In vitro evolution of enzymes represents a powerful device to evolve new or to improve weak enzymatic functions. In the present work a semi-rational engineering approach has been used to design an efficient and thermostable organophosphate hydrolase, starting from a lactonase scaffold (SsoPox from Sulfolobus solfataricus). In particular, by in vitro evolution of the SsoPox ancillary promiscuous activity, the triple mutant C258L/I261F/W263A has been obtained which, retaining its inherent stability, showed an enhancement of its hydrolytic activity on paraoxon up to 300-fold, achieving absolute values of catalytic efficiency up to 10(5) M(-1) s(-1). The kinetics and structural determinants of this enhanced activity were thoroughly investigated and, in order to evaluate its potential biotechnological applications, the mutant was tested in formulations of different solvents (methanol or ethanol) or detergents (SDS or a commercial soap) for the cleaning of pesticide-contaminated surfaces.

PON1, PON2, and PON3 belong to a family of lactone hydrolyzing enzymes endowed with various substrate specificities. Among PONs, PON2 shows the highest hydrolytic activity toward many acyl-homoserine lactones (acyl-HL) involved in bacterial quorum-sensing signaling. Accordingly, defense against pathogens, such as Brevundimonas aeruginosa (B. aeruginosa), was postulated to be the principal function of PON2. However, recent findings have highlighted the importance of PON2 in oxidative stress control, inhibition of apoptosis, and the progression of various types of malignancies. This review focuses on all of these aspects of PON2.

The activity of human paraoxonase 2 (PON2) is rapidly reduced in cells incubated with the bacterial quorormone 3-Oxo-dodecanoyl Homoserine Lactone (3OC12HSL), an observation that led to hypothesize a fast PON2 posttranslational modification (PTM). Recently, we detected a 3OC12HSL-induced PTM in a cell-free system in which a crude extract from 3OC12HSL-treated HeLa cells was able to inactivate and ubiquitinate at position 144 a recombinant PON2. Here we show the occurrence of this and new PTMs on PON2 in HeLa cells. PTMs were found to gather nearby the two SNPs, A148G, and S311C, that are related to type-2 diabetes and its complications. Furthermore, we detected a PTM nearby a 12 amino acids region that is deleted in PON2 Isoform 2. An in vitro mutation analysis showed that the SNPs and the deletion are involved in PON2 activity and suggested a role of PTMs on its modulation, while a SAXS analysis pointed to Isoform 2 as being largely unstructured, compared to the wild type. Besides, we discovered a control of PON2 expression via a putative mRNA operon involving the Wilms tumor 1 associated protein (WTAP) and the E3 ubiquitin ligase (E3UbL) baculoviral IAP repeat-containing 3 (BIRC3).