Overcoming the Challenges of Enzyme Evolution To Adapt Phosphotriesterase for V-Agent Decontamination

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(cat)/K-m 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-p enantiomer of VX. Multiple variants that were highly active for (S-P)-VR were identified, the best of which has a k(cat)/K-m values that is improved 13400-fold relative to that of wild-type PTE.