Anti-biofilm efficiency and substrate specificity of recombinantly produced human paraoxonases.

The human paraoxonase (PON) gene family, comprising PON1, PON2, and PON3, has evolved through parallel evolutionary trajectories leading to distinct substrate selectivity, thereby enabling these enzymes to hydrolyze a range of substrates such as lactones, arylesters, and organophosphates. This study explores the evolutionary divergence of PON family members and their varied anti-biofilm activities through in silico molecular docking and in vitro analysis. The huPON1, huPON2, and huPON3 genes were cloned and expressed in the E. coli expression system. These overexpressed proteins formed inclusion bodies, which were further subjected to in vitro refolding and biochemical characterization. The enzymatic studies revealed that huPON1 has unique paraoxonase activity and high arylesterase activity, while huPON2 demonstrated the highest lactonase activity, followed by huPON3 and huPON1. A comparative assessment of the anti-biofilm potential of recombinant huPONs was conducted against Mycobacterium smegmatis, which revealed that all three paraoxonases inhibited biofilm formation; however, huPON2 displayed the highest anti-biofilm activity, followed by huPON3 and huPON1. Additionally, all the huPONs showed a synergistic effect with the conventional TB drug, Rifampicin, in enhancing biofilm disruption. These findings contribute valuable insights into the application of huPONs as a therapeutic tool against drug-resistant bacterial infections.