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, including suicidal attempts. For in vivo detoxification, the application of hydrolytic enzymes potentially offers a promising treatment. A well-studied example is the phosphotriesterase of the bacterium Brevundimonas diminuta (BdPTE). Whereas wild-type BdPTE can hydrolyse pesticides like paraoxon, chlorpyrifos-oxon and mevinphos with high catalytic efficiencies, kcat/KM > 2 × 107 M-1 min-1, degradation of malaoxon is unsatisfactory (kcat/KM ≈ 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/KM = 4.6 × 105 M-1 min-1), together with enhanced expression yield, improved thermal stability (with reference to wt-BdPTE) and reduced susceptibility to oxidation. Therefore, this BdPTE mutant constitutes a powerful candidate-possibly after further engineering-to develop a biocatalytic antidote for the detoxification of this common pesticide metabolite as well as related OP compounds.