Engineering of a phosphotriesterase with improved stability and enhanced activity for detoxification of the pesticide metabolite malaoxon

Organophosphorus (OP) pesticides are still widely applied but pose a severe toxicological threat if misused. For in vivo detoxification, the application of hydrolytic enzymes potentially offers a promising treatment. A well-studied example is the phosphotriesterase of Brevundimonas diminuta (BdPTE). Whereas wild-type BdPTE can hydrolyse pesticides like paraoxon, chlorpyrifos-oxon and mevinphos with high catalytic efficiencies, kcat/K_M >2 × 10⁷ M⁻¹ min⁻¹, degradation of malaoxon is unsatisfactory (kcat/K_M ≈ 1 × 10⁴ M⁻¹ min⁻¹). Here, we report the rational engineering of BdPTE mutants with improved properties and their efficient production in Escherichia coli. As result, the mutant BdPTE(VRNVVLARY) exhibits 37-fold faster malaoxon hydrolysis (kcat/K_M = 4.6 × 10⁵ M⁻¹ min⁻¹), together with enhanced expression yield, improved thermal stability and reduced susceptibility to oxidation. Therefore, this BdPTE mutant constitutes a powerful candidate to develop a biocatalytic antidote for the detoxification of this common pesticide metabolite as well as related OP compounds.