Abstract
The human paraoxonase 2 (PON2) has been described as a highly specific lactonase hydrolysing the quorum sensing molecule N-(3-oxododecanoyl)-L-homoserine lactone (3OC12-HSL) and having secondary esterase but not phosphotriesterase activity, in contrast with the related enzymes PON1 and PON3. It has been suggested that PON2 enzyme activity is dependent on glycosylation and its N-terminal region has been recently demonstrated to be a transmembrane domain mediating association to membranes. In the present study we describe a mutated form of PON2, lacking the above N-terminal region, which has been further stabilized by the insertion of six amino acidic substitutions. The engineered version, hence forth called rPON2, has been over-expressed in E.coli, refolded from inclusion bodies and purified, yielding an enzyme with the same characteristics as the full length enzyme. Therefore the first conclusion of this work was that the catalytic activity is independent from the N-terminus and protein glycosylation. The kinetic characterization confirmed the primary activity on 3OC12-HSL; accordingly, in vitro experiments of inhibition of the biofilm formed by Pseudomonas aeruginosa (PAO1) have demonstrated that rPON2 is more effective than PON1. In addition, we observed small but significant activity against organophosphorothiotes pesticides, m-parathion, coumaphos and malathion.The availability of fair amount of active protein allowed to pinpoint, by mass-spectrometry, ubiquitination of Lys 168 induced in rPON2 by HeLa extract and to correlate such post-translational modification to the modulation of catalytic activity. A mutational analysis of the modified residue confirmed the result.
Highlights
In the last few decades there has been an increased interest in human paraoxonases (PONs), because a number of diseases has been related to these proteins [1, 2]
In the present study we have analyzed the possibility to produce a recombinant paraoxonase 2 (PON2) in E. coli to better explore the main biochemical features of the enzyme, and above all we have focused on the relationship between PON2 hydrolytic activity and its post-translational modifications; in particular we have investigated the relationship between glycosylation and catalytic activity [21, 22], as well as the second modification postulated to modulate PON2 activity by Horke and colleagues [23]
We confirmed that the best substrate was 3OC12-HSL (Tables 1–3), in full agreement with the in vitro evidence of inhibition of PAO1 biofilm formation; the recombinant PON2 behaved better the other human paraoxonase paraoxonase 1 (PON1) (Fig 3), indicating that its physiological role could be the attenuation of infection of some pathogens by 3OC12-HSL hydrolysis, as previously hypothesized [17]
Summary
In the last few decades there has been an increased interest in human paraoxonases (PONs), because a number of diseases has been related to these proteins [1, 2]. The family comprises three members: paraoxonase 1 (PON1), paraoxonase 2 (PON2), and paraoxonase 3 (PON3), encoded by three different genes located in a cluster of chromosome 7 [3]; these genes share
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