Promiscuous Phosphotriesterase Activity Research Articles

Harnessing hyperthermostable lactonase from Sulfolobus solfataricus for biotechnological applications.

Extremozymes have gained considerable interest as they could meet industrial requirements. Among these, SsoPox is a hyperthermostable enzyme isolated from the archaeon Sulfolobus solfataricus. This enzyme is a lactonase catalyzing the hydrolysis of acyl-homoserine lactones; these molecules are involved in Gram-negative bacterial communication referred to as quorum sensing. SsoPox exhibits promiscuous phosphotriesterase activity for the degradation of organophosphorous chemicals including insecticides and chemical warfare agents. Owing to its bi-functional catalytic abilities as well as its intrinsic stability, SsoPox is appealing for many applications, having potential uses in the agriculture, defense, food and health industries. Here we investigate the biotechnological properties of the mutant SsoPox-W263I, a variant with increased lactonase and phosphotriesterase activities. We tested enzyme resistance against diverse process-like and operating conditions such as heat resistance, contact with organic solvents, sterilization, storage and immobilization. Bacterial secreted materials from both Gram-negative and positive bacteria were harmless on SsoPox-W263I activity and could reactivate heat-inactivated enzyme. SsoPox showed resistance to harsh conditions demonstrating that it is an extremely attractive enzyme for many applications. Finally, the potential of SsoPox-W263I to be active at subzero temperature is highlighted and discussed in regards to the common idea that hyperthermophile enzymes are nearly inactive at low temperatures.

Biochemical basis for hydrolysis of organophosphorus by a marine bacterial prolidase

Abstract Extensive application of synthesized organophosphorus compounds (OPs) leads to pollutant accumulation and enhanced eco-toxicity. Hydrolysis of phosphotriester bonds catalyzed by evolved microbial enzymes is a key step for detoxification of OPs. Here, a new marine bacterial prolidase OPAA4301 exhibiting promiscuous phosphotriesterase activity was isolated and systematically characterized. The homo-tetrameric enzyme OPAA4301 can catalyze the hydrolysis of both amido bond and phosphotriester bond. Manganese ions were observed to be essential for its catalytic integrity, and in vitro substitution of manganese ions by different metal cofactors led to decreased activity. We also revealed cooperation pattern of metal ligands and substrate-binding residues on OP hydrolysis by mutational analysis. Metal-binding sites together with Arg418 in the large-binding pocket of the enzyme were found to be indispensable for catalytic ability. Substitution mutation of small- and large-binding pocket residues caused significant variation in phosphotriesterase activity, and leaving group sites appeared to be involved in the catalytic process as substrate affinity regulators. Our study gave an overall biochemical understanding on the organophosphorus hydrolysis pattern of the newly identified marine bacterial prolidase and provided ideas for protein engineering to expand its application in the bioremediation field.

Active site loop conformation regulates promiscuous activity in a lactonase from Geobacillus kaustophilus HTA426.

Enzyme promiscuity is a prerequisite for fast divergent evolution of biocatalysts. A phosphotriesterase-like lactonase (PLL) from Geobacillus kaustophilus HTA426 (GkaP) exhibits main lactonase and promiscuous phosphotriesterase activities. To understand its catalytic and evolutionary mechanisms, we investigated a “hot spot” in the active site by saturation mutagenesis as well as X-ray crystallographic analyses. We found that position 99 in the active site was involved in substrate discrimination. One mutant, Y99L, exhibited 11-fold improvement over wild-type in reactivity (kcat/Km) toward the phosphotriesterase substrate ethyl-paraoxon, but showed 15-fold decrease toward the lactonase substrate δ-decanolactone, resulting in a 157-fold inversion of the substrate specificity. Structural analysis of Y99L revealed that the mutation causes a ∼6.6 Å outward shift of adjacent loop 7, which may cause increased flexibility of the active site and facilitate accommodation and/or catalysis of organophosphate substrate. This study provides for the PLL family an example of how the evolutionary route from promiscuity to specificity can derive from very few mutations, which promotes alteration in the conformational adjustment of the active site loops, in turn draws the capacity of substrate binding and activity.

