Engineering V‑Type Nerve Agents Detoxifying Enzymes Using Computationally Focused Libraries

VX
and its Russian (RVX) and Chinese (CVX) analogues rapidly inactivate
acetylcholinesterase and are the most toxic stockpile nerve agents.
These organophosphates have a thiol leaving group with a choline-like
moiety and are hydrolyzed very slowly by natural enzymes. We used
an integrated computational and experimental approach to increase Brevundimonas diminuta phosphotriesterase’s (PTE)
detoxification rate of V-agents by 5000-fold. Computational models
were built of the complex between PTE and V-agents. On the basis of
these models, the active site was redesigned to be complementary in
shape to VX and RVX and to include favorable electrostatic interactions
with their choline-like leaving group. Small libraries based on designed
sequences were constructed. The libraries were screened by a direct
assay for V-agent detoxification, as our initial studies showed that
colorimetric surrogates fail to report the detoxification rates of
the actual agents. The experimental results were fed back to improve
the computational models. Overall, five rounds of iterating between
experiment and model refinement led to variants that hydrolyze the
toxic SP isomers of all three V-agents
with kcat/KM values of up to 5 × 106 M–1 min–1 and also efficiently detoxify G-agents. These new
catalysts provide the basis for broad spectrum nerve agent detoxification.