Luigi Mandrich

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.

Denaturing action of urea and guanidine hydrochloride towards two thermophilic esterases

The stability of two thermophilic esterases, AFEST from Archaeoglobus fulgidus and EST2 from Alicyclobacillus acidocaldarius, against the denaturing action of urea and guanidine hydrochloride has been investigated by means of steady-state fluorescence and circular dichroism measurements. Experimental results indicate that the two enzymes, even though very resistant to temperature and urea, show a resistance to guanidine hydrochloride weaker than expected on the basis of data collected so far for a large set of globular proteins. Structural information available for AFEST and EST2 and ideas that emerged from studies on the molecular origin of the greater thermal stability of thermophiles allow the suggestion of a reliable rationale. The present results may be an indication that the optimization of charge-charge interactions on the protein surface is a key factor for the stability of the two esterases.

Structural determinants of the high thermal stability of SsoPox from the hyperthermophilic archaeon Sulfolobus solfataricus

Organophosphates (OPs) constitute the largest class of insecticides used worldwide and certain of them are potent nerve agents. Consequently, enzymes degrading OPs are of paramount interest, as they could be used as bioscavengers and biodecontaminants. Looking for a stable OPs catalyst, able to support industrial process constraints, a hyperthermophilic phosphotriesterase (PTE) (SsoPox) was isolated from the archaeon Sulfolobus solfataricus and was found to be highly thermostable. The solved 3D structure revealed that SsoPox is a noncovalent dimer, with lactonase activity against “quorum sensing signals”, and therefore could represent also a potential weapon against certain pathogens. The structural basis of the high thermostability of SsoPox has been investigated by performing a careful comparison between its structure and that of two mesophilic PTEs from Pseudomonas diminuta and Agrobacterium radiobacter. In addition, the conformational stability of SsoPox against the denaturing action of temperature and GuHCl has been determined by means of circular dichroism and fluorescence measurements. The data suggest that the two fundamental differences between SsoPox and the mesophilic counterparts are: (a) a larger number of surface salt bridges, also involved in complex networks; (b) a tighter quaternary structure due to an optimization of the interactions at the interface between the two monomers.

The hormone-sensitive lipase from Psychrobacter sp. TA144: New insight in the structural/functional characterization

Cold-adapted esterases and lipases have been found to be dominant activities throughout the cold marine environment, indicating their importance in bacterial degradation of the organic matter. lip2 Gene from Psychrobacter sp. TA144, a micro-organism isolated from the Antarctic sea water, was cloned and over-expressed in Escherichia coli. The recombinant protein (PsyHSL) accumulated in the insoluble fraction from which it was recovered in active form, purified to homogeneity and deeply characterised. Temperature dependence of PsyHSL activity was typical of psychrophilic enzymes, with an optimal temperature of 35 degrees C at pH 8.0. The enzyme resulted to be active on pNP-esters of fatty acids with acyl chain length from C(2) to C(12) and the preferred substrate was pNP-pentanoate showing a k(cat) = 26.2 +/- 0.1 s(-1), K(M) = 0.122 +/- 0.006 mM and a k(cat)/K(M) = 215 +/- 11 mM(-1) s(-1). The enzyme was strongly inhibited by Hg(2+), Zn(2+), Cu(2+), Fe(3+), Mn(2+) ions and it resulted to be activated in presence of methanol and acetonitrile, with calculated C(50) values of 1.98 M and 0.92 M, respectively. The region surrounding PsyHSL catalytic site showed an unexpected homology with the human HSL. Further, both enzymes are characterised by the presence of an extra N-terminal domain, which role in the human protein has been related to regulative function. To clarify the function of PsyHSL N-terminal domain, a 97 amino acids deleted version of the enzyme was produced in E. coli in soluble form, purified and characterised. This mutant was inactive towards all tested substrates, indicating the involvement of this region in the catalytic process.

Functional and structural features of the oxyanion hole in a thermophilic esterase from Alicyclobacillus acidocaldarius.

Recent mutagenic and molecular modelling studies suggested a role for glycine 84 in the putative oxyanion loop of the carboxylesterase EST2 from Alicyclobacillus acidocaldarius. A 114 times decrease of the esterase catalytic activity of the G84S mutant was observed, without changes in the thermal stability. The recently solved three‐dimensional (3D) structure of EST2 in complex with a HEPES molecule permitted to demonstrate that G84 (together with G83 and A156) is involved in the stabilization of the oxyanion through a hydrogen bond from its main chain NH group. The structural data in this case did not allowed us to rationalize the effect of the mutation, since this hydrogen bond was predicted to be unaltered in the mutant. Since the mutation could shed light on the role of the oxyanion loop in the HSL family, experiments to elucidate at the mechanistic level the reasons of the observed drop in k cat were devised. In this work, the kinetic and structural features of the G84S mutant were investigated in more detail. The optimal temperature and pH for the activity of the mutated enzyme were found significantly changed (T = 65°C and pH = 5.75). The catalytic constants K M and Vmax were found considerably altered in the mutant, with ninefold increased K M and 14‐fold decreased Vmax, at pH 5.75. At pH 7.1, the decrease in k cat was much more dramatic. The measurement of kinetic constants for some steps of the reaction mechanism and the resolution of the mutant 3D structure provided evidences that the observed effects were partly due to the steric hindrance of the S84‐OH group towards the ester substrate and partly to its interference with the nucleophilic attack of a water molecule on the second tetrahedral intermediate. Proteins 2008.

