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Literature summary for 6.1.1.2 extracted from
- Selective inhibition of bacterial tryptophanyl-tRNA synthetases by indolmycin is mechanism-based (2016), J. Biol. Chem., 291, 255-265 .
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| recombinant expression of His-tagged enzyme in Escherichia coli strain BL21(DE3)pLysS by autoinduction | Geobacillus stearothermophilus |
Crystallization (Commentary)
| Crystallization (Comment) | Organism |
|---|---|
| purified recombinant selenomethionine-substituted BsTrpRS in complex with ATP, Mg2+, and indolmycin are grown by vapor diffusion against a reservoir of 1.4 M potassium citrate and 0.1 M Hepes, pH 7.4, X-ray diffraction structure determination and analysis at 1.9 A resolution, PDB ID 5DK4 | Geobacillus stearothermophilus |
Inhibitors
| Inhibitors | Comment | Organism | Structure |
|---|---|---|---|
| indolmycin | competitive inhibitor, produced by Streptomyces griseus, causes selective, mechanism-based inhibition of the bacterial enzyme, which preferentially binds indolmycin about 1500fold more tightly than its tryptophan substrate, binding structure analysis overview. Long range coupling to residues within an allosteric region called the D1 switch of BsTrpRS positions the Mg2+ ion in a manner that allows it to assist in transition state stabilization. The Mg2+ ion in the inhibited complex forms significantly closer contacts with non-bridging oxygen atoms from each phosphate group of ATP and three water molecules than occur in the (presumably catalytically competent) pre-transition state (preTS) crystal structures. This altered coordination stabilizes a ground state Mg2+-ATP-configuration, accounting for the high affinity inhibition of BsTrpRS by indolmycin | Geobacillus stearothermophilus |  |
| additional information | indolmycin and ATP form a ternary complex with BsTrpRS | Geobacillus stearothermophilus |
Metals/Ions
| Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|
| Mg2+ | required | Geobacillus stearothermophilus | |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| ATP + L-tryptophan + tRNATrp | Geobacillus stearothermophilus | - |
AMP + diphosphate + L-tryptophyl-tRNATrp | - |
? | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Geobacillus stearothermophilus | P00953 | - |
- |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3)pLysS by nickel affinity chromatography, followed by cleavage fo the tag by TEV protease, and dialysis | Geobacillus stearothermophilus |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| ATP + L-tryptophan + tRNATrp | - |
Geobacillus stearothermophilus | AMP + diphosphate + L-tryptophyl-tRNATrp | - |
? | |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| BsTRpRS | - |
Geobacillus stearothermophilus |
| TrpRS | - |
Geobacillus stearothermophilus |
| Tryptophanyl-tRNA synthetase | - |
Geobacillus stearothermophilus |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 37 | - |
assay at | Geobacillus stearothermophilus |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 8 | - |
assay at | Geobacillus stearothermophilus |
Cofactor
| Cofactor | Comment | Organism | Structure |
|---|---|---|---|
| ATP | - |
Geobacillus stearothermophilus | |
| additional information | indolmycin and ATP form a ternary complex with BsTrpRS | Geobacillus stearothermophilus |
Ki Value [mM]
| Ki Value [mM] | Ki Value maximum [mM] | Inhibitor | Comment | Organism | Structure |
|---|---|---|---|---|---|
| additional information | - |
additional information | indolmycin Michaelis-Menten inhibition kinetics | Geobacillus stearothermophilus |
IC50 Value
| IC50 Value | IC50 Value Maximum | Comment | Organism | Inhibitor | Structure |
|---|---|---|---|---|---|
| 0.000002 | - |
pH 8.0, 37°C, recombinant enzyme | Geobacillus stearothermophilus | indolmycin | |
General Information
| General Information | Comment | Organism |
|---|---|---|
| additional information | both the ATP configuration and Mg2+ coordination in the human cytosolic (Hc)TrpRS preTS structure differ greatly from the BsTrpRS preTS structure. The effect of these differences is that catalysis occurs via a different transition state stabilization mechanism in HcTrpRS with a yet-to-be determined role for Mg2+ | Geobacillus stearothermophilus |
| physiological function | aminoacyl-tRNA synthetases maintain the fidelity of the genetic code by ensuring the charging of tRNA with its cognate amino acid | Geobacillus stearothermophilus |
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