Crystallization and preliminary X-ray diffraction analysis of the lactonaseVmoLac fromVulcanisaeta moutnovskia

Phosphotriesterase-like lactonases (PLLs) are native lactonases that are capable of hydrolyzing lactones such as aliphatic lactones or acyl-homoserine lactones, which are involved in bacterial quorum sensing. Previously characterized PLLs are moreover endowed with a promiscuous phosphotriesterase activity and are therefore able to detoxify organophosphate insecticides. A novel PLL representative, dubbed VmoLac, has been identified from the hyperthermophilic crenarchaeon Vulcanisaeta moutnovskia. Because of its intrinsic high thermal stability, VmoLac may constitute an appealing candidate for engineering studies with the aim of producing an efficient biodecontaminant for organophosphorus compounds and a bacterial antivirulence agent. In combination with biochemical studies, structural information will allow the identification of the residues involved in substrate specificity and an understanding of the enzymatic catalytic mechanisms. Here, the expression, purification, crystallization and X-ray data collection at 2.4 Å resolution of VmoLac are reported.

Differential active site loop conformations mediate promiscuous activities in the lactonase SsoPox.

Enzymes are proficient catalysts that enable fast rates of Michaelis-complex formation, the chemical step and products release. These different steps may require different conformational states of the active site that have distinct binding properties. Moreover, the conformational flexibility of the active site mediates alternative, promiscuous functions. Here we focused on the lactonase SsoPox from Sulfolobus solfataricus. SsoPox is a native lactonase endowed with promiscuous phosphotriesterase activity. We identified a position in the active site loop (W263) that governs its flexibility, and thereby affects the substrate specificity of the enzyme. We isolated two different sets of substitutions at position 263 that induce two distinct conformational sampling of the active loop and characterized the structural and kinetic effects of these substitutions. These sets of mutations selectively and distinctly mediate the improvement of the promiscuous phosphotriesterase and oxo-lactonase activities of SsoPox by increasing active-site loop flexibility. These observations corroborate the idea that conformational diversity governs enzymatic promiscuity and is a key feature of protein evolvability.

A Further Biochemical Characterization of DrPLL the Thermophilic Lactonase from Deinococcus radiodurans

Recently, the cloning of the ORF Dr0930 from Deinococcus radiodurans displaying, as primary activity, a lactonase activity and a promiscuous phosphotriesterase activity was reported. The crystal structure of the resulting recombinant enzyme has been solved, and many mutants have been generated in order to increase the phosphotriesterase activity, with the aim to reach the level of activity of the related pPTE from Pseudomonas diminuta. In this paper we report an additional biochemical characterization of DrPLL and show that this enzyme has an optimal temperature for catalysis of 85 °C and possesses promiscuous carboxylesterase, phophodiesterase and thioesterase activities which were not previously described. A metal analysis was performed on the purified protein by inductively coupled plasma mass spectrometry (ICP-MS ELAN DRC-e), which confirmed the presence of Ni(2+) as a main metal in the recombinant protein. Surprisingly, the specificity constants (s=k(cat)/K(M)) for the pNP-decanoate and pNP-dodecanoate esters were only one order of magnitude lower than that for the lactone substrate thio-buthyl-γ-butyric-lactone (TBBL), and the K(M) value for TBBL was more than ten-fold higher than those for the esters. We named this enzyme DrPLL, based on its structural and biochemical features it belongs to the Phosphotriesterase Like Lactonase group, a small protein family belonging to the amidohydrolase superfamily.

Characterisation of the organophosphate hydrolase catalytic activity of SsoPox

SsoPox is a lactonase endowed with promiscuous phosphotriesterase activity isolated from Sulfolobus solfataricus that belongs to the Phosphotriesterase-Like Lactonase family. Because of its intrinsic thermal stability, SsoPox is seen as an appealing candidate as a bioscavenger for organophosphorus compounds. A comprehensive kinetic characterisation of SsoPox has been performed with various phosphotriesters (insecticides) and phosphodiesters (nerve agent analogues) as substrates. We show that SsoPox is active for a broad range of OPs and remains active under denaturing conditions. In addition, its OP hydrolase activity is highly stimulated by anionic detergent at ambient temperature and exhibits catalytic efficiencies as high as kcat/KM of 105 M−1s−1 against a nerve agent analogue. The structure of SsoPox bound to the phosphotriester fensulfothion reveals an unexpected and non-productive binding mode. This feature suggests that SsoPox's active site is sub-optimal for phosphotriester binding, which depends not only upon shape but also on localised charge of the ligand.

