Overcoming an optimization plateau in the directed evolution of highly efficient nerve agent bioscavengers

Abstract

Improving an enzyme's initially low catalytic efficiency with a new target substrate by an order of magnitude or two may require only a few rounds of mutagenesis and screening or selection. However, subsequent rounds of optimization tend to yield decreasing degrees of improvement (diminishing returns) eventually leading to an optimization plateau. We aimed to optimize the catalytic efficiency of bacterial phosphotriesterase (PTE) toward V-type nerve agents. Previously, we improved the catalytic efficiency of wild-type PTE toward the nerve agent VX by 500-fold, to a catalytic efficiency (kcat/KM) of 5 × 106 M-1 min-1. However, effective in vivo detoxification demands an enzyme with a catalytic efficiency of >107 M-1 min-1. Here, following eight additional rounds of directed evolution and the computational design of a stabilized variant, we evolved PTE variants that detoxify VX with a kcat/KM ≥ 5 × 107 M-1 min-1 and Russian VX (RVX) with a kcat/KM ≥ 107 M-1 min-1. These final 10-fold improvements were the most time consuming and laborious, as most libraries yielded either minor or no improvements. Stabilizing the evolving enzyme, and avoiding tradeoffs in activity with different substrates, enabled us to obtain further improvements beyond the optimization plateau and evolve PTE variants that were overall improved by >5000-fold with VX and by >17 000-fold with RVX. The resulting variants also hydrolyze G-type nerve agents with high efficiency (GA, GB at kcat/KM > 5 × 107 M-1 min-1) and can thus serve as candidates for broad-spectrum nerve-agent prophylaxis and post-exposure therapy using low enzyme doses.

Bacterial Expression of Human Butyrylcholinesterase as a Tool for Nerve Agent Bioscavengers Development

“…Probably a major point of interest of this prokaryotic expression system is its possible use as a tool for high-throughput screening of BChE mutants for the development of catalytic bioscavengers, such as what have been done with bacterial phosphotriesterases [ 39 ]. Indeed, BChE variants, such as the Gly117His and Gly117His/Glu197Gln human BChE mutants [ 40 , 41 ] or swine BChE (manuscript in preparation) can catalyze OP hydrolysis.…”

Directed evolution methods for overcoming trade‐offs between protein activity and stability

“…Next, protein mutants containing combinations of these mutations are then designed using Rosetta combinatorial sequence optimization to identify variants with improved native‐state energy. Such an approach was employed during directed evolution to convert bacterial phosphotriesterases into efficient nerve agent bioscavengers with high activity against both VX and Russian VX . Prior efforts to engineer these phosphotriesterases had reached an optimization plateau short of the catalytic design goals.…”

“…Prior efforts to engineer these phosphotriesterases had reached an optimization plateau short of the catalytic design goals. The PROSS design tool was used to guide library design for stabilization of the phosphotriesterases, and this enabled additional productive rounds of directed evolution and incorporation of previously identified beneficial mutations that were not well tolerated when introduced into the less stable phosphotriesterases . PROSS has also been used to stabilize the malaria invasion protein PfRH5, a promising malaria vaccine candidate, without disrupting its immunogenicity or ligand binding .…”

Optimization of Cholinesterase-Based Catalytic Bioscavengers Against Organophosphorus Agents

“…Other mutants of this enzyme, made by directed evolution and computer design, display a wide spectrum of activity. Not only do they hydrolyze V agents with high efficiency, but also G agents with k cat / K m > 5 × 10 7 M −1 ·min −1 for tabun and sarin (Goldsmith et al, 2017 ). The evolved mutant of PON-1, C23AL, has k cat / K m = 1.2 × 10 7 M −1 ·min −1 for racemic VX (Goldsmith et al, 2016 ).…”