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Literature summary for 6.1.1.1 extracted from
- Redesigning the stereospecificity of tyrosyl-tRNA synthetase (2016), Proteins, 84, 240-253 .
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| gene tyrS, recombinant expression of His-tagged wild-type enzyme in Escherichia coli strain BL21(DE3), recombinant expression of His-tagged mutant enzymes in Saccharomyces cerevisiae strain BY4742 | Escherichia coli |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| D41N | site-directed mutagenesis | Escherichia coli |
| D81H/Q179E/Q201D | site-directed mutagenesis, the mutant shows no activity for L-Tyr and D-Tyr | Escherichia coli |
| D81K/Q179E | site-directed mutagenesis, the mutant shows no activity for L-Tyr and D-Tyr | Escherichia coli |
| D81K/Q179E/Q201D | site-directed mutagenesis, the mutant shows no activity for L-Tyr and D-Tyr | Escherichia coli |
| D81N | site-directed mutagenesis, the mutant shows a preference for L-Tyr that is much stronger than for the wild-type TyrRS. The ligand ammonium is coordinated at the L-Tyr endpoint by Asp41 (which replaces Asp81 in the coordination shell) and Tyr175 but not Gln179. At the D-Tyr endpoint, the ligand ammonium is coordinated by a mixture of Gln201, Tyr175, Asp41, and sometimes weakly by Gln179 | Escherichia coli |
| D81R | site-drected mutagenesis, the mutant shows low activity for L-Tyr and D-Tyr, and the same KM value for L-Tyr compared to wild-type enzyme, the mutant is D-Tyr specific, but with low activity | Escherichia coli |
| additional information | computational mutant design using the crystal structure of Escherichia coli TyrRS bound to a tyrosyl adenylate analogue, PDB ID 1VBM | Escherichia coli |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| additional information | - |
additional information | calculation of the L-Tyr/D-Tyr binding free energy difference. Michaelis-Menten kinetics of wild-type and mutant enzymes | Escherichia coli |
Metals/Ions
| Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|
| Mg2+ | required | Escherichia coli |  |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| ATP + L-tyrosine + tRNATyr | Escherichia coli | - |
AMP + diphosphate + L-tyrosyl-tRNATyr | - |
? | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Escherichia coli | P0AGJ9 | - |
- |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| recombinant enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography to homogeneity, recombinant His-tagged mutant enzymes from Saccharomyces cerevisiae strain BY4742 by nickel affinity chromatography to over 90% purity | Escherichia coli |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| ATP + D-tyrosine + tRNATyr | enzyme TyrRS has a detectable, natural activity for the D-tyrosine stereoisomer, only tenfold less than for L-Tyr | Escherichia coli | AMP + diphosphate + D-tyrosyl-tRNATyr | - |
? | |
| ATP + L-tyrosine + tRNATyr | - |
Escherichia coli | AMP + diphosphate + L-tyrosyl-tRNATyr | - |
? | |
| additional information | residues Asp81, Tyr175, Gln179, and Gln201 coordinate the ammonium group of the L-Tyr ligand | Escherichia coli | ? | - |
? | |
Subunits
| Subunits | Comment | Organism |
|---|---|---|
| homodimer | - |
Escherichia coli |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| Tyrosyl-tRNA synthetase | - |
Escherichia coli |
| TyrRS | - |
Escherichia coli |
| tyrS | - |
Escherichia coli |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 37 | - |
assay at | Escherichia coli |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 7.8 | - |
assay at | Escherichia coli |
Cofactor
| Cofactor | Comment | Organism | Structure |
|---|---|---|---|
| ATP | - |
Escherichia coli | |
General Information
| General Information | Comment | Organism |
|---|---|---|
| malfunction | of six mutants tested, two are active towards D-Tyr; one of these has an inverted stereospecificity, with a large preference for D-Tyr, but its activity is low | Escherichia coli |
| additional information | the TyrRS stereospecificity is robust towards charge rearrangements near the ligand. Whereas most aminoacyl-tRNA synthetases (aaRSs) have a strong preference for their L-amino acid substrate, TyrRS has a detectable, natural activity for the D-tyrosine stereoisomer, only tenfold less than for L-Tyr; additional protection against D-Tyr is usually provided by another enzyme, D-aminoacyl-tRNA hydrolase. Enzyme molecular dynamics simulations using the crystal structure of Escherichia coli TyrRS bound to a tyrosyl adenylate analogue, PDB ID 1VBM | Escherichia coli |
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