Rational designing of human senescence marker protein 30 for enhanced hydrolysis of V-type nerve agent analog

Organophosphate (OP) nerve agents are a class of compounds known for high toxicity and rapid action, posing a significant threat to human health. Catalytic bio-scavengers have gained substantial attention due to the limited protective efficacy and adverse side effects of chemical drugs in treating OP poisoning. However, the low catalytic efficiency remains a key limitation to their effectiveness and broader applications. Unlike existing human catalytic bio-scavengers such as huPON1, which lack activity against V-type nerve agents, the human Senescence Marker protein 30 (huSMP30) can detoxify a surrogate V-type nerve agent, i.e., Demeton-S; however, its hydrolytic activity is too low to be utilized as a drug. Therefore, in this study, the huSMP30 has been engineered through a rational protein designing approach to enhance its catalytic activity for Demeton-S. Initially, in silico analysis identified six key amino acid residues (E18, A62, K106, N154, D157, and D204), which were targeted for mutational analysis. Fourteen mutants, including single, double, and triple mutants, were created by site-directed mutagenesis, recombinantly produced, and analyzed in vitro. Substitution mutation at position 106 significantly enhanced the catalytic efficiency (kcat/Km) by up to ∼26-fold. In conclusion, the engineered huSMP30 may act as an effective catalytic bio-scavenger for the hydrolysis of Demeton-S; therefore, it could be utilized for the treatment of V-type nerve agent poisoning.