Merone L

References (9)

Title : An efficient thermostable organophosphate hydrolase and its application in pesticide decontamination - Del Giudice_2016_Biotechnol.Bioeng_113_724
Author(s) : Del Giudice I , Coppolecchia R , Merone L , Porzio E , Carusone TM , Mandrich L , Worek F , Manco G
Ref : Biotechnol Bioeng , 113 :724 , 2016
Abstract : 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. Biotechnol. Bioeng. 2016;113: 724-734. (c) 2015 Wiley Periodicals, Inc.
ESTHER : Del Giudice_2016_Biotechnol.Bioeng_113_724
PubMedSearch : Del Giudice_2016_Biotechnol.Bioeng_113_724
PubMedID: 26416557

Title : Enzyme Promiscuity in the Hormone-Sensitive Lipase Family of Proteins - Manco_2012_Protein.Pept.Lett_19_144
Author(s) : Manco G , Merone L , Porzio E , Feng Y , Mandrich L
Ref : Protein Pept Lett , 19 :144 , 2012
Abstract : The number of enzymes endowed with the capacity to catalyse other reactions than the main, physiological one, a feature that has been called promiscuity, is increasing at a fast pace. Promiscuity is a highly pervasive phenomenon that is present at each level of life complexity. For enzymes, promiscuity encompasses interesting aspects related to their physiological role, evolution and biotechnological applications. Herein, at first we will describe some general aspects of enzyme promiscuity and then we will report some examples from the alpha/beta hydrolase superfamily of proteins, with particular emphasis to the hormone-sensitive lipase family.
ESTHER : Manco_2012_Protein.Pept.Lett_19_144
PubMedSearch : Manco_2012_Protein.Pept.Lett_19_144
PubMedID: 21933124
Gene_locus related to this paper: human-LIPE

Title : Hyperthermophilic phosphotriesterases\/lactonases for the environment and human health - Mandrich_2010_Environ.Technol_31_1115
Author(s) : Mandrich L , Merone L , Manco G
Ref : Environ Technol , 31 :1115 , 2010
Abstract : In the last decades the idea to use enzymes for environmental bioremediation has been more and more proposed and, in the light of this, new solutions have been suggested and detailed studies on some classes of enzymes have been performed. In particular, our attention in the last few years has been focused on the enzymes belonging to the amidohydrolase superfamily. Several members of this superfamily are endowed with promiscuous activities. The term 'catalytic promiscuity' describes the capability of an enzyme to catalyse different chemical reactions, called secondary activities, at the active site responsible for the main activity. Recently, a new family of microbial lactonases with promiscuous phosphotriesterase activity, dubbed PTE-Like Lactonase (PLL), has been ascribed to the amidohydrolase superfamily. Among members of this family are enzymes found in the archaea Sulfolobus solfataricus and Sulfolobus acidocaldarius, which show high thermophilicity and thermal resistance. Enzymes showing phosphotriesterase activity are attractive from a biotechnological point of view because they are capable of hydrolysing the organophosphate phosphotriesters (OPs), a class of synthetic compounds employed worldwide both as insecticides and chemical warfare agents. Furthermore, from a basic point of view, studies of catalytic promiscuity offer clues to understand natural evolution of enzymes and to translate this into in vitro adaptation of enzymes to specific human needs. Thermostable enzymes able to hydrolyse OPs are considered good candidates for the set-up of efficient detoxification tools.
ESTHER : Mandrich_2010_Environ.Technol_31_1115
PubMedSearch : Mandrich_2010_Environ.Technol_31_1115
PubMedID: 20718294

