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 kcat/Km 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 SP enantiomer of VX. Multiple variants that were highly
active for (SP)-VR were identified, the
best of which has a kcat/Km values that is improved 13400-fold relative to that
of wild-type PTE.