Department of Chemistry , Texas A&M University , College Station , Texas 77843 , United States.
U.S. Army Edgewood Chemical Biological Center , 5183 Blackhawk Road , Aberdeen Proving Ground , Maryland 21010 , United States.
Biochemistry. 2019 Apr 16;58(15):2039-2053. doi: 10.1021/acs.biochem.9b00097. Epub 2019 Apr 1.
The bacterial enzyme phosphotriesterase (PTE) is noted for its ability to hydrolyze many organophosphate compounds, including insecticides and chemical warfare agents. PTE has been the subject of multiple enzyme evolution attempts, which have been highly successful against specific insecticides and the G-type nerve agents. Similar attempts targeting the V-type nerve agents have failed to achieve the same degree of success. Enzyme evolution is an inherently complex problem, which is complicated by synergistic effects, the need to use analogues in high-throughput screening, and a lack of quantitative data to direct future efforts. Previous evolution experiments with PTE have assumed an absence of synergy and minimally screened large libraries, which provides no quantitative information about the effects of individual mutations. Here a systemic approach has been applied to a 28800-member six-site PTE library. The library is screened against multiple V-agent analogues, and a combination of sequence and quantitative activity analysis is used to extract data about the effects of individual mutations. We demonstrate that synergistic relationships dominate the evolutionary landscape of PTE and that analogue activity profiles can be used to identify variants with high activity for substrates. Using these approaches, multiple variants with k/ K values for the hydrolysis of VX that were improved >1500-fold were identified, including one variant that is improved 9200-fold relative to wild-type PTE and is specific for the S enantiomer of VX. Multiple variants that were highly active for ( S)-VR were identified, the best of which has a k/ K values that is improved 13400-fold relative to that of wild-type PTE.
细菌酶磷酸三酯酶 (PTE) 以其能够水解多种有机磷化合物的能力而闻名，包括杀虫剂和化学战剂。PTE 一直是多次酶进化尝试的主题，这些尝试针对特定的杀虫剂和 G 型神经毒剂取得了高度成功。针对 V 型神经毒剂的类似尝试未能取得同样程度的成功。酶进化是一个固有的复杂问题，协同作用、在高通量筛选中使用类似物的需要以及缺乏定量数据来指导未来的努力使其变得复杂。以前的 PTE 进化实验假设不存在协同作用，并且最小限度地筛选了大型文库，这没有提供关于单个突变影响的定量信息。在这里，我们采用了一种系统的方法来处理一个包含 28800 个成员的六位点 PTE 文库。该文库针对多种 V 型毒剂类似物进行筛选，并结合序列和定量活性分析，提取关于单个突变影响的数据。我们证明协同关系主导了 PTE 的进化景观，并且类似物活性谱可用于识别对底物具有高活性的变体。使用这些方法，鉴定出了多个水解 VX 的 k/ K 值提高 >1500 倍的变体，包括一个相对于野生型 PTE 提高 9200 倍的变体，并且对 VX 的 S 对映体具有特异性。鉴定出了多个对（S）-VR 具有高活性的变体，其中最好的变体的 k/ K 值相对于野生型 PTE 提高了 13400 倍。
