Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06511.
Department of Chemistry, Boston College, Chestnut Hill, Massachusetts 02467.
J Biol Chem. 2019 Aug 23;294(34):12855-12865. doi: 10.1074/jbc.RA119.008219. Epub 2019 Jul 11.
Selenocysteine (Sec) is the 21st genetically encoded amino acid in organisms across all domains of life. Although structurally similar to cysteine (Cys), the Sec selenol group has unique properties that are attractive for protein engineering and biotechnology applications. Production of designer proteins with Sec (selenoproteins) at desired positions is now possible with engineered translation systems in However, obtaining pure selenoproteins at high yields is limited by the accumulation of free Sec in cells, causing undesired incorporation of Sec at Cys codons due to the inability of cysteinyl-tRNA synthetase (CysRS) to discriminate against Sec. Sec misincorporation is toxic to cells and causes protein aggregation in yeast. To overcome this limitation, here we investigated a CysRS from the selenium accumulator plant that is reported to reject Sec Sequence analysis revealed a rare His → Asn variation adjacent to the CysRS catalytic pocket. Introducing this variation into and CysRS increased resistance to the toxic effects of selenite and selenomethionine (SeMet), respectively. Although the CysRS variant could still use Sec as a substrate , we observed a reduction in the frequency of Sec misincorporation at Cys codons We surmise that the His → Asn variation can be introduced into any CysRS to provide a fitness advantage for strains burdened by Sec misincorporation and selenium toxicity. Our results also support the notion that the CysRS variant provides higher specificity for Cys as a mechanism for plants to grow in selenium-rich soils.
硒代半胱氨酸（Sec）是所有生命领域的生物中第 21 种遗传编码氨基酸。虽然结构上与半胱氨酸（Cys）相似，但 Sec 的硒醇基团具有独特的性质，这使其成为蛋白质工程和生物技术应用的理想选择。通过工程化的翻译系统，现在可以在期望的位置生产含有 Sec（硒蛋白）的设计蛋白。然而，由于细胞中游离 Sec 的积累，高产量获得纯硒蛋白受到限制，这会导致由于半胱氨酸 tRNA 合成酶（CysRS）无法区分 Sec，Cys 密码子的 Sec 错误掺入。Sec 错误掺入对细胞有毒，并导致酵母中的蛋白质聚集。为了克服这一限制，我们研究了一种来自硒积累植物的 CysRS，据报道该酶可以拒绝 Sec。序列分析揭示了 CysRS 催化口袋附近一个罕见的 His→Asn 变异。将这种变异引入 和 CysRS 分别提高了对亚硒酸盐和硒代蛋氨酸（SeMet）的毒性作用的抗性。尽管 CysRS 变体仍然可以将 Sec 用作底物，但我们观察到 Cys 密码子上 Sec 错误掺入的频率降低。我们推测，可以将 His→Asn 变异引入任何 CysRS 中，为受到 Sec 错误掺入和硒毒性负担的菌株提供适应性优势。我们的结果还支持这样一种观点，即 CysRS 变体对 Cys 具有更高的特异性，这是植物在富含硒的土壤中生长的一种机制。
