Cloned (Comment) | Organism |
---|---|
expression of His-tagged wild-type and mutant enzymes in strain BL21(DE3), subcloning in strain DH5alpha | Escherichia coli |
Protein Variants | Comment | Organism |
---|---|---|
A293D | site-directed mutagenesis, the mutant activity is similar to the wild-type enzyme | Escherichia coli |
A293E | site-directed mutagenesis, the mutant activity is similar to the wild-type enzyme | Escherichia coli |
A293K | site-directed mutagenesis, the post-transfer editing activity of the isolated CP1-domain is enhanced compared to the wild-type enzyme's domain | Escherichia coli |
A293R | site-directed mutagenesis, the post-transfer editing activity of the isolated CP1-domain is enhanced compared to the wild-type enzyme's domain | Escherichia coli |
D345A | site-directed mutagenesis, the mutation in the isolated CP1-domain abolishes hydrolytic post-transfer editing activity | Escherichia coli |
additional information | isolated LeuRS CP1 domain requires idiosyncratic adaptations to confer editing activity independent of the full-length enzyme, the beta-strands, which link the CP1 domain to the aminoacylation core of LeuRS, are required for editing of mischarged tRNALeu, hydrolytic activity is also enhanced by inclusion of short flexible peptides, called hinges, at the end of both LeuRS beta-strands, overview | Escherichia coli |
T247V | site-directed mutagenesis, hydrolysis of Ile-tRNALeu is completely abolished | Escherichia coli |
T247V/T248V | site-directed mutagenesis, the double mutation abolishes post-transfer editing activity | Escherichia coli |
T248V | site-directed mutagenesis, hydrolysis of Ile-tRNALeu is completely abolished | Escherichia coli |
T252A | site-directed mutagenesis, the mutation in the full-length LeuRS uncouples specificity and hydrolyzes correctly charged LeutRNALeu | Escherichia coli |
T252Y | site-directed mutagenesis, the mutation occupies the amino acid binding pocket and blocks the binding of substrate to abolish editing activity | Escherichia coli |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | - |
Escherichia coli |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + L-leucine + tRNALeu | Escherichia coli | - |
AMP + diphosphate + L-leucyl-tRNALeu | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Escherichia coli | - |
- |
- |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged wild-type and mutant enzymes from strain BL21(DE3) by nickel affiniry chromatography | Escherichia coli |
Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|
additional information | - |
hydrolytic post-transfer editing activity of wild-type and D345A mutant LeuRS CP1 domains, activities of truncation mutants, overview | Escherichia coli |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + L-leucine + tRNALeu | - |
Escherichia coli | AMP + diphosphate + L-leucyl-tRNALeu | - |
? | |
ATP + L-leucine + tRNALeu | the editing domain called CP1 is required for hydrolyzing the incorrectly misaminoacylated noncognate amino acids Ile and Val, the beta-strands, which link the CP1 domain to the aminoacylation core of LeuRS, are required for editing of mischarged tRNALeu, hydrolytic activity is also enhanced by inclusion of short flexible peptides, called hinges, at the end of both LeuRS beta-strands, overview | Escherichia coli | AMP + diphosphate + L-leucyl-tRNALeu | - |
? | |
additional information | isolated LeuRS CP1 domain requires idiosyncratic adaptations to confer editing activity independent of the full-length enzyme, overview | Escherichia coli | ? | - |
? |
Synonyms | Comment | Organism |
---|---|---|
Leucyl-tRNA synthetase | - |
Escherichia coli |
LeuRS | - |
Escherichia coli |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
37 | - |
assay at | Escherichia coli |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | - |
assay at | Escherichia coli |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
ATP | - |
Escherichia coli |