechanism, inactivation mechanism, detailed overview. Binding kinetics of wild-type and mutant enzymes | Helicobacter pylori |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.039 | - |
shikimate | enzyme mutant E114A, pH 7.5, 25°C | Helicobacter pylori | |
0.06 | - |
shikimate | wild-type enzyme, pH 7.5, 25°C | Helicobacter pylori | |
0.101 | - |
ATP | wild-type enzyme, pH 7.5, 25°C | Helicobacter pylori | |
0.101 | - |
ATP | enzyme mutant M10A, pH 7.5, 25°C | Helicobacter pylori | |
0.135 | - |
shikimate | enzyme mutant M10A, pH 7.5, 25°C | Helicobacter pylori | |
0.143 | - |
ATP | enzyme mutant E114A, pH 7.5, 25°C | Helicobacter pylori | |
0.231 | - |
ATP | enzyme mutant F48Y, pH 7.5, 25°C | Helicobacter pylori | |
0.291 | - |
shikimate | enzyme mutant F48Y, pH 7.5, 25°C | Helicobacter pylori |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | required | Helicobacter pylori |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + shikimate | Helicobacter pylori | - |
ADP + 3-phosphoshikimate | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Helicobacter pylori | P56073 | - |
- |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + shikimate | - |
Helicobacter pylori | ADP + 3-phosphoshikimate | - |
? |
Subunits | Comment | Organism |
---|---|---|
dimer | detailed structure-activity relationship analysis, overview | Helicobacter pylori |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Helicobacter pylori |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | - |
assay at | Helicobacter pylori |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
ATP | - |
Helicobacter pylori |
IC50 Value | IC50 Value Maximum | Comment | Organism | Inhibitor | Structure |
---|---|---|---|---|---|
0.0049 | - |
pH 7.5, 25°C | Helicobacter pylori | NSC162535 |
General Information | Comment | Organism |
---|---|---|
metabolism | shikimate kinase catalyzes the fifth step of the shikimate pathway for biosynthesis of aromatic amino acids | Helicobacter pylori |
additional information | detailed structure-activity relationship analysis, overview. The critical conserved residues D33, F48, R57, R116, and R132 interact with shikimate. A characteristic three-layer architecture and a conformationally elastic region consisting of F48, R57, R116, and R132 are occupied by shikimate | Helicobacter pylori |