A Further Biochemical Characterization of DrPLL the Thermophilic Lactonase from Deinococcus radiodurans

Recently, the cloning of the ORF Dr0930 from Deinococcus radiodurans displaying, as primary activity, a lactonase activity and a promiscuous phosphotriesterase activity was reported. The crystal structure of the resulting recombinant enzyme has been solved, and many mutants have been generated in order to increase the phosphotriesterase activity, with the aim to reach the level of activity of the related pPTE from Pseudomonas diminuta. In this paper we report an additional biochemical characterization of DrPLL and show that this enzyme has an optimal temperature for catalysis of 85 °C and possesses promiscuous carboxylesterase, phophodiesterase and thioesterase activities which were not previously described. A metal analysis was performed on the purified protein by inductively coupled plasma mass spectrometry (ICP-MS ELAN DRC-e), which confirmed the presence of Ni2+ as a main metal in the recombinant protein. Surprisingly, the specificity constants (s=kcat/KM) for the pNP-decanoate and pNP-dodecanoate esters were only one order of magnitude lower than that for the lactone substrate thio-buthyl-γ-butyric-lactone (TBBL), and the KM value for TBBL was more than ten-fold higher than those for the esters. We named this enzyme DrPLL, based on its structural and biochemical features it belongs to the Phosphotriesterase Like Lactonase group, a small protein family belonging to the amidohydrolase superfamily. Keywords: Amidohydrolase superfamily, deinococcus radiodurans, lactonases, phophotriesterases, promiscuous activities, thermophilic enzymes, crystal structure, mutants, carboxylesterase, thioesterase

A Further Biochemical Characterization of DrPLL the Thermophilic Lactonase from Deinococcus radiodurans

Recently, the cloning of the ORF Dr0930 from Deinococcus radiodurans displaying, as primary activity, a lactonase activity and a promiscuous phosphotriesterase activity was reported. The crystal structure of the resulting recombinant enzyme has been solved, and many mutants have been generated in order to increase the phosphotriesterase activity, with the aim to reach the level of activity of the related pPTE from Pseudomonas diminuta. In this paper we report an additional biochemical characterization of DrPLL and show that this enzyme has an optimal temperature for catalysis of 85 °C and possesses promiscuous carboxylesterase, phophodiesterase and thioesterase activities which were not previously described. A metal analysis was performed on the purified protein by inductively coupled plasma mass spectrometry (ICP-MS ELAN DRC-e), which confirmed the presence of Ni(2+) as a main metal in the recombinant protein. Surprisingly, the specificity constants (s=k(cat)/K(M)) for the pNP-decanoate and pNP-dodecanoate esters were only one order of magnitude lower than that for the lactone substrate thio-buthyl-γ-butyric-lactone (TBBL), and the K(M) value for TBBL was more than ten-fold higher than those for the esters. We named this enzyme DrPLL, based on its structural and biochemical features it belongs to the Phosphotriesterase Like Lactonase group, a small protein family belonging to the amidohydrolase superfamily.

Structural and Enzymatic characterization of the lactonase SisLac from Sulfolobus islandicus

BackgroundA new member of the Phosphotriesterase-Like Lactonases (PLL) family from the hyperthermophilic archeon Sulfolobus islandicus (SisLac) has been characterized. SisLac is a native lactonase that exhibits a high promiscuous phosphotriesterase activity. SisLac thus represents a promising target for engineering studies, exhibiting both detoxification and bacterial quorum quenching abilities, including human pathogens such as Pseudomonas aeruginosa.Methodology/Principal FindingsHere, we describe the substrate specificity of SisLac, providing extensive kinetic studies performed with various phosphotriesters, esters, N-acyl-homoserine lactones (AHLs) and other lactones as substrates. Moreover, we solved the X-ray structure of SisLac and structural comparisons with the closely related SsoPox structure highlighted differences in the surface salt bridge network and the dimerization interface. SisLac and SsoPox being close homologues (91% sequence identity), we undertook a mutational study to decipher these structural differences and their putative consequences on the stability and the catalytic properties of these proteins.Conclusions/SignificanceWe show that SisLac is a very proficient lactonase against aroma lactones and AHLs as substrates. Hence, data herein emphasize the potential role of SisLac as quorum quenching agent in Sulfolobus. Moreover, despite the very high sequence homology with SsoPox, we highlight key epistatic substitutions that influence the enzyme stability and activity.

