Elena Porzio

Structural basis for natural lactonase and promiscuous phosphotriesterase activities.

Organophosphates are the largest class of known insecticides, several of which are potent nerve agents. Consequently, organophosphate-degrading enzymes are of great scientific interest as bioscavengers and biodecontaminants. Recently, a hyperthermophilic phosphotriesterase (known as SsoPox), from the Archaeon Sulfolobus solfataricus, has been isolated and found to possess a very high lactonase activity. Here, we report the three-dimensional structures of SsoPox in the apo form (2.6 A resolution) and in complex with a quorum-sensing lactone mimic at 2.0 A resolution. The structure also reveals an unexpected active site topology, and a unique hydrophobic channel that perfectly accommodates the lactone substrate. Structural and mutagenesis evidence allows us to propose a mechanism for lactone hydrolysis and to refine the catalytic mechanism established for phosphotriesterases. In addition, SsoPox structures permit the correlation of experimental lactonase and phosphotriesterase activities and this strongly suggests lactonase activity as the cognate function of SsoPox. This example demonstrates that promiscuous activities probably constitute a large and efficient reservoir for the creation of novel catalytic activities.

An efficient thermostable organophosphate hydrolase and its application in pesticide decontamination

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 105 M−1s−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. Biotechnol. Bioeng. 2016;113: 724–734.

Mn2+ modulates the kinetic properties of an archaeal member of the PLL family.

Recently we reported on the characterization of an archaeal member of the amidohydrolase superfamily, namely Sulfolobus acidocaldarius lactonase, showing low but significant and extremely thermostable paraoxonase activity. This enzyme, that we have named SacPox, is a member of the new described family of phosphotriesterase-like lactonases (PLLs). In this family the binuclear metal centre, which is involved in the catalytic machinery, has been poorly studied up to now. In this work we describe the expression of the protein in presence of different metals showing Mn(2+) to support the higher activity. The enzyme has been over-expressed, purified and characterized as a Mn(2+)-containing enzyme by inductive plasma coupled mass spectrometry (ICP-MS), showing also surprising kinetic differences in comparison with the cadmium-containing enzyme. The Mn(2+) containing enzyme was about 30-fold more efficient with paraoxon as substrate and more stable than the Cd(2+) counterpart, even though the Mn(2+) affinity for the binuclear metal centre is apparently lower. These results increase our knowledge of the biochemical characteristics of SacPox mainly with regard to the metal-ions modulation of function.

Purification of the Poly-ADP-Ribose Polymerase-Like Thermozyme from the Archaeon Sulfolobus solfataricus

Several different protocols have been developed to purify the ADP-ribosylating enzyme from Sulfolobus solfataricus. A number of techniques have been applied in regard to the crude homogenate preparation and protein extraction. Either mechanical cell lysis with DNAase digestion or freeze-thawing with sonication allowed to obtain fairly similar amounts of the thermozyme in the homogenate. While similar recovery of thermozyme was obtained by employing both purification protocols, the proteins were solubilized with different methods, and the affinity chromatography on NAD-Agarose of the first protocol was replaced by a gel filtration step in the second protocol. When enzyme activity was compared with electrophoresis and anti-poly-ADP-ribose polymerase 1 antibody immunoblotting results, it was noticed that lysis by sonication induces aggregation of monomeric PARP-like thermozyme at least in a dimeric form. The dimeric aggregate is also evidenced by treatment of cells with sonication followed by different protein extraction (Method III). Finally, we describe the third purification protocol that allows fast recovery of small amounts of purified ADP-ribosylating enzyme.

Engineering of Extremophilic Phosphotriesterase-Like Lactonases for Biotechnological Applications

Organophosphate compounds, as most pesticides and chemical warfare agents, first appeared in the US after 1930s and became widespread after World War II. At present, enzymatic detoxification of organophosphate compounds represents an important issue worldwide, due to their permanent and excessive use that has led in many places to the contamination of soil and water. In the last years our research group focused the attention on the enzymes belonging to amidohydrolase superfamily. In particular, a new family of lactonases with promiscuous phosphotriesterase activity, dubbed PTE-like Lactonases (PLLs), has been discovered. We report here an overview of the actual use of organophosphate compounds and the hydrolytic enzymes able to degrade them. In the PLL family there are enzymes that hydrolyze pesticides, show high thermal resistance and, therefore, are very attractive from a biotechnology point of view. The combination of different in vitro evolution methods represents a successful approach to increase their promiscuous phosphotriesterase activity in order to obtain efficient detoxification enzymatic tools.

