Shared promiscuous activities and evolutionary features in various members of the amidohydrolase superfamily
Shared promiscuous activities and evolutionary features in various members of the amidohydrolase superfamily
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DOI：
10.1021/bi051021e
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发表时间：
2005-09-27
期刊：
影响因子：
2.9
通讯作者：
Tawfik, DS
中科院分区：
文献类型：
--
作者：
Roodveldt, C;Tawfik, DS
酰胺水解酶超家族包括数百种具有单核或双核活性中心的(β/α)(8)桶折叠的水解酶，以及不同的底物和反应谱。同一超家族不同成员之间的混杂活动或交叉反应可能提供关于进化和机制关系的重要线索。我们研究了三个成员：二氢水解酶(DHO)、磷酸三酯酶(PTE)和PTE同源蛋白(PHP)。特别令人感兴趣的是PTE，它被认为是在过去几十年中进化的，以及PHP，一个功能未知的氨基水解酶超家族成员，也是PTE已知的最接近的同源物。我们在这些酶中发现了不同的和部分重叠的混杂活性模式，包括PTE中显著的内酯酶活性，PTE和PHP中的酯酶活性，以及DHO中的弱PTE活性。采用定向进化的方法提高了PTE和PHP的酯酶活性。值得注意的是，在PTE或PHP中，最频繁的增加酯酶活性的突变映射到它们叠加的3D结构中的相同位置。进化的变异体还表现出新获得的未被选择的混杂活动，包括非常弱但可测量的PHP中的对氧磷酶活性。我们的结果阐明了这些酶之间的机制、结构和进化联系，并突出了研究实验室进化中间体的重要性，这些中间体可能类似于节点中间体，沿着导致酶超家族差异的进化路径。
The amidohydrolase superfamily comprises hundreds of hydrolytic enzymes of the (beta/alpha)(8) barrel fold with mono- or binuclear active-site metal centers, and a diverse spectrum of substrates and reactions. Promiscuous activities, or cross-reactivities, between different members of the same superfamily may provide important hints regarding evolutionary and mechanistic relationships. We examined three members: dihydroorotase (DHO), phosphotriesterase (PTE), and PTE-homology protein (PHP). Of particular interest are PTE, which is thought to have evolved within the last several decades, and PHP, an amidohydrolase superfamily member of unknown function, and the closest known homologue of PTE. We found a diverse and partially overlapping pattern of promiscuous activities in these enzymes, including a significant lactonase activity in PTE, esterase activities in both PTE and PHP, and a weak PTE activity in DHO. Directed evolution was applied to improve the promiscuous esterase activities of PTE and PHP. Remarkably, the most recurrent mutation increasing esterase activity in PTE, or PHP, maps to the same location in their superposed 3D structures. The evolved variants also exhibit newly acquired promiscuous activities that were not selected for, including very weak, yet measurable, paraoxonase activity in PHP. Our results illustrate the mechanistic, structural, and evolutionary links between these enzymes, and highlight the importance of studying laboratory evolution intermediates that might resemble node intermediates along the evolutionary pathways leading to the divergence of enzyme superfamilies.