Enhancing the Promiscuous Phosphotriesterase Activity of a Thermostable Lactonase (GkaP) for the Efficient Degradation of Organophosphate Pesticides

The phosphotriesterase-like lactonase (PLL) enzymes in the amidohydrolase superfamily hydrolyze various lactones and exhibit latent phosphotriesterase activities. These enzymes serve as attractive templates for in vitro evolution of neurotoxic organophosphates (OPs) with hydrolytic capabilities that can be used as bioremediation tools. Here, a thermostable PLL from Geobacillus kaustophilus HTA426 (GkaP) was targeted for joint laboratory evolution with the aim of enhancing its catalytic efficiency against OP pesticides. By a combination of site saturation mutagenesis and whole-gene error-prone PCR approaches, several improved variants were isolated. The most active variant, 26A8C, accumulated eight amino acid substitutions and demonstrated a 232-fold improvement over the wild-type enzyme in reactivity (k(cat)/K(m)) for the OP pesticide ethyl-paraoxon. Concomitantly, this variant showed a 767-fold decrease in lactonase activity with δ-decanolactone, imparting a specificity switch of 1.8 × 10(5)-fold. 26A8C also exhibited high hydrolytic activities (19- to 497-fold) for several OP pesticides, including parathion, diazinon, and chlorpyrifos. Analysis of the mutagenesis sites on the GkaP structure revealed that most mutations are located in loop 8, which determines substrate specificity in the amidohydrolase superfamily. Molecular dynamics simulation shed light on why 26A8C lost its native lactonase activity and improved the promiscuous phosphotriesterase activity. These results permit us to obtain further insights into the divergent evolution of promiscuous enzymes and suggest that laboratory evolution of GkaP may lead to potential biological solutions for the efficient decontamination of neurotoxic OP compounds.

Catalytic Versatility and Backups in Enzyme Active Sites: The Case of Serum Paraoxonase 1

The origins of enzyme specificity are well established. However, the molecular details underlying the ability of a single active site to promiscuously bind different substrates and catalyze different reactions remain largely unknown. To better understand the molecular basis of enzyme promiscuity, we studied the mammalian serum paraoxonase 1 (PON1) whose native substrates are lipophilic lactones. We describe the crystal structures of PON1 at a catalytically relevant pH and of its complex with a lactone analogue. The various PON1 structures and the analysis of active-site mutants guided the generation of docking models of the various substrates and their reaction intermediates. The models suggest that promiscuity is driven by coincidental overlaps between the reactive intermediate for the native lactonase reaction and the ground and/or intermediate states of the promiscuous reactions. This overlap is also enabled by different active-site conformations: the lactonase activity utilizes one active-site conformation whereas the promiscuous phosphotriesterase activity utilizes another. The hydrolysis of phosphotriesters, and of the aromatic lactone dihydrocoumarin, is also driven by an alternative catalytic mode that uses only a subset of the active-site residues utilized for lactone hydrolysis. Indeed, PON1's active site shows a remarkable level of networking and versatility whereby multiple residues share the same task and individual active-site residues perform multiple tasks (e.g., binding the catalytic calcium and activating the hydrolytic water). Overall, the coexistence of multiple conformations and alternative catalytic modes within the same active site underlines PON1's promiscuity and evolutionary potential.

Crystallization and preliminary X-ray diffraction analysis of the hyperthermophilic Sulfolobus islandicus lactonase.

