期刊
ARCHIVES OF TOXICOLOGY
卷 95, 期 8, 页码 2815-2823出版社
SPRINGER HEIDELBERG
DOI: 10.1007/s00204-021-03094-0
关键词
Bispecific bioscavenger; Detoxification; Mutant; Nerve agent; PASylation; Phosphotriesterase
类别
资金
- Projekt DEAL
The study investigated the catalytic efficacies of five BdPTE mutants and modified versions with delayed plasma clearance, as well as a bispecific BdPTE with broadened substrate spectrum. The newly developed enzymes BdPTE-4 and BdPTE-7 exhibited high catalytic efficacy towards structurally different nerve agents and stereoselectivity towards the toxic P(-) VX enantiomer in vitro.
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 k(cat)/K-M values were as high as 6.5 and 1.5 x 10(8) 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.
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