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Mikael Elias - One of the best experts on this subject based on the ideXlab platform.
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Effects of Signal Disruption Depends on the Substrate Preference of the Lactonase.
Frontiers in microbiology, 2020Co-Authors: Kathleen Mahan, Ryan Martinmaki, Isabel Larus, Rakesh Sikdar, Jordan M. Dunitz, Mikael EliasAbstract:Many bacteria produce and use extracellular signaling molecules such as acyl homoserine lactones (AHLs) to communicate and coordinate behavior in a cell-density dependent manner, via a communication system called quorum sensing (QS). This system regulates behaviors including but not limited to virulence and biofilm formation. We focused on Pseudomonas aeruginosa, a human opportunistic pathogen that is involved in acute and chronic lung infections and which disproportionately affects people with cystic fibrosis. P. aeruginosa infections are becoming increasingly challenging to treat with the spread of antibiotic resistance. Therefore, QS disruption approaches, known as quorum quenching, are appealing due to their potential to control the virulence of resistant strains. Interestingly, P. aeruginosa is known to simultaneously utilize two main QS circuits, one based on C4-AHL, the other with 3-oxo-C12-AHL. Here, we evaluated the effects of signal disruption on 39 cystic fibrosis clinical isolates of P. aeruginosa, including drug resistant strains. We used two enzymes capable of degrading AHLs, known as Lactonases, with distinct substrate preference: one degrading 3-oxo-C12-AHL, the other degrading both C4-AHL and 3-oxo-C12-AHL. Two Lactonases were used to determine the effects of signal disruption on the clinical isolates, and to evaluate the importance of the QS circuits by measuring effects on virulence factors (elastase, protease, and pyocyanin) and biofilm formation. Signal disruption results in at least one of these factors being inhibited for most isolates (92%). Virulence factor activity or production were inhibited by up to 100% and biofilm was inhibited by an average of 2.3 fold. Remarkably, the treatments led to distinct inhibition profiles of the isolates; the treatment with the Lactonase degrading both signaling molecules resulted in a higher fraction of inhibited isolates (77% vs. 67%), and the simultaneous inhibition of more virulence factors per strain (2 vs. 1.5). This finding suggests that the Lactonase AHL preference is key to its inhibitory spectrum and is an essential parameter to improve quorum quenching strategies.
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Lactonase Specificity Is Key to Quorum Quenching in Pseudomonas aeruginosa
FRONTIERS IN MICROBIOLOGY, 2020Co-Authors: Benjamin Remy, Mikael Elias, Laure Plener, Philippe Decloquement, Nicholas Armstrong, David Daudé, Eric ChabriereAbstract:The human opportunistic pathogen Pseudomonas aeruