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Literature summary for 6.1.1.24 extracted from
- Synthesis of Glu-tRNAGln by engineered and natural aminoacyl-tRNA synthetases (2010), Biochemistry, 49, 6727-6736.
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| expression of C-terminally His-tagged mutant enzymes containing an N-terminal signal sequence tag directing the protein to the periplasm, without effect on the steady-state kinetic parameters of GlnRS. The mutants are expressed as N-terminal fusions with the leader sequence of the bacterial fd gene III protein in Escherichia coli | Escherichia coli |
| expression of GluRSND using a pCYB1 vector in Escherichia coli pLysS Rosetta cells | Methanothermobacter thermautotrophicus |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| L1L2 | site-directed mutagenesis, construction of a hybrid enzyme, kinetic comparison to wild-type enzyme and other hybrid enzyme mutants | Escherichia coli |
| additional information | construction of a hybrid enzyme in which 23 amino acids from the catalytic domain of Escherichia coli glutaminyl-tRNA synthetase, GlnRS, are replaced with the corresponding residues of human glutamyl-tRNA synthetase, GluRS. Further introduction of two distal surface loops bridging core secondary structural elements of the Rossmann fold then produces a hybrid enzyme GlnRS S1/L1/L2. The engineered hybrid GlnRS S1/L1/L2 synthesizes Glu-tRNAGln over 104fold more efficiently than GlnRS, overview. The simultaneous optimization of paired amino acid and tRNA binding sites found in a naturally occurring enzyme is not recapitulated in a hybrid that is successfully engineered for amino acid complementarity. Design and characterization of four additional hybrids identify further residues involved in improving complementarity for glutamate and in communicating between amino acid and tRNA binding sites, complementarity for tRNA, mutant enzyme structure, overview. Relationship between tRNA and amino acid binding sites in the hybrid enzymes, overview | Escherichia coli |
| T231L | site-directed mutagenesis, construction of a hybrid enzyme, kinetic comparison to wild-type enzyme and other hybrid enzyme mutants | Escherichia coli |
| V218S | site-directed mutagenesis, construction of a hybrid enzyme, kinetic comparison to wild-type enzyme and other hybrid enzyme mutants | Escherichia coli |
| W256Y | site-directed mutagenesis, construction of a hybrid enzyme, kinetic comparison to wild-type enzyme and other hybrid enzyme mutants | Escherichia coli |
| Y240D/D241F | site-directed mutagenesis, construction of a hybrid enzyme, kinetic comparison to wild-type enzyme and other hybrid enzyme mutants | Escherichia coli |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| additional information | - |
additional information | detailed comparison of kinetic parameters between Methanothermobacter thermautotrophicus GluRSND and Escherichia coli recombinant hybrid GlnRS mutants, which are also capable of Glu-tRNAGln synthesis, overview | Methanothermobacter thermautotrophicus | |
| additional information | - |
additional information | detailed comparison of kinetic parameters between recombinant hybrid GlnRS S1/L1/L2 and other recombinant hybrid mutant enzymes, with the naturally occurring Methanothermobacter thermautotrophicus GluRSND, which is also capable of Glu-tRNAGln synthesis, overview. Both kcat and Km for glutamate are recapitulated in the engineered enzyme, but Km for tRNA is 200fold higher | Escherichia coli | |
| 0.000038 | - |
tRNAGln | recombinant hybrid GlnRS S1/L1/L2, pH not specified in the publication, temperature not specified in the publication | Methanothermobacter thermautotrophicus |  |
| 0.0076 | - |
tRNAGln | recombinant hybrid GlnRS S1/L1/L2, pH not specified in the publication, temperature not specified in the publication | Escherichia coli | |
| 0.62 | - |
ATP | recombinant hybrid GlnRS S1/L1/L2, pH not specified in the publication, temperature not specified in the publication | Escherichia coli | |
