Ultrahigh-throughput directed evolution of a metal-free a/ß-hydrolase with a Cys-His-Asp triad into an efficient phosphotriesterase
Ultrahigh-throughput directed evolution of a metal-free a/ß-hydrolase with a Cys-His-Asp triad into an efficient phosphotriesterase
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具有 Cys-His-Asp 三联体的无金属 a/α-水解酶超高通量定向进化为高效磷酸三酯酶
DOI：
10.1101/2022.02.14.480337
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发表时间：
2022
期刊：
影响因子：
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通讯作者：
Schnettler Fernández D
中科院分区：
文献类型：
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作者：
Schnettler Fernández D
最近大量新的人造物质释放到环境中，需要生物修复，但可供使用的酶数量非常有限。当环境在其进化历史中没有遇到潜在的有害物质时，现有的酶必须被重新利用。偶然的，通常是低水平的混杂活动的招募提供了一个良好的开端，在基因复制后，可以适应并提供可选择的优势。这种进化的情况提出了一个问题，即是否有可能适应性地提高从非生物体中招募的酶的低水平活性。（或只是最近）污染的环境迅速发展到进化的生物修复剂的水平。（用于水解有机磷农药或化学战剂的酶）在这种情况下：在先前的功能宏基因组学筛选中，我们已经鉴定了α/β-水解酶P91的混杂磷酸三酯酶活性，其具有意想不到的Cys-His-Asp催化三联体作为活性位点基序。我们现在使用微流控液滴中的超高通量筛选来探测P91的进化性，并首次测试含半胱氨酸的三联体的独特催化基序是否可以适应以实现与现有磷酸三酯酶竞争的速率。这些机制不同的酶实现其高速率的基础上催化涉及金属离子辅因子。通过定量反应产物荧光素，在微流控液滴中在芯片上筛选P91（> 105个成员）的聚焦组合文库。仅在两轮进化中，P91的磷酸三酯酶活性就增加了400倍，达到106 M − 1 s −1的akcat/Km，与自然进化的金属依赖性磷酸三酯酶的催化效率相匹配。与其遭受自杀抑制的同系物乙酰胆碱酯酶相比，P91显示出快速的去磷酸化速率，并且通过共价加合物的形成而不是通过其水解来限制速率。我们的分析强调了如何将聚焦的组合文库与液滴微流体的微通量相结合，以识别和增强在自然界中尚未达到高效率的机制策略，从而产生具有新型催化机制的替代试剂。图形摘要
The recent massive release of new, man-made substances into the environment requires bioremediation, but a very limited number of enzymes evolved in response are available. When environments have not encountered the potentially hazardous materials in their evolutionary history, existing enzymes have to be repurposed. The recruitment of accidental, typically low-level promiscuous activities provides a head start that, after gene duplication, can adapt and provide a selectable advantage. This evolutionary scenario raises the question whether it is possible to adaptively improve the low-level activity of enzymes recruited from non- (or only recently) contaminated environments quickly to the level of evolved bioremediators.Here we address the evolution of phosphotriesterases (enzymes for hydrolysis of organophosphate pesticides or chemical warfare agents) in such a scenario: In a previous functional metagenomics screening we had identified a promiscuous phosphotriesterase activity of the α/β-hydrolase P91, with an unexpected Cys-His-Asp catalytic triad as the active site motif. We now probe evolvability of P91 using ultrahigh-throughput screening in microfluidic droplets, and test for the first time whether the unique catalytic motif of a cysteine-containing triad can adapt to achieve rates that rival existing phosphotriesterases. These mechanistically distinct enzymes achieve their high rates based on catalysis involving a metal-ion cofactor. A focussed, combinatorial library of P91 (> 105members) was screened on-chip in microfluidic droplets by quantification of the reaction product, fluorescein. Within only two rounds of evolution P91’s phosphotriesterase activity was increased ≈ 400-fold to akcat/KMof ≈ 106M−1s−1, matching the catalytic efficiencies of naturally evolved metal-dependent phosphotriesterases. In contrast to its homologue acetylcholinesterase that suffers suicide inhibition, P91 shows fast de-phosphorylation rates and is rate-limited by the formation of the covalent adduct rather than by its hydrolysis. Our analysis highlights how the combination of focussed, combinatorial libraries with the ultrahigh throughput of droplet microfluidics can be leveraged to identify and enhance mechanistic strategies that have not reached high efficiency in Nature, resulting in alternative reagents with a novel catalytic machinery.GRAPHICAL ABSTRACT