Title : Improving the promiscuous nerve agent hydrolase activity of a thermostable archaeal lactonase - Merone_2010_Bioresour.Technol_101_9204
Author(s) : Merone L , Mandrich L , Porzio E , Rossi M , Muller S , Reiter G , Worek F , Manco G
Ref : Bioresour Technol , 101 :9204 , 2010
Abstract : The thermostable Phosphotriesterase-Like Lactonase from Sulfolobus solfataricus (SsoPox) hydrolyzes lactones and, at a lower rate, neurotoxic organophosphorus compounds. The persistent demand of detoxification tools in the field of agricultural wastes and restoring of conditions after terrorist acts prompted us to exploit SsoPox as a "starter" to evolve its ancillary nerve agents hydrolytic capability. A directed evolution strategy yielded, among several variants, the single mutant W263F with k(cat) and specificity constant against paraoxon 16- and 6-fold enhanced, respectively, compared to the wild type. Furthermore, a phenomenon of enzyme activation by SDS has been observed, which allowed to increase those values 150- and 28-fold, respectively. The activity of SsoPox against the deadly nerve gas Cyclosarin has been reported for the first time and proved to be substantially unaffected for variant W263F. Finally, outperforming efficiency of W263F was demonstrated, under severe stressing conditions, with respect to the best known phosphotriesterase PTE from Brevundimonas diminuta.
ESTHER : Merone_2010_Bioresour.Technol_101_9204
PubMedSearch : Merone_2010_Bioresour.Technol_101_9204
PubMedID: 20667718

Title : Structural and kinetic overview of the carboxylesterase EST2 from alicyclobacillus acidocaldarius: a comparison with the other members of the HSL family - Mandrich_2009_Protein.Pept.Lett_16_1189
Author(s) : Mandrich L , Merone L , Manco G
Ref : Protein Pept Lett , 16 :1189 , 2009
Abstract : Thermophilic and hyperthermophilic carboxylesterases (EC 3.1.1.1) are excellent model systems for studying structure function relationships as well as in vitro and in vivo evolution and possible biotechnological applications. In this paper we review the main aspect of one of most studied microbial representative of the hormone sensitive lipase family (HSL), namely carboxylesterase 2 (EST2) from Alicyclobacillus acidocaldarius.
ESTHER : Mandrich_2009_Protein.Pept.Lett_16_1189
PubMedSearch : Mandrich_2009_Protein.Pept.Lett_16_1189
PubMedID: 19508183
Gene_locus related to this paper: aliac-est2

Title : Irreversible inhibition of the thermophilic esterase EST2 from Alicyclobacillus acidocaldarius - Febbraio_2008_Extremophiles_12_719
Author(s) : Febbraio F , D'Andrea SE , Mandrich L , Merone L , Rossi M , Nucci R , Manco G
Ref : Extremophiles , 12 :719 , 2008
Abstract : Kinetic studies of irreversible inhibition in recent years have received growing attention owing to their relevance to problems of basic scientific interest as well as to their practical importance. Our studies have been devoted to the characterization of the effects that well-known acetylcholinesterase irreversible inhibitors exert on a carboxylesterase (EST2) from the thermophilic eubacterium Alicyclobacillus acidocaldarius. In particular, sulfonyl inhibitors and the organophosphorous insecticide diethyl-p-nitrophenyl phosphate (paraoxon) have been studied. The incubation of EST2 with sulfonyl inhibitors resulted in a time-dependent inactivation according to a pseudo-first-order kinetics. On the other hand, the EST2 inactivation process elicited by paraoxon, being the inhibition reaction completed immediately after the inhibitor addition, cannot be described as a pseudo-first-order kinetics but is better considered as a high affinity inhibition. The values of apparent rate constants for paraoxon inactivation were determined by monitoring the enzyme/substrate reaction in the presence of the inhibitor, and were compared with those of the sulfonyl inhibitors. The protective effect afforded by a competitive inhibitor on the EST2 irreversible inhibition, and the reactivation of a complex enzyme/irreversible-inhibitor by hydroxylamine and 2-PAM, were also investigated. The data have been discussed in the light of the recently described dual substrate binding mode of EST2, considering that the irreversible inhibitors employed were able to discriminate between the two different binding sites.
ESTHER : Febbraio_2008_Extremophiles_12_719
PubMedSearch : Febbraio_2008_Extremophiles_12_719
PubMedID: 18622571
Gene_locus related to this paper: aliac-est2

Title : Structural basis for natural lactonase and promiscuous phosphotriesterase activities - Elias_2008_J.Mol.Biol_379_1017
Author(s) : Elias M , Dupuy J , Merone L , Mandrich L , Porzio E , Moniot S , Rochu D , Lecomte C , Rossi M , Masson P , Manco G , Chabriere E
Ref : Journal of Molecular Biology , 379 :1017 , 2008
Abstract : 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.
ESTHER : Elias_2008_J.Mol.Biol_379_1017
PubMedSearch : Elias_2008_J.Mol.Biol_379_1017
PubMedID: 18486146

