Active site engineering of recombinant human paraoxonase 1 for enhancing its catalytic efficiency against V-type nerve agent analog- Demeton S

Human Paraoxonase 1 (HuPON1) is a calcium-dependent serum enzyme with broad substrate specificity and therapeutic potential for detoxifying the organophosphates, including nerve agents. The V-type nerve agents are the most toxic synthetic organophosphates, with high lethality and environmental persistence, necessitating the development of an efficient catalytic bioscavenger for their detoxification. However, HuPON1 exhibits promiscuous hydrolytic activity towards various organophosphates; its catalytic efficiency against V-type nerve agents and its analogs remains extremely low. In this study, a structure-guided active site engineering approach was used to enhance the catalytic efficiency of recombinant HuPON1 against Demeton S, a surrogate for the V-type nerve agent. The in silico analysis has identified key amino acid residues (H115, Q192, F222, and L240) for a mutagenesis study, and the residue F77 in the catalytic lid was selected to influence substrate accessibility and binding. Since the wild-type HuPON1 exhibits very low activity, the engineering approach was initiated using the active site H115W mutant, which showed ∼2.86-fold higher enzymatic activity against Demeton S, bringing its hydrolytic activity within a measurable range. The combinatorial mutations were constructed progressively, resulting in the generation of double, triple, quadruple, and quintuple mutants. These engineered mutants displayed a stepwise improvement in catalytic activity, with the quintuple mutant (H115W, F77I, Q192N, F222S and L240S) showing ∼10.6-fold higher hydrolytic activity and a ∼ 28.89-fold improvement in catalytic efficiency ( k cat / k m ). Furthermore, the protein retained 58% residual acetylcholinesterase activity and remained structurally and functionally stable in human serum. This engineered HuPON1 mutant demonstrated enhanced hydrolysis of Demeton S, a surrogate V-type nerve agent, supporting its development as a broad-spectrum catalytic bioscavenger against various organophosphorus compounds.