| 3.1 | - |
ATP | recombinant hybrid GlnRS S1/L1/L2, pH not specified in the publication, temperature not specified in the publication | Methanothermobacter thermautotrophicus | |
| 5.8 | - |
L-Glu | recombinant hybrid GlnRS S1/L1/L2, pH not specified in the publication, temperature not specified in the publication | Escherichia coli | |
| 6.2 | - |
L-Glu | recombinant hybrid GlnRS S1/L1/L2, pH not specified in the publication, temperature not specified in the publication | Methanothermobacter thermautotrophicus | |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| ATP + L-Glu + tRNAGln | Methanothermobacter thermautotrophicus | - |
AMP + diphosphate + Glu-tRNAGln | - |
? | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Escherichia coli | - |
- |
- |
| Methanothermobacter thermautotrophicus | - |
- |
- |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| recombinant GluRSND from Escherichia coli pLysS Rosetta cells | Methanothermobacter thermautotrophicus |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| ATP + L-Glu + tRNAGln | - |
Methanothermobacter thermautotrophicus | AMP + diphosphate + Glu-tRNAGln | - |
? | |
| ATP + L-Glu + tRNAGln | synthesis of Glu-tRNAGln by engineered, not natural GlnRS, overview | Escherichia coli | AMP + diphosphate + Glu-tRNAGln | - |
? | |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| GluRSND | - |
Methanothermobacter thermautotrophicus |
| GlxRS | - |
Methanothermobacter thermautotrophicus |
| nondiscriminating GluRS | - |
Methanothermobacter thermautotrophicus |
Turnover Number [1/s]
| Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.04 | - |
tRNAGln | recombinant hybrid GlnRS S1/L1/L2, pH not specified in the publication, temperature not specified in the publication | Escherichia coli | |
| 0.09 | - |
L-Glu | recombinant hybrid GlnRS S1/L1/L2, pH not specified in the publication, temperature not specified in the publication | Escherichia coli | |
| 0.09 | - |
L-Glu | recombinant hybrid GlnRS S1/L1/L2, pH not specified in the publication, temperature not specified in the publication | Methanothermobacter thermautotrophicus | |
| 0.1 | - |
ATP | recombinant hybrid GlnRS S1/L1/L2, pH not specified in the publication, temperature not specified in the publication | Escherichia coli | |
| 0.1 | - |
ATP | recombinant hybrid GlnRS S1/L1/L2, pH not specified in the publication, temperature not specified in the publication | Methanothermobacter thermautotrophicus | |
| 0.1 | - |
tRNAGln | recombinant hybrid GlnRS S1/L1/L2, pH not specified in the publication, temperature not specified in the publication | Methanothermobacter thermautotrophicus | |
Cofactor
| Cofactor | Comment | Organism | Structure |
|---|---|---|---|
| ATP | - |
Escherichia coli | |
| ATP | - |
Methanothermobacter thermautotrophicus | |
General Information
| General Information | Comment | Organism |
|---|---|---|
| additional information | a hybrid enzyme, in which 23 amino acids from the catalytic domain of Escherichia coli glutaminyl-tRNA synthetase, GlnRS, are replaced with the corresponding residues of human glutamyl-tRNA synthetase, GluRS, synthesizes Glu-tRNAGln over 104fold more efficiently than GlnRS. Identification of residues involved in improving complementarity for glutamate and in communicating between amino acid and tRNA binding sites, overview | Escherichia coli |
kcat/KM [mM/s]
| kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.0155 | - |
L-Glu | recombinant hybrid GlnRS S1/L1/L2, pH not specified in the publication, temperature not specified in the publication | Escherichia coli | |
| 0.16 | - |
ATP | recombinant hybrid GlnRS S1/L1/L2, pH not specified in the publication, temperature not specified in the publication | Escherichia coli | |
| 5.2 | - |
tRNAGln | recombinant hybrid GlnRS S1/L1/L2, pH not specified in the publication, temperature not specified in the publication | Escherichia coli | |
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