Mechanism of discrimination of isoleucyl‐tRNA synthetase against nonproteinogenic α‐aminobutyrate and its fluorinated analogues
| Autor: | Beate Koksch, Johann Moschner, Igor Zivkovic, Ita Gruić-Sovulj |
|---|---|
| Rok vydání: | 2019 |
| Předmět: |
Isoleucine-tRNA Ligase
Models Molecular Protein Conformation alpha-Helical 0301 basic medicine Halogenation Valine-tRNA Ligase Stereochemistry Aminobutyrate Gene Expression Biochemistry Substrate Specificity 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Valine Escherichia coli Protein Interaction Domains and Motifs Cloning Molecular Methylene Molecular Biology chemistry.chemical_classification Binding Sites Chemistry Aminoacyl tRNA synthetase Aminobutyrates Escherichia coli Proteins Cell Biology aminoacyl-tRNA synthetase fluorinated amino acids hydrophobicity nonproteinogenic amino acids proofreading Recombinant Proteins Amino acid Kinetics 030104 developmental biology Enzyme 030220 oncology & carcinogenesis Thermodynamics Leucine-tRNA Ligase Protein Conformation beta-Strand Norvaline Isoleucine Protein Binding |
| Zdroj: | The FEBS Journal. 287:800-813 |
| ISSN: | 1742-4658 1742-464X |
| DOI: | 10.1111/febs.15053 |
| Popis: | Isoleucyl-tRNA synthetase (IleRS) is a paradigm for understanding how specificity against smaller hydrophobic substrates evolved in both the synthetic and editing reactions. IleRS misactivates nonproteinogenic norvaline (Nva) and proteinogenic valine (Val), with a 200-fold lower efficiency than the cognate isoleucine (Ile). Translational errors are, however, prevented by IleRS hydrolytic editing. Nva and Val are both smaller than Ile by a single methylene group. How does the removal of one additional methylene group affects IleRS specificity? We found that the nonproteinogenic α-aminobutyrate (Abu) is activated 30-fold less efficiently than Nva and Val, indicating that the removal of the second methylene group comes with a lower penalty. As with Nva and Val, discrimination against Abu predominantly originated from a higher KM . To examine whether increased hydrophobicity could compensate for the loss of van der Waals interactions, we tested fluorinated Abu analogues. We found that fluorination further hampered activation by IleRS, and even more so by the evolutionary-related ValRS. This suggests that hydrophobicity is not a main driving force of substrate binding in these enzymes. Finally, a discrimination factor of 7100 suggests that IleRS is not expected to edit Abu. However, we found that the IleRS editing domain hydrolyzes Abu-tRNAIle with a rate of 40 s-1 and the introduction of fluorine did not slow down the hydrolysis. This raises interesting questions regarding the mechanism of specificity of the editing domain and its evolution. Understanding what shapes IleRS specificity is also of importance for reengineering translation to accommodate artificial substrates including fluorinated amino acids. ENZYMES: Isoleucyl-tRNA synthetase (EC 6.1.1.5), leucyl-tRNA synthetase (EC 6.1.1.4), valyl-tRNA synthetase (EC 6.1.1.9). |
| Databáze: | OpenAIRE |
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