Fluorescence Spectroscopy Approaches for the Development of a Real-Time Organophosphate Detection System Using an Enzymatic Sensor

Organophosphates are organic substances that contain a phosphoryl or a thiophosphoryl bond. They are mainly used around the world as pesticides, but can also be used as chemical warfare agents. Their detection is normally entrusted to techniques like GC- and LC-MS that, although sensitive, do not allow their identification on site and in real time. We have approached their identification by exploiting the high-affinity binding of these compounds with the esterase 2 from Alicyclobacillus acidocaldarius. Using an in silico analysis to evaluate the binding affinities of the enzyme with organophosphate inhibitors, like paraoxon, and other organophosphate compounds, like parathion, chlorpyriphos, and other organophosphate thio-derivatives, we have designed fluorescence spectroscopy experiments to study the quenching of the tryptophan residues after esterase 2 binding with the organophosphate pesticides. The changes in the fluorescence signals permitted an immediate and quantitative identification of these compounds from nano- to picomolar concentrations. A fluorescence based polarity-sensitive probe (ANS) was also employed as a means to understand the extent of the interactions involved, as well as to explore other ways to detect organophosphate pesticides. Finally, we designed a framework for the development of a biosensor that exploits fluorescence technology in combination with a sensitive and very stable bio-receptor.

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.

Alicyclobacillus acidocaldarius thermophilic esterase EST2's activity in milk and cheese models.

ABSTRACT The aim of this work was to investigate the behavior of thermophilic esterase EST2 from Alicyclobacillus acidocaldarius in milk and cheese models. The pure enzyme was used to compare the EST2 hydrolytic activity to the activity of endogenous esterase EstA from Lactococcus lactis. The results indicate that EST2 exhibits 30-fold-higher esterase activity than EstA. As EstA has thioesterase activity, EST2 was assayed for this activity under the optimal conditions determined for EstA (namely, 30°C and pH 7.5). Although it is a thermophilic enzyme, EST2 exhibited eightfold-higher thioesterase activity than EstA with S-methyl thiobutanoate. The abilities of EST2 and EstA to synthesize short-chain fatty acid esters were compared. Two methods were developed to do this. In the first method a spectrophotometric assay was used to monitor the synthesis of esters by the pure enzymes using p-nitrophenol as the alcohol substrate. The synthetic activities were also evaluated under conditions that mimicked those present in milk and/or cheese. The second method involved evaluation of the synthetic abilities of the enzymes when they were directly added to a model cheese matrix. Substantial ester synthesis by EST2 was observed under both conditions. Finally, esterase and thioesterase activities were evaluated in milk using the purified EST2 enzyme and in the model cheese matrix using a strain of L. lactis NZ9000 harboring the EST2 gene and thus overproducing EST2. Both the esterase and thioesterase activities measured in milk and in the cheese matrix were much greater than the activities of the controls.

Comprehensive analysis of surface charged residues involved in thermal stability in Alicyclobacillus acidocaldarius esterase 2

Here we report a comprehensive analysis through alanine-scanning mutagenesis of the contribution of surface ion pairs to the thermal stability of Alicyclobacillus acidocaldarius esterase 2 (EST2). We produced 16 single mutants, 4 double mutants corresponding to selected ion pairs R31/E118, E43/K102, R58/D130, D145/R148, 2 double mutants (R63A/R98A and E50A/D94A) involving residues of a large ion network on the protein surface and the double-mutant R98A/R148A meant to disrupt the R98 interactions within the said network and, contextually, the interaction between R148 and D145. The double-mutant E43A/E273K was obtained by chance. All selected residues were replaced with alanine except E91, which was mutated to a glycine and K102, which was changed to a glutamine. All 24 proteins were over-expressed in Escherichia coli, purified and characterized with respect to the main features. Structural stability data were compared with an in silico prediction of ΔΔG values. Our study of the individual factors involved in thermostability and their structural interpretation reveals that the great stability of this thermophilic protein can be explained by the contribution of a few residues at the protein surface.

Hyperthermophilic phosphotriesterases/lactonases for the environment and human health

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.