Phosphotriesterase-like lactonases (PLLs) constitute an interesting family of enzymes that are of paramount interest in biotechnology with respect to their catalytic functions. As natural lactonases, they may act against pathogens such asPseudomonas aeruginosa by shutting down their quorum-sensing system (quorum quenching) and thus decreasing pathogen virulence. Owing totheir promiscuous phosphotriesterase activity, which can inactivate toxic organophosphorus compounds such as pesticides and nerve agents, they are equally appealing as potent bioscavengers. A new representative of the PLL family has been identified (SisPox) and its gene was cloned from the hyperthermophilic archeon Sulfolobus islandicus. Owing to its hyperthermostable architecture, SisPox appears to be a good candidate for engineering studies. Here, production, purification, crystallization conditions and data collection to 2.34 Å resolution are reported for this lactonase from the hyperthermophilic S.islandicus.

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.

Structural basis for natural lactonase and promiscuous phosphotriesterase activities

Structural basis for natural lactonase and promiscuous phosphotriesterase activities

Evolution in the Amidohydrolase Superfamily: Substrate-Assisted Gain of Function in the E183K Mutant of a Phosphotriesterase-like Metal-Carboxylesterase

The recent specialization for utilization of pesticides reported for Pseudomonas diminuta phosphotriesterase (pPTE) strongly suggests that this activity evolved from an enzyme endowed with promiscuous phosphotriesterase activity. Such a putative "generalist" enzyme was recently proposed to be a member of the new phoshotriesterase-like lactonase family (PLL). The promiscuous carboxylesterase and phosphodiesterase activities detected in pPTE and PLLs in turn paved the way for the prediction of the existence in nature of PTE-like enzymes with predominant carboxylesterase or phosphodiesterase activities. An "in silico" analysis of the related Mesorhizobium loti ORF MLL7664 and the biochemical characterization demonstrated its prominent carboxylesterase and low phosphotriesterase specificity. On the basis of sequence similarity with the phosphotriesterase homology protein from Escherichia coli and the carboxylesterase activity, we called it phosphotriesterase-like carboxylesterase (MloPLC). The carboxylesterase activity is strictly dependent on divalent cations, and as such MloPLC is the first phosphotriesterase-like metal-carboxylesterase characterized to date. In related enzymes of the amidohydrolase superfamily either glutamate or carboxylated lysine substitutes for MloPLC glutamate 183 and the residue appear invariantly involved in maintaining the structural integrity of the binuclear metal center. Accordingly, we changed Glu-183 to lysine or glutamine. All the tested activities were completely abolished in the E183Q mutant, while only a residual phosphotriesterase activity could be detected in the E183K mutant. Surprisingly, in the latter mutant a parallel 650-fold specificity increase in bis-p-nitrophenyl-phosphate (BpNP-P) was observed, turning MloPLC from a carboxylesterase into a phosphodiesterase. Chemical, structural, and kinetic data strongly suggested that K183 is not carboxylated and that the gain of the new function is assisted by the substrate.

Structural basis for natural lactonase and promiscuous phosphotriesterase activities

Structural basis for natural lactonase and promiscuous phosphotriesterase activities

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 Å resolution) and in complex with a quorum-sensing lactone mimic at 2.0 Å 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.

The Latent Promiscuity of Newly Identified Microbial Lactonases Is Linked to a Recently Diverged Phosphotriesterase

In essence, evolutionary processes occur gradually, while maintaining fitness throughout. Along this line, it has been proposed that the ability of a progenitor to promiscuously catalyze a low level of the evolving activity could facilitate the divergence of a new function by providing an immediate selective advantage. To directly establish a role for promiscuity in the divergence of natural enzymes, we attempted to trace the origins of a bacterial phosphotriesterase (PTE), an enzyme thought to have evolved for the purpose of degradation of a synthetic insecticide introduced in the 20th century. We surmised that PTE's promiscuous lactonase activity may be a vestige of its progenitor and tested homologues annotated as "putative PTEs" for lactonase and phosphotriesterase activity. We identified three genes that define a new group of microbial lactonases dubbed PTE-like lactonases (PLLs). These enzymes proficiently hydrolyze various lactones, and in particular quorum-sensing N-acyl homoserine lactones (AHLs), and exhibit much lower promiscuous phosphotriesterase activities. PLLs share key sequence and active site features with PTE and differ primarily by an insertion in one surface loop. Given their biochemical and biological function, PLLs are likely to have existed for many millions of years. PTE could have therefore evolved from a member of the PLL family while utilizing its latent promiscuous paraoxonase activity as an essential starting point.