Catalytic activity and stereoselectivity of engineered phosphotriesterases towards structurally different nerve agents in vitro
| Autor: | Horst Thiermann, Benjamin Escher, Laura Job, Marianne Koller, Anja Köhler, Franz Worek, Arne Skerra |
|---|---|
| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
Stereochemistry Health Toxicology and Mutagenesis Population Toxicology Catalysis Organ Toxicity and Mechanisms Substrate Specificity 03 medical and health sciences In vivo Tandem Mass Spectrometry medicine Enzyme kinetics education Nerve agent chemistry.chemical_classification education.field_of_study 030102 biochemistry & molecular biology Hydrolysis Mutant Substrate (chemistry) Stereoisomerism General Medicine Caulobacteraceae Bispecific bioscavenger ddc 030104 developmental biology Enzyme PASylation Phosphoric Triester Hydrolases chemistry Mutation Phosphotriesterase Stereoselectivity Enantiomer Detoxification Nerve Agents medicine.drug Chromatography Liquid |
| Zdroj: | Archives of Toxicology |
| Popis: | Highly toxic organophosphorus nerve agents, especially the extremely stable and persistent V-type agents such as VX, still pose a threat to the human population and require effective medical countermeasures. Engineered mutants of the Brevundimonas diminuta phosphotriesterase (BdPTE) exhibit enhanced catalytic activities and have demonstrated detoxification in animal models, however, substrate specificity and fast plasma clearance limit their medical applicability. To allow better assessment of their substrate profiles, we have thoroughly investigated the catalytic efficacies of five BdPTE mutants with 17 different nerve agents using an AChE inhibition assay. In addition, we studied one BdPTE version that was fused with structurally disordered PAS polypeptides to enable delayed plasma clearance and one bispecific BdPTE with broadened substrate spectrum composed of two functionally distinct subunits connected by a PAS linker. Measured kcat/KM values were as high as 6.5 and 1.5 × 108 M−1 min−1 with G- and V-agents, respectively. Furthermore, the stereoselective degradation of VX enantiomers by the PASylated BdPTE-4 and the bispecific BdPTE-7 were investigated by chiral LC–MS/MS, resulting in a several fold faster hydrolysis of the more toxic P(−) VX stereoisomer compared to P(+) VX. In conclusion, the newly developed enzymes BdPTE-4 and BdPTE-7 have shown high catalytic efficacy towards structurally different nerve agents and stereoselectivity towards the toxic P(−) VX enantiomer in vitro and offer promise for use as bioscavengers in vivo. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|