Title : A new phosphotriesterase from Sulfolobus acidocaldarius and its comparison with the homologue from Sulfolobus solfataricus - Porzio_2007_Biochimie_89_625
Author(s) : Porzio E , Merone L , Mandrich L , Rossi M , Manco G
Ref : Biochimie , 89 :625 , 2007
Abstract : The phosphotriesterase PTE, identified in the soil bacterium Pseudomonas diminuta, is thought to have evolved in the last several decades to degrade the pesticide paraoxon with proficiency approaching the limit of substrate diffusion (k(cat)/K(M) of 4 x 10(7)M(-1)s(-1)). It belongs to the amidohydrolase superfamily, but its evolutionary origin remains obscure. The enzyme has important potentiality in the field of the organophosphate decontamination. Recently we reported on the characterization of an archaeal member of the amidohydrolase superfamily, namely Sulfolobus solfataricus, showing low but significant and extremely thermostable paraoxonase activity (k(cat)/K(M) of 4 x 10(3)M(-1)s(-1)). Looking for other thermostable phosphotriesterases we assayed, among others, crude extracts of Sulfolobus acidocaldarius and detected activity. Since the genome of S. acidocaldarius has been recently reported, we identified there an open reading frame highly related to the S. solfataricus enzyme. The gene was cloned, the protein overexpressed in Escherichia coli, purified, and proven to have paraoxonase activity. A comparative analysis detected some significant differences between the two archaeal enzymes.
ESTHER : Porzio_2007_Biochimie_89_625
PubMedSearch : Porzio_2007_Biochimie_89_625
PubMedID: 17337320

Title : Role of the N terminus in enzyme activity, stability and specificity in thermophilic esterases belonging to the HSL family - Mandrich_2005_J.Mol.Biol_345_501
Author(s) : Mandrich L , Merone L , Pezzullo M , Cipolla L , Nicotra F , Rossi M , Manco G
Ref : Journal of Molecular Biology , 345 :501 , 2005
Abstract : A superposition between the structures of Alicyclobacillus acidocaldarius esterase 2 (EST2) and Burkholderia cepacia lipase, the latter complexed with a phosphonate inhibitor, allowed us to hypothesize for the EST2 N terminus a role in restricting the access to the active site and therefore in modulating substrate specificity. In order to test this hypothesis we generated by site-directed mutagenesis some truncated versions of EST2 and its double mutant M211S/R215L (S/L) at the N terminus. In parallel, an analysis of the Sulfolobus solfataricus P2 genome allowed us to identify a gene coding for a putative esterase of the HSL family having a natural deletion of the corresponding region. The product of this gene and the above-mentioned EST2 mutants were expressed in Escherichia coli, purified and characterised. These studies support the notion that the N terminus affects substrate specificity other than several other enzyme parameters. Although the deletions afforded a tenfold and 550-fold decrease in catalytic efficiency towards the best substrate pNP-hexanoate at 50 degrees C for EST2 and S/L, respectively, the analysis of the specific activities with different triacylglycerols with respect to pNP-hexanoate showed that their ratios were higher for deleted versus non-deleted enzymes, on all tested substrates. In particular, the above ratios for glyceryl tridecanoate were 30-fold and 14-fold higher in S/L and EST2 deleted forms, respectively, compared with their full-length versions. This behaviour was confirmed by the analysis of the S.solfataricus esterase, which showed similar specific activities on pNP-hexanoate and triacylglycerols; in addition, higher activities on the latter substrates were observed in comparison with EST2, S/L and their deleted forms. Finally, a dramatic effect on thermophilicity and thermostability in the EST2 deleted forms was observed. This is the first report highlighting the importance of the "cap" domain in the HSL family, since the N terminus partly contributes to the building up of this structure.
ESTHER : Mandrich_2005_J.Mol.Biol_345_501
PubMedSearch : Mandrich_2005_J.Mol.Biol_345_501
PubMedID: 15581894
Gene_locus related to this paper: burce-lipaa