Transthyretin was discovered in the 1940s, named after its ability to bind thyroid hormones and retinol. In the genomic era, transthyretins were found to be part of a larger family with homologs of no obvious function, then called transthyretin-related proteins. Thus, it was proposed that the transthyretin gene could be the result of gene duplication of an ancestral of this newly identified homolog, later found out to be an enzyme involved in uric acid degradation, then named HIUase (5-hydroxy-isourate hydrolase). Here, we sought to re-enact the evolutionary history of this protein family by reconstructing, from a phylogeny inferred from 123 vertebrate sequences, three ancestors corresponding to key moments in their evolution—before duplication; the common transthyretin ancestor after gene duplication and the common ancestor of Eutheria transthyretins. Experimental and computational characterization showed the reconstructed ancestor before duplication was unable to bind thyroxine and likely presented the modern HIUase reaction mechanism, while the substitutions after duplication prevented that activity and were enough to provide stable thyroxine binding, as confirmed by calorimetry and x-ray diffraction. The Eutheria transthyretin ancestor was less prone to characterization, but limited data suggested thyroxine binding as expected. Sequence/structure analysis suggests an early ability to bind the Retinol Binding Protein. We solved the X-ray structures from the two first ancestors, the first at 1.46 resolution, the second at 1.55 resolution with well-defined electron density for thyroxine, providing a useful tool for the understanding of structural adaptation from enzyme to hormone distributor.

转甲状腺素蛋白于 1940 年代被发现，以其结合甲状腺激素和视黄醇的能力命名。在基因组时代，转甲状腺素蛋白被发现是一个更大家族的一部分，具有没有明显功能的同源物，然后被称为转甲状腺素蛋白相关蛋白。因此，有人提出转甲状腺素蛋白基因可能是这种新鉴定的同源物的祖先基因复制的结果，后来发现它是一种参与尿酸降解的酶，然后命名为 HIUase（5-羟基异尿酸水解酶）。在这里，我们试图通过从 123 种脊椎动物序列推断出的系统发育中重建对应于其进化关键时刻的三个祖先——复制之前，重现这个蛋白质家族的进化历史；基因复制后的共同转甲状腺素蛋白祖先和 Eutheria 转甲状腺素蛋白的共同祖先。实验和计算表征表明，复制前重建的祖先无法结合甲状腺素，可能呈现现代 HIUase 反应机制，而复制后的取代阻止了这种活性，足以提供稳定的甲状腺素结合，如量热法和 X 射线衍射所证实. Eutheria 转甲状腺素蛋白祖先不太容易被表征，但有限的数据表明甲状腺素与预期的结合。序列/结构分析表明结合视黄醇结合蛋白的早期能力。我们解决了两个第一个祖先的 X 射线结构，第一个分辨率为 1.46，第二个分辨率为 1.55，具有明确定义的甲状腺素电子密度，