Human paraoxonases (PON1, PON2, and PON3) are lactonases with overlapping and distinct substrate specificities

Abstract

The paraoxonase (PON) gene family in humans has three members, PON1 , PON2 , and PON3 . Their physiological role(s) and natural substrates are uncertain. We developed a baculovirus-mediated expression system, suitable for all three human PONs, and optimized procedures for their purification. The recombinant PONs are glycosylated with high-mannose-type sugars, which are important for protein stability but are not essential for their enzymatic activities. Enzymatic characterization of the purified PONs has revealed them to be lactonases/lactonizing enzymes, with some overlapping substrates (e.g., aromatic lactones), but also to have distinctive substrate specificities. All three PONs metabolized very efficiently 5-hydroxy-eicosatetraenoic acid 1,5-lactone and 4-hydroxy-docosahexaenoic acid, which are products of both enzymatic and nonenzymatic oxidation of arachidonic acid and docosahexaenoic acid, respectively, and may represent the PONs' endogenous substrates. Organophosphates are hydrolyzed almost exclusively by PON1, whereas bulky drug substrates such as lovastatin and spironolactone are hydrolyzed only by PON3. Of special interest is the ability of the human PONs, especially PON2, to hydrolyze and thereby inactivate N -acyl-homoserine lactones, which are quorum-sensing signals of pathogenic bacteria.

Exploiting Quorum Sensing To Confuse Bacterial Pathogens

“…Interestingly, in addition to differential expression at various sites within the host (PON1 and PON3 are expressed in the liver and sera, whereas PON2 is a cell-associated enzyme expressed in various tissues [103]), the different PON enzymes appear to have unique enzymatic activities. All three purified PONs had AHL lactonase activity in vitro, but each enzyme was determined to have a distinct specificity against a range of substrates, with PON1 and PON3 acting on the widest range of substrates but PON2 being the most active against AHLs (76,(103)(104)(105). In vitro results have supported a role for PON2 in AHL degradation by murine tracheal epithelial cells (104), and other experiments have demonstrated that both serum PON1 and purified PON1 reduce P. aeruiginosa biofilm formation in vitro (103).…”

“…CHO cells expressing any of the murine PON genes were able to degrade 3OC12HSL at levels comparable to those in cells expressing the known AHL lactonase gene aiiA (102), and treatment of serum with PON inhibitors reduced 3OC12HSL degradation (103). Evolutionary relationship analysis of PONs and studies of PON1 reactivity with more than 50 different substrates demonstrated that PON1 can hydrolyze lactones, esters, and phosphotriesters using the same active site, but its primary activity appears to be as a lactonase (76,77). Interestingly, in addition to differential expression at various sites within the host (PON1 and PON3 are expressed in the liver and sera, whereas PON2 is a cell-associated enzyme expressed in various tissues [103]), the different PON enzymes appear to have unique enzymatic activities.…”

Oxidative stress and anti-oxidative defense in schoolchildren residing in a petrochemical industry environment

“…Protective HDL function is primarily via the enzyme paraoxonase 1 (PON1) which hydrolyzes lipid peroxides in human atherosclerotic lesions (31)(32)(33). PON1 has a common coding region polymorphism, a glutamine (Q) to arginine (R) substitution at position 192, which is associated with a number of pathophysiological conditions such as CVD and some others (31).…”

Serum PON1 arylesterase activity in relation to hyperhomocysteinaemia and oxidative stress in young adult central retinal venous occlusion patients

“…4 In this context, the levels of PON1, which acts as lactonase as well as an antioxidant, could play an important role as a protective agent against the deleterious effect of elevated Hcys. The physiological substrate of PON1 is unknown, 21 and measurements of PON1 activity are done by monitoring the rates of hydrolysis of exogenous substrates such as phenylacetate and paraoxon, although this may not quantify the degree of the antiatherogenic or antioxidant potential of PON1. As a strong correlation between the PON1 levels assessed by ELISA and the PON1 activity measurements has been reported, PON1 activity measurements qualify as markers for PON1 levels in serum as reported by Himbergen et al 22 In this study, the PON1 activity is measured in terms of its ARE activity using phenylacetate as the substrate.…”