Department of Chemistry, Faculty of Science, University of Zagreb, Croatia.
Institute of Chemistry and Biochemistry - Organic Chemistry, Freie Universitat Berlin, Germany.
FEBS J. 2020 Feb;287(4):800-813. doi: 10.1111/febs.15053. Epub 2019 Sep 18.
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 K . 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-tRNA with a rate of 40 s 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).
异亮氨酰-tRNA 合成酶 (IleRS) 是理解在合成和编辑反应中针对较小疏水性底物的特异性如何进化的范例。IleRS 错误激活非蛋白源的正缬氨酸 (Nva) 和蛋白源的缬氨酸 (Val)，其效率比相应的异亮氨酸 (Ile) 低 200 倍。然而，IleRS 的水解编辑防止了翻译错误。Nva 和 Val 都比 Ile 小一个亚甲基。额外的亚甲基去除如何影响 IleRS 的特异性？我们发现，非蛋白源的α-氨基丁酸 (Abu) 的激活效率比 Nva 和 Val 低 30 倍，这表明第二个亚甲基的去除带来的惩罚较低。与 Nva 和 Val 一样，对 Abu 的歧视主要源于更高的 K 值。为了研究增加疏水性是否可以弥补范德华相互作用的损失，我们测试了氟化 Abu 类似物。我们发现，氟取代进一步阻碍了 IleRS 的激活，而对进化相关的 ValRS 的阻碍更大。这表明疏水性不是这些酶中底物结合的主要驱动力。最后，7100 的判别因子表明 IleRS 预计不会编辑 Abu。然而，我们发现 IleRS 编辑结构域以 40 s 的速度水解 Abu-tRNA，并且氟取代并没有减缓水解。这就提出了关于编辑结构域特异性及其进化的机制的有趣问题。理解塑造 IleRS 特异性的原因对于重新设计翻译以适应包括氟化氨基酸在内的人工底物也很重要。酶：异亮氨酰-tRNA 合成酶（EC 6.1.1.5），亮氨酰-tRNA 合成酶（EC 6.1.1.4），缬氨酰-tRNA 合成酶（EC 6.1.1.9）。
