Accurate translation of genetic information into proteins is vital for cell sustainability. ProXp-ala prevents proteome-wide Pro-to-Ala mutations by hydrolyzing misacylated Ala-tRNAPro, which is synthesized by prolyl-tRNA synthetase. Bacterial ProXp-ala was previously shown to combine a size-based exclusion mechanism with conformational and chemical selection for the recognition of the alanyl moiety, whereas tRNAPro is selected via recognition of tRNA acceptor-stem elements G72 and A73. The identity of these critical bases changed during evolution with eukaryotic cytosolic tRNAPro possessing a cytosine at the corresponding positions. The mechanism by which eukaryotic ProXp-ala adapted to these changes remains unknown. In this work, recognition of the aminoacyl moiety and tRNA acceptor stem by human (Homo sapiens, or Hs) ProXp-ala was examined. Enzymatic assays revealed that Hs ProXp-ala requires C72 and C73 in the context of Hs cytosolic tRNAPro for efficient deacylation of mischarged Ala-tRNAPro. The strong dependence on these bases prevents cross-species deacylation of bacterial Ala-tRNAPro or of Hs mitochondrial Ala-tRNAPro by the human enzyme. Similar to the bacterial enzyme, Hs ProXp-ala showed strong tRNA acceptor-stem recognition but differed in its amino acid specificity profile relative to bacterial ProXp-ala. Changes at conserved residues in both the Hs and bacterial ProXp-ala substrate-binding pockets modulated this specificity. These results illustrate how the mechanism of substrate selection diverged during the evolution of the ProXp-ala family, providing the first example of a trans-editing domain whose specificity evolved to adapt to changes in its tRNA substrate.

中文翻译：

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人类反式编辑酶显示tRNA受体干特异性和轻松的氨基酸选择性

将遗传信息准确翻译为蛋白质对于细胞可持续性至关重要。ProXp-ala通过水解由脯氨酰基-tRNA合成酶合成的错误酰化的Ala-tRNAPro来防止整个蛋白质组的Pro-to-Ala突变。先前显示细菌ProXp-ala将基于大小的排阻机制与构象和化学选择相结合，以识别丙氨酰基部分，而通过识别tRNA受体-干元件G72和A73选择tRNAPro。这些关键碱基的身份在进化过程中发生了变化，因为真核细胞质tRNAPro在相应位置具有胞嘧啶。真核ProXp-ala适应这些变化的机制仍然未知。在这项工作中，检查了人类（智人或Hs）ProXp-ala对氨基酰基部分和tRNA受体茎的识别。酶促测定显示，Hs ProXp-ala在Hs胞质tRNAPro的背景下需要C72和C73才能使带错电荷的Ala-tRNAPro有效脱酰。对这些碱基的强烈依赖性阻止了人类酶对细菌Ala-tRNAPro或Hs线粒体Ala-tRNAPro的种间去酰化作用。与细菌酶类似，Hs ProXp-ala显示出强大的tRNA受体-茎识别能力，但其氨基酸特异性谱相对于细菌ProXp-ala不同。Hs和细菌ProXp-ala底物结合口袋中保守残基的变化调节了这种特异性。这些结果说明了ProXp-ala家族进化过程中底物选择的机制是如何变化的，这提供了反转录域的第一个例子，其特异性进化为适应其tRNA底物的变化。