Reconstructing a Missing Link in the Evolution of a Recently Diverged Phosphotriesterase by Active-Site Loop Remodeling
Reconstructing a Missing Link in the Evolution of a Recently Diverged Phosphotriesterase by Active-Site Loop Remodeling
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DOI：
10.1021/bi300694t
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发表时间：
2012-08-07
期刊：
影响因子：
2.9
通讯作者：
Tawfik, Dan S.
中科院分区：
文献类型：
--
作者：
Afriat-Jurnou, Livnat;Jackson, Colin J.;Tawfik, Dan S.
在有机磷农药引入几十年后，细菌磷酸三酯酶（PTE）已经进化到以显着的效率催化其降解。它们最近的已知亲戚，具有混杂磷酸三酯酶活性的内酯酶，被称为PTE样内酯酶（PLL），只有30%的序列相同性，并且在活性位点环的构型上也不同。因此，推测PTE是从尚未知晓的PLL进化而来的，其主要活性是群体感应高丝氨酸内酯（HSL）的水解（Afriat等人（2006）Biochemistry 45，13677-13686）。然而，PTE如何偏离这个假定的PLL仍然是一个谜。在这项研究中，我们研究了环重塑作为一种手段，重建高丝氨酸内酯酶的祖先，涉及到PTE的几个突变步骤。虽然在自然界中，环重塑是酶功能分歧的常见机制，但在实验室中再现该过程是一个挑战。结构和系统发育分析使我们能够改造的PTE的活性位点循环成一个PLL样配置之一。环7中的缺失与相邻的高度上位性点突变相结合，导致出现在PTE中检测不到的HSLAse活性（k（cat）/K-M值高达2 X 10（4））。HSL酶活性的出现伴随着PTE的对氧磷酶活性的轻微下降。这种特异性变化表明了双功能中间体在新酶功能分化中的潜在作用，并突出了环重塑对新酶功能快速分化的关键贡献。
Only decades after the introduction of organophosphate pesticides, bacterial phosphotriesterases (PTEs) have evolved to catalyze their degradation with remarkable efficiency. Their closest known relatives, lactonases, with promiscuous phosphotriasterase activity, dubbed PTE-like lactonases (PLLs), share only 30% sequence identity and also differ in the configuration of their active-site loops. PTE was therefore presumed to have evolved from a yet unknown PLL whose primary activity was the hydrolysis of quorum sensing homoserine lactones (HSLs) (Afriat et al. (2006) Biochemistry 45, 13677-13686). However, how PTEs diverged from this presumed PLL remains a mystery. In this study we investigated loop remodeling as a means of reconstructing a homoserine lactonase ancestor that relates to PTE by few mutational steps. Although, in nature, loop remodeling is a common mechanism of divergence of enzymatic functions, reproducing this process in the laboratory is a challenge. Structural and phylogenetic analyses enabled us to remodel one of PTE's active-site loops into a PLL-like configuration. A deletion in loop 7, combined with an adjacent, highly epistatic, point mutation led to the emergence of an HSLase activity that is undetectable in PTE (k(cat)/K-M values of up to 2 X 10(4)). The appearance of the HSLase activity was accompanied by only a minor decrease in PTE's paraoxonase activity. This specificity change demonstrates the potential role of bifunctional intermediates in the divergence of new enzymatic functions and highlights the critical contribution of loop remodeling to the rapid divergence of new enzyme functions.