Cloned (Comment) | Organism |
---|---|
recombinant expression of FLAG- and His-tagged enzyme in Escherichia coli strains BL21 and HSM174 in inclusion bodies, HSM174 cells produce about 5fold more enzyme protein | Geobacillus stearothermophilus |
General Stability | Organism |
---|---|
TrpRS Urzyme has substantially reduced hydrophobic stabilization with non-cooperative melting and cold-denaturation | Geobacillus stearothermophilus |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | required | Geobacillus stearothermophilus |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + L-tryptophan + tRNATrp | Geobacillus stearothermophilus | - |
AMP + diphosphate + L-tryptophyl-tRNATrp | - |
? | |
additional information | Geobacillus stearothermophilus | an ancestral tryptophanyl-tRNA synthetase precursor achieves high catalytic rate enhancement without ordered ground-state tertiary structures | ? | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Geobacillus stearothermophilus | - |
- |
- |
Purification (Comment) | Organism |
---|---|
recombinant solubilized FLAG- and His-tagged enzyme from Escherichia coli strains BL21 and HSM174 by nickel affinity chromatography, gel filtration, and ultrafiltration | Geobacillus stearothermophilus |
Renatured (Comment) | Organism |
---|---|
solubilization and refolding of recombinant FLAG- and His-tagged enzyme from Escherichia coli strains BL21 and HSM174 by homogenization in 100 mM sodium phosphate, and 6 M guanidinium hydrochloride, pH 7.2 before purification | Geobacillus stearothermophilus |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + L-tryptophan + tRNATrp | - |
Geobacillus stearothermophilus | AMP + diphosphate + L-tryptophyl-tRNATrp | - |
? | |
additional information | an ancestral tryptophanyl-tRNA synthetase precursor achieves high catalytic rate enhancement without ordered ground-state tertiary structures | Geobacillus stearothermophilus | ? | - |
? |
Subunits | Comment | Organism |
---|---|---|
More | 15N tryptophanyl-tRNA synthetase Urzyme structure analysis by heteronuclear single quantum coherence (HSQC) NMR spectroscopy supplemented by circular dichroism, thermal melting, and induced fluorescence of bound dye. Circular dichroism analysis demonstrates reversible folding of the alpha-helix formed by 61 of 130 residues | Geobacillus stearothermophilus |
Synonyms | Comment | Organism |
---|---|---|
TrpRS Urzyme | - |
Geobacillus stearothermophilus |
Tryptophanyl-tRNA synthetase | - |
Geobacillus stearothermophilus |
Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
---|---|---|---|
additional information | - |
TrpRS Urzyme has substantially reduced hydrophobic stabilization with non-cooperative melting and cold-denaturation | Geobacillus stearothermophilus |
35 | - |
a broad maximum stability is centered at about 35°C | Geobacillus stearothermophilus |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
ATP | - |
Geobacillus stearothermophilus |
General Information | Comment | Organism |
---|---|---|
evolution | an ancestral tryptophanyl-tRNA synthetase precursor achieves high catalytic rate enhancement without ordered ground-state tertiary structures. The TrpRS Urzyme catalytic activity arises neither from tiny amounts of wild-type enzyme, nor from a separate population of folded and highly active Urzyme molecules not in equilibrium with the general population. AaRS Urzymes lack much of the mass of modern aaRS, retaining only a small portion of the hydrophobic cores of the full-length enzymes. AaRS Urzymes contain 120-130 amino acids, and consist of little more than is required to form intact active sites. They retain over 60% of the transition-state stabilization free energy for amino acid activation and the ability to aminoacylate tRNA. Further, they preserve about 20% of the Gibbs energies necessary to discriminate between competing amino acid substrates and preferentially activate amino acids from within, rather than outside, their own class. A major fraction of TrpRS Urzyme molecules contribute to the rate acceleration by transiently forming tight transition-state complexes | Geobacillus stearothermophilus |
additional information | 15N tryptophanyl-tRNA synthetase Urzyme structure analysis by heteronuclear single quantum coherence (HSQC) NMR spectroscopy supplemented by circular dichroism, thermal melting, and induced fluorescence of bound dye. TrpRS Urzyme is not a typical protein domain. Transition state stabilization and catalytic activity from molten globules, overview | Geobacillus stearothermophilus |