Human paraoxonases are multifunctional enzymes considered to be antioxidant, anti-inflammatory, antimicrobial, and anti-atherogenic [1]. Although the members of the PON family, PON1, PON2, and PON3, have similar amino acid sequences and the same origin, they are quite different in their functions and locations [2]. PON1 and PON3 are found in the blood plasma and remain associated with high-density lipoproteins (HDLs), while PON2 is ubiquitous in occurrence [3], [4]. PON1 and PON3 seem to evolve from PON2, which is the oldest member of the paraoxonase family [5]. They have native lactonase activity but show different and overlapping substrate specificities [6]. They are highly promiscuous enzymes, and the true physiological substrate is still unknown. Kinetic studies and protein engineering approaches provide a comprehensive knowledge of the enzyme’s active site and the catalytic mechanism [7], [8]. The huPONs are calcium-dependent enzymes characterized by a conserved six-bladed β propeller fold, which coordinates catalytic calcium ions for their enzymatic functions [8]. Among them, huPON1 has been most extensively studied and known to catalyze hydrolytic reactions via a well-defined catalytic mechanism. The reaction proceeds through substrate binding near the calcium ion, which polarizes the carbonyl group. A water molecule activated by conserved His residues (His115-His134) performs a nucleophilic attack to form a tetrahedral intermediate, which subsequently breaks down to release hydrolyzed products [9], [10]. Although detailed mechanistic studies on huPON2 and huPON3 are limited, structural and sequence conservation suggest they share a similar catalytic architecture and mechanism. Therefore, a comparative analysis of the paraoxonase gene family is crucial for uncovering the unique functional roles and substrate specificities of each member, enhancing our understanding of their biological activities and evolutionary adaptations.

The biofilms are complex communities of microorganisms that are embedded in the self-produced extracellular polymeric substances and adhere to the surface [11]. These structures contribute to anti-microbial resistance, persistent infection, and protection against host immune response [12]. The Mycobacterium biofilms are significant in both environmental and clinical settings concerning pathogenic species like Mycobacterium tuberculosis and non-tuberculosis mycobacteria (NTMs) [13], [14], [15]. They have a specialized cell wall structure consisting of unique mycolic acid that creates a hydrophobic environment, facilitating the aggregation of bacterial cells [16], [17]. A previous study showed that the extracellular polymeric surface of Mycobacterium biofilms is lipid-rich, majorly comprising short keto-mycolic acid and glycolpeptidolipids [13]. More recent findings indicate that the secondary messenger, cyclic di GMP, promotes keto mycolic acid and biofilm formation, suggesting that targeting lipid-associated signaling pathways could be an effective strategy to inhibit mycobacterial biofilm formation [18].

Human lactonases have shown anti-biofilm potential against various pathogenic bacteria. The huPONs have been previously investigated for their ability to disrupt biofilms formed by gram-negative bacteria, such as Pseudomonas aeruginosa, by targeting the quorum-sensing signaling molecules [19], [20], [21], [22], [23]. Recent studies have identified human lactonases, such as huSMP30 and huPON2, as anti-biofilm agents against Mycobacterium smegmatis [24], [25]. However, the primary quorum-sensing molecule for mycobacterium biofilms has not been fully explored [26], [27]. While the paraoxonases have potential as anti-infective agents; however, their direct effects against Mycobacteria are not well-established [28]. Reduced PON1 activity has been observed in children with pulmonary tuberculosis compared to healthy controls, suggesting a link between PON1 and mycobacterial infection [29]. Previous studies have shown that the human PONs are capable of hydrolyzing a variety of lipid substrates. They efficiently hydrolyze bioactive δ-lactones derived from arachidonic acid, such as 5,6-dihydroxy-eicosatrienoic acid lactone and cyclo-epoxy cyclopentenone [30], [31]. Based on these substrate preferences, we hypothesized that PONs could interact with EPS, particularly mycolic acid within the biofilm matrix, potentially hydrolyzing it and thereby inhibiting Mycobacterium biofilm formation. In addition to the enzyme versatility, PONs hold significant clinical relevance due to their detoxification, cardiovascular and pharmacological roles [32]. The specificity of PON-substrate interaction directly influences the disease risk and drug response. Recent studies have demonstrated that these enzymes are influenced by the co-administered drugs; understanding such interactions might be essential for ensuring the safety and efficacy of PON-based therapeutic strategies [33], [34], [35]. While PON1 has been the primary focus of most studies, the less studied PON2 and PON3 members also hold significant potential, and these lesser-known enzymes might be a good asset for therapeutic development.

The heterologous expression of mammalian proteins is quite challenging due to the formation of inclusion bodies (IBs) [36]. The in vitro refolding of inactive proteins from IBs to produce active recombinant proteins has emerged as a favorable alternative to the soluble expression of these proteins, and this procedure is designed for the particular enzyme due to its unique amino acid sequence [37]. In this study, the huPON1, huPON2, and huPON3 were cloned and overexpressed as IBs in the E. coli expression system. The inactive protein was refolded into active enzymes using an in vitro refolding method. To further characterize these enzymes, a comparative analysis was conducted through enzymatic assays. Mycobacterium smegmatis was chosen as a model organism to study the anti-biofilm potential due to its non-pathogenic nature and short generation time. The refolded enzymes were then used to study their ability to inhibit biofilm formation. The in vitro enzymatic studies were further complemented by in silico analysis to explore the evolutionary trajectories of the enzymes, providing deeper insights into their functional adaptations and substrate specificities.