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Literature summary extracted from

  • Cvetesic, N.; Dulic, M.; Bilus, M.; Sostaric, N.; Lenhard, B.; Gruic-Sovulj, I.
    Naturally occurring isoleucyl-tRNA synthetase without tRNA-dependent pre-transfer editing (2016), J. Biol. Chem., 291, 8618-8631 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
6.1.1.5 gene ileS, phylogenetic analysis, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) Streptomyces griseus
6.1.1.5 gene ileS, phylogenetic analysis, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain Rosetta (DE3) Saccharomyces cerevisiae
6.1.1.5 genes ileS1 and ileS2, phylogenetic analysis Escherichia coli

Protein Variants

EC Number Protein Variants Comment Organism
6.1.1.5 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
6.1.1.5 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

EC Number Inhibitors Comment Organism Structure
6.1.1.5 mupirocin inhibition of isoleucine activation by mupirocin, competitive inhibition, analyzed with ATP-diphosphate exchange reaction Saccharomyces cerevisiae
6.1.1.5 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]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
6.1.1.5 additional information
-
 additional information single-turnover kinetic analysis Escherichia coli
6.1.1.5 additional information
-
 additional information single-turnover kinetic analysis, activation kinetics of isoleucine and valine by ScIleRS at 30°C Saccharomyces cerevisiae
6.1.1.5 additional information
-
 additional information single-turnover kinetic analysis, activation kinetics of isoleucine and valine by SgIleRS at 30°C Streptomyces griseus

Localization

EC Number Localization Comment Organism GeneOntology No. Textmining
6.1.1.5 cytoplasm
-
 Saccharomyces cerevisiae 5737
-

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
6.1.1.5 Mg2+ required Streptomyces griseus
6.1.1.5 Mg2+ required Escherichia coli
6.1.1.5 Mg2+ required Saccharomyces cerevisiae
6.1.1.5 Zn2+ required Streptomyces griseus
6.1.1.5 Zn2+ required Escherichia coli
6.1.1.5 Zn2+ required Saccharomyces cerevisiae

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
6.1.1.5 ATP + L-isoleucine + tRNAIle Streptomyces griseus
-
 AMP + diphosphate + L-isoleucyl-tRNAIle
-
 r
6.1.1.5 ATP + L-isoleucine + tRNAIle Escherichia coli
-
 AMP + diphosphate + L-isoleucyl-tRNAIle
-
 r
6.1.1.5 ATP + L-isoleucine + tRNAIle Saccharomyces cerevisiae
-
 AMP + diphosphate + L-isoleucyl-tRNAIle
-
 r
6.1.1.5 ATP + L-isoleucine + tRNAIle Saccharomyces cerevisiae ATCC 204508 / S288c
-
 AMP + diphosphate + L-isoleucyl-tRNAIle
-
 r
6.1.1.5 additional information Streptomyces griseus the enzyme is also active with L-valine instead of L-isoleucine ?
-
 ?
6.1.1.5 additional information Saccharomyces cerevisiae the enzyme is also active with L-valine instead of L-isoleucine ?
-
 ?
6.1.1.5 additional information Saccharomyces cerevisiae ATCC 204508 / S288c the enzyme is also active with L-valine instead of L-isoleucine ?
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
6.1.1.5 Escherichia coli P00956 gene ileS
-
6.1.1.5 Saccharomyces cerevisiae P09436
-
-
6.1.1.5 Saccharomyces cerevisiae ATCC 204508 / S288c P09436
-
-
6.1.1.5 Streptomyces griseus
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
6.1.1.5 recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography Streptomyces griseus
6.1.1.5 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)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
6.1.1.5 ATP + L-isoleucine + tRNAIle
-
 Streptomyces griseus AMP + diphosphate + L-isoleucyl-tRNAIle
-
 r
6.1.1.5 ATP + L-isoleucine + tRNAIle
-
 Escherichia coli AMP + diphosphate + L-isoleucyl-tRNAIle
-
 r
6.1.1.5 ATP + L-isoleucine + tRNAIle
-
 Saccharomyces cerevisiae AMP + diphosphate + L-isoleucyl-tRNAIle
-
 r
6.1.1.5 ATP + L-isoleucine + tRNAIle
-
 Saccharomyces cerevisiae ATCC 204508 / S288c AMP + diphosphate + L-isoleucyl-tRNAIle
-
 r
6.1.1.5 additional information the enzyme is also active with L-valine instead of L-isoleucine Streptomyces griseus ?
-
 ?
6.1.1.5 additional information the enzyme is also active with L-valine instead of L-isoleucine Saccharomyces cerevisiae ?
-
 ?
6.1.1.5 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 ?
-
 ?
6.1.1.5 additional information the enzyme is also active with L-valine instead of L-isoleucine, kinetics Streptomyces griseus ?
-
 ?
6.1.1.5 additional information the enzyme is also active with L-valine instead of L-isoleucine Saccharomyces cerevisiae ATCC 204508 / S288c ?
-
 ?
6.1.1.5 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

EC Number Synonyms Comment Organism
6.1.1.5 EcIleRS
-
 Escherichia coli
6.1.1.5 IleRS
-
 Streptomyces griseus
6.1.1.5 IleRS
-
 Escherichia coli
6.1.1.5 IleRS
-
 Saccharomyces cerevisiae
6.1.1.5 ileS
-
 Streptomyces griseus
6.1.1.5 ileS
-
 Escherichia coli
6.1.1.5 ileS
-
 Saccharomyces cerevisiae
6.1.1.5 ileS1
-
 Escherichia coli
6.1.1.5 ileS2
-
 Escherichia coli
6.1.1.5 Isoleucyl-tRNA synthetase
-
 Streptomyces griseus
6.1.1.5 Isoleucyl-tRNA synthetase
-
 Escherichia coli
6.1.1.5 Isoleucyl-tRNA synthetase
-
 Saccharomyces cerevisiae
6.1.1.5 ScIleRS
-
 Saccharomyces cerevisiae
6.1.1.5 SgIleRS
-
 Streptomyces griseus

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
6.1.1.5 30
-
 aminoacylation assay at Streptomyces griseus
6.1.1.5 30
-
 aminoacylation assay at Saccharomyces cerevisiae
6.1.1.5 37
-
 aminoacylation assay at Escherichia coli

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
6.1.1.5 0.03
-
 tRNAIle pH 7.5, 30°C, deacylation, recombinant mutant D334A Saccharomyces cerevisiae
6.1.1.5 0.038
-
 AMP pH 7.5, 30°C, deacylation, recombinant mutant D334A Saccharomyces cerevisiae
6.1.1.5 0.13
-
 tRNAIle pH 7.5, 30°C, deacylation, recombinant wild-type enzyme Saccharomyces cerevisiae
6.1.1.5 0.19
-
 tRNAIle pH 7.5, 30°C, deacylation, recombinant mutant D334A Streptomyces griseus
6.1.1.5 0.2
-
 AMP pH 7.5, 30°C, deacylation, recombinant mutant D334A Streptomyces griseus
6.1.1.5 0.2
-
 AMP pH 7.5, 30°C, deacylation, recombinant wild-type enzyme Saccharomyces cerevisiae
6.1.1.5 0.64
-
 AMP pH 7.5, 30°C, deacylation, recombinant wild-type enzyme Streptomyces griseus
6.1.1.5 0.65
-
 tRNAIle pH 7.5, 30°C, deacylation, recombinant wild-type enzyme Streptomyces griseus

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
6.1.1.5 7.5
-
 aminoacylation assay at Streptomyces griseus
6.1.1.5 7.5
-
 aminoacylation assay at Escherichia coli
6.1.1.5 7.5
-
 aminoacylation assay at Saccharomyces cerevisiae

Cofactor

EC Number Cofactor Comment Organism Structure
6.1.1.5 ATP
-
 Streptomyces griseus
6.1.1.5 ATP
-
 Escherichia coli
6.1.1.5 ATP
-
 Saccharomyces cerevisiae

General Information

EC Number General Information Comment Organism
6.1.1.5 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
6.1.1.5 evolution phylogenetic analysis, the origin of discrimination against valine in the synthetic reaction is evolutionarily conserved in IleRS, overview Streptomyces griseus
6.1.1.5 evolution phylogenetic analysis, the origin of discrimination against valine in the synthetic reaction is evolutionarily conserved in IleRS, overview Saccharomyces cerevisiae
6.1.1.5 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
6.1.1.5 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
6.1.1.5 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
6.1.1.5 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