The DINGGG thermoprotein is membrane bound in the Crenarchaeon Sulfolobus solfataricus

BackgroundSulfolobus solfataricus N-terminus and other regions of the partial amino acid sequence of a thermoprotein exhibiting poly(ADP-ribose) polymerase activity suggest that it belongs to the DINGGG class of proteins that are often described as membrane bound. Our previous biochemical studies demonstrated that the thermoprotein is also strictly associated with DNA, and is only partially solubilized from cell homogenate. The present research is focused on the analysis of the sulfolobal DING thermozyme localization within the archaeal cell.ResultsImmunofluorescence microscopy evidenced the peripheral cell localization of Sulfolobal DING protein, along the plasma membrane hedge. Less intense, but clearly occurring, is the merge of Sulfolobus poly (ADP-ribose) polymerase with nucleoid. Anti-poly(ADP-ribose) polymerase immunoblottings clearly showed the occurrence of Sulfolobus thermozyme in membrane fractions as well as they confirmed its association with nucleoid DNA.ConclusionsFluorescent anti-PARP-1 antibodies showed that the PARPSso immunosignal localizes close to the membrane, at the periphery of cell, and that PARPSso green signal is also overlapping or strictly close to the nucleoid. Biochemical analyses confirmed that the thermozyme occurs in both membrane and nucleoid preparations.Graphical abstractIntracellular localization of DING thermozyme by fluorescence microscopy

Innovative Biocatalysts as Tools to Detect and Inactivate Nerve Agents

Pesticides and warfare nerve agents are frequently organophosphates (OPs) or related compounds. Their acute toxicity highlighted more than ever the need to explore applicable strategies for the sensing, decontamination and/or detoxification of these compounds. Herein, we report the use of two different thermostable enzyme families capable to detect and inactivate OPs. In particular, mutants of carboxylesterase-2 from Alicyclobacillus acidocaldarius and of phosphotriesterase-like lactonases from Sulfolobus solfataricus and Sulfolobus acidocaldarius, have been selected and assembled in an optimized format for the development of an electrochemical biosensor and a decontamination formulation, respectively. The features of the developed tools have been tested in an ad-hoc fabricated chamber, to mimic an alarming situation of exposure to a nerve agent. Choosing ethyl-paraoxon as nerve agent simulant, a limit of detection (LOD) of 0.4 nM, after 5 s of exposure time was obtained. Furthermore, an optimized enzymatic formulation was used for a fast and efficient environmental detoxification (>99%) of the nebulized nerve agent simulants in the air and on surfaces. Crucial, large-scale experiments have been possible thanks to production of grams amounts of pure (>90%) enzymes.

Phosphotriesterase-Magnetic Nanoparticle Bioconjugates with Improved Enzyme Activity in a Biocatalytic Membrane Reactor

The need to find alternative bioremediation solutions for organophosphate degradation pushed the research to develop technologies based on organophosphate degrading enzymes, such as phosphotriesterase. The use of free phosphotriesterase poses limits in terms of enzyme reuse, stability, and process development. The heterogenization of enzyme on a support and their use in bioreactors implemented by membranes seems a suitable strategy, thanks to the ability of membranes to compartmentalize, to govern mass transfer, and to provide a microenvironment with tuned physicochemical and structural properties. Usually, hydrophilic membranes are used since they easily guarantee the presence of water molecules needed for the enzyme catalytic activity. However, hydrophobic materials exhibit a larger shelf life and are preferred for the construction of filters and masks. Therefore, in this work, hydrophobic polyvinylidene fluoride (PVDF) porous membranes were used to develop biocatalytic membrane reactors (BMR). The phosphotriesterase-like lactonase (PLL) enzyme ( SsoPox triple mutant from S. solfataricus) endowed with thermostable phosphotriesterase activity was used as model biocatalyst. The enzyme was covalently bound directly to the PVDF hydrophobic membrane or it was bound to magnetic nanoparticles and then positioned on the hydrophobic membrane surface by means of an external magnetic field. Investigation of kinetic properties of the two BMRs and the influence of immobilized enzyme amount revealed that the performance of the BMR was mostly dependent on the amount of enzyme and its distribution on the immobilization support. Magnetic nanocomposite mediated immobilization showed a much better performance, with an observed specific activity higher than 90% compared to grafting of the enzyme on the membrane. Even though the present work focused on phosphotriesterase, it can be easily translated to other classes of enzymes and related applications.