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Literature summary for 6.1.1.5 extracted from
- Naturally occurring isoleucyl-tRNA synthetase without tRNA-dependent pre-transfer editing (2016), J. Biol. Chem., 291, 8618-8631 .
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| gene ileS, phylogenetic analysis, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) | Streptomyces griseus |
| gene ileS, phylogenetic analysis, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain Rosetta (DE3) | Saccharomyces cerevisiae |
| genes ileS1 and ileS2, phylogenetic analysis | Escherichia coli |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| D333A | site-directed mutagenesis, solution-based Val-AMP hydrolysis is 25fold slower than the rate of AMP formation assigned to editing in mutant D333A ScIleRS, non-enzymatic hydrolysis only weakly contributes to AMP accumulation | Saccharomyces cerevisiae |
| D334A | site-directed mutagenesis, the post-transfer editing-defective mutant of SgIleRS displays the similar rates of aminoacylation and AMP formation in the presence of valine, exhibiting a kAMP/kVal-tRNA ratio of 1.1. Stoichiometric ATP consumption in Val-tRNAIle synthesis demonstrates the lack of proofreading by D334A SgIleRS, arguing against hydrolysis of Val-AMP alongside aminoacylation within the synthetic site, SgIleRS naturally lacks tRNA-dependent pre-transfer editing | Streptomyces griseus |
Inhibitors
| Inhibitors | Comment | Organism | Structure |
|---|---|---|---|
| mupirocin | inhibition of isoleucine activation by mupirocin, competitive inhibition, analyzed with ATP-diphosphate exchange reaction | Saccharomyces cerevisiae |  |
| mupirocin | poor inhibition of isoleucine activation by mupirocin, competitive inhibition, analyzed with ATP-diphosphate exchange reaction. SgIleRS synthetic site is highly resistant to mupirocin | Streptomyces griseus | |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| additional information | - |
additional information | single-turnover kinetic analysis | Escherichia coli | |
| additional information | - |
additional information | single-turnover kinetic analysis, activation kinetics of isoleucine and valine by ScIleRS at 30°C | Saccharomyces cerevisiae | |
| additional information | - |
additional information | single-turnover kinetic analysis, activation kinetics of isoleucine and valine by SgIleRS at 30°C | Streptomyces griseus |
Localization
| Localization | Comment | Organism | GeneOntology No. | Textmining |
|---|---|---|---|---|
| cytoplasm | - |
Saccharomyces cerevisiae | 5737 | - |
Metals/Ions
| Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|
| Mg2+ | required | Streptomyces griseus | |
| Mg2+ | required | Escherichia coli | |
| Mg2+ | required | Saccharomyces cerevisiae | |
| Zn2+ | required | Streptomyces griseus | |
| Zn2+ | required | Escherichia coli | |
| Zn2+ | required | Saccharomyces cerevisiae | |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| ATP + L-isoleucine + tRNAIle | Streptomyces griseus | - |
AMP + diphosphate + L-isoleucyl-tRNAIle | - |
r | |
| ATP + L-isoleucine + tRNAIle | Escherichia coli | - |
AMP + diphosphate + L-isoleucyl-tRNAIle | - |
r | |
| ATP + L-isoleucine + tRNAIle | Saccharomyces cerevisiae | - |
AMP + diphosphate + L-isoleucyl-tRNAIle | - |
r | |
| ATP + L-isoleucine + tRNAIle | Saccharomyces cerevisiae ATCC 204508 / S288c | - |
AMP + diphosphate + L-isoleucyl-tRNAIle | - |
r | |
| additional information | Streptomyces griseus | the enzyme is also active with L-valine instead of L-isoleucine | ? | - |
? | |
| additional information | Saccharomyces cerevisiae | the enzyme is also active with L-valine instead of L-isoleucine | ? | - |
? | |
| additional information | Saccharomyces cerevisiae ATCC 204508 / S288c | the enzyme is also active with L-valine instead of L-isoleucine | ? | - |
? | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Escherichia coli | P00956 | gene ileS | - |
| Saccharomyces cerevisiae | P09436 | - |
- |
| Saccharomyces cerevisiae ATCC 204508 / S288c | P09436 | - |
- |
| Streptomyces griseus | - |
- |
- |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography | Streptomyces griseus |
| recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain Rosetta (DE3) by nickel affinity chromatography and gel filtration in stable monomeric form | Saccharomyces cerevisiae |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| ATP + L-isoleucine + tRNAIle | - |
Streptomyces griseus | AMP + diphosphate + L-isoleucyl-tRNAIle | - |
r | |
| ATP + L-isoleucine + tRNAIle | - |
Escherichia coli | AMP + diphosphate + L-isoleucyl-tRNAIle | - |
r | |
| ATP + L-isoleucine + tRNAIle | - |
Saccharomyces cerevisiae | AMP + diphosphate + L-isoleucyl-tRNAIle | - |
r | |
| ATP + L-isoleucine + tRNAIle | - |
Saccharomyces cerevisiae ATCC 204508 / S288c | AMP + diphosphate + L-isoleucyl-tRNAIle | - |
r | |
| additional information | the enzyme is also active with L-valine instead of L-isoleucine | Streptomyces griseus | ? | - |
? | |
| additional information | the enzyme is also active with L-valine instead of L-isoleucine | Saccharomyces cerevisiae | ? | - |
? | |
| additional information | analysed are ATP-PPi exchange assay, aminoacylation, and editing in the presence of tRNA of the recombinant wild-type and mutant enzymes. The enzyme is also active with L-valine instead of L-isoleucine, kinetics | Saccharomyces cerevisiae | ? | - |
? | |
| additional information | the enzyme is also active with L-valine instead of L-isoleucine, kinetics | Streptomyces griseus | ? | - |
? | |
| additional information | the enzyme is also active with L-valine instead of L-isoleucine | Saccharomyces cerevisiae ATCC 204508 / S288c | ? | - |
? | |
| additional information | analysed are ATP-PPi exchange assay, aminoacylation, and editing in the presence of tRNA of the recombinant wild-type and mutant enzymes. The enzyme is also active with L-valine instead of L-isoleucine, kinetics | Saccharomyces cerevisiae ATCC 204508 / S288c | ? | - |
? | |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| EcIleRS | - |
Escherichia coli |
| IleRS | - |
Streptomyces griseus |
| IleRS | - |
Escherichia coli |
| IleRS | - |
Saccharomyces cerevisiae |
| ileS | - |
Streptomyces griseus |
| ileS | - |
Escherichia coli |
| ileS | - |
Saccharomyces cerevisiae |
| ileS1 | - |
Escherichia coli |
| ileS2 | - |
Escherichia coli |
| Isoleucyl-tRNA synthetase | - |
Streptomyces griseus |
| Isoleucyl-tRNA synthetase | - |
Escherichia coli |
| Isoleucyl-tRNA synthetase | - |
Saccharomyces cerevisiae |
| ScIleRS | - |
Saccharomyces cerevisiae |
| SgIleRS | - |
Streptomyces griseus |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 30 | - |
aminoacylation assay at | Streptomyces griseus |
| 30 | - |
aminoacylation assay at | Saccharomyces cerevisiae |
| 37 | - |
aminoacylation assay at | Escherichia coli |
Turnover Number [1/s]
| Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.03 | - |
tRNAIle | pH 7.5, 30°C, deacylation, recombinant mutant D334A | Saccharomyces cerevisiae | |
| 0.038 | - |
AMP | pH 7.5, 30°C, deacylation, recombinant mutant D334A | Saccharomyces cerevisiae | |
| 0.13 | - |
tRNAIle | pH 7.5, 30°C, deacylation, recombinant wild-type enzyme | Saccharomyces cerevisiae | |
| 0.19 | - |
tRNAIle | pH 7.5, 30°C, deacylation, recombinant mutant D334A | Streptomyces griseus | |
| 0.2 | - |
AMP | pH 7.5, 30°C, deacylation, recombinant mutant D334A | Streptomyces griseus | |
| 0.2 | - |
AMP | pH 7.5, 30°C, deacylation, recombinant wild-type enzyme | Saccharomyces cerevisiae | |
| 0.64 | - |
AMP | pH 7.5, 30°C, deacylation, recombinant wild-type enzyme | Streptomyces griseus | |
| 0.65 | - |
tRNAIle | pH 7.5, 30°C, deacylation, recombinant wild-type enzyme | Streptomyces griseus | |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 7.5 | - |
aminoacylation assay at | Streptomyces griseus |
| 7.5 | - |
aminoacylation assay at | Escherichia coli |
| 7.5 | - |
aminoacylation assay at | Saccharomyces cerevisiae |
Cofactor
| Cofactor | Comment | Organism | Structure |
|---|---|---|---|
| ATP | - |
Streptomyces griseus | |
| ATP | - |
Escherichia coli | |
| ATP | - |
Saccharomyces cerevisiae | |
General Information
| General Information | Comment | Organism |
|---|---|---|
| evolution | enzyme IleRS is a class I aaRS enzyme built around the conserved N-terminal Rossmann fold catalytic domain, which encloses the synthetic site. Phylogenetic analysis suggests that the ileS1 and ileS2 genes of contemporary bacteria are the descendants of genes that might have arisen by an ancient duplication event before the separation of bacteria and archaea. The accuracy of Ile-tRNAIle synthesis may be entirely ensured by the powerful post-transfer editing domain, which is absolutely conserved through evolution. The origin of discrimination against valine in the synthetic reaction is evolutionarily conserved in IleRS, overview | Escherichia coli |
| evolution | phylogenetic analysis, the origin of discrimination against valine in the synthetic reaction is evolutionarily conserved in IleRS, overview | Streptomyces griseus |
| evolution | phylogenetic analysis, the origin of discrimination against valine in the synthetic reaction is evolutionarily conserved in IleRS, overview | Saccharomyces cerevisiae |
| malfunction | under error-prone conditions Streptomyces griseus IleRS is able to rescue the growth of an Escherichia coli lacking functional IleRS, providing the first evidence that tRNA-dependent pre-transfer editing in IleRS is not essential for cell viability | Escherichia coli |
| physiological function | isoleucyl-tRNA synthetase (IleRS) is responsible for decoding of isoleucine codons in all three domains of life. Besides isoleucine, IleRS also activates non-cognate valine with a discrimination factor as low as 200 and thus it requires editing to enhance accuracy of isoleucyltRNAIle (Ile-tRNAIle) synthesis. Enzyme IleRS is unusual among aminoacyl-tRNA synthetases in having a tRNA-dependent pre-transfer editing activity as an optional property. Some bacteria also have the enzymes (eukaryote-like) that cluster with eukaryotic IleRSs and exhibit low sensitivity to the antibiotic mupirocin. tRNA-dependent pre-transfer editing in IleRS is not essential for cell viability. Specificity of the editing pathways, overview | Escherichia coli |
| physiological function | isoleucyl-tRNA synthetase (IleRS) is responsible for decoding of isoleucine codons in all three domains of life. Besides isoleucine, IleRS also activates non-cognate valine with a discrimination factor as low as 200 and thus it requires editing to enhance accuracy of isoleucyltRNAIle (Ile-tRNAIle) synthesis. Enzyme IleRS is unusual among aminoacyl-tRNA synthetases in having a tRNA-dependent pre-transfer editing activity. The main tRNA-dependent pre-transfer editing pathway in ScIleRS is the enzyme-based aa-AMP hydrolysis. Specificity of the editing pathways, overview | Saccharomyces cerevisiae |
| physiological function | isoleucyl-tRNA synthetase (IleRS) is responsible for decoding of isoleucine codons in all three domains of life. Besides isoleucine, IleRS also activates non-cognate valine with a discrimination factor as low as 200 and thus it requires editing to enhance accuracy of isoleucyltRNAIle (Ile-tRNAIle) synthesis. The eukaryote-like enzyme from Streptomyces griseus IleRS lacks the tRNA-dependent pre-transfer editing activity, an unusual capacity of isoleucyl-tRNA synthetases (IleRS). At the same time, its synthetic site displays the 103fold drop in sensitivity to antibiotic mupirocin relative to the yeast enzyme. Under error-prone conditions Streptomyces griseus IleRS is able to rescue the growth of an Escherichia coli lacking functional IleRS, providing the first evidence that tRNA-dependent pre-transfer editing in IleRS is not essential for cell viability | Streptomyces griseus |
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