A catalytic bioscavenger with improved stability and reduced susceptibility to oxidation for treatment of acute poisoning with neurotoxic organophosphorus compounds. (15th March 2020)

• Record Type: Journal Article
• Authors: Job, Laura; Köhler, Anja; Escher, Benjamin; Worek, Franz; Skerra, Arne
• Is Part Of: Toxicology letters. Volume 321(2020)
• Page Start: 138
• Page End: 145
• Publication Date: 2020-03-15
• Subjects: Bioscavenger -- Enzyme engineering -- Organophosphate hydrolase -- Organophosphorus compound -- Phosphotriesterase -- Protein oxidation; Toxicology -- Periodicals; 363.179
• DOI: 10.1016/j.toxlet.2019.12.030
• Languages: English
• ISSNs: 0378-4274

Abstract

Highlights: The phosphotriesterase from Brevundimonas diminuta can hydrolyze organophosphates. Engineered BdPTE offers a catalytic bioscavenger of nerve agents and pesticides. However, its biochemical stability needs optimization for therapeutic application. We describe the replacement of two unpaired cysteine residues by inert side chains. This leads both to high stability and catalytic activity towards toxic substrates. Abstract: Organophosphorus (OP) 1 nerve agents pose a severe toxicological threat, both after dissemination in military conflicts and by terrorists. Hydrolytic enzymes, which may be administered into the blood stream of victims by injection and can decompose the circulating nerve agent into non-toxic metabolites in vivo, could offer a treatment. Indeed, for the phosphotriesterase found in the bacterium Brevundimonas diminuta (BdPTE), 2 engineered versions with improved catalytic efficiencies have been described; yet, their biochemical stabilities are insufficient for therapeutic use. Here, we describe the application of rational protein design to develop novel mutants of BdPTE that are less susceptible to oxidative damage. In particular, the replacement of two unpaired cysteine residues by more inert amino acids led to higher stability while maintaining high catalytic activity towards a broad spectrum of substrates, including OP pesticides and V-type nerve agents. The mutant BdPTE enzymes were produced in Escherichia coli, purified to homogeneity, and their biochemical and enzymological properties were assessed. Several candidates both revealed enhanced thermal stability and were less susceptible to oxidative stress, as demonstrated by mass spectrometry. These mutants of BdPTE may show promise for the treatment of acute intoxications by nerve agents as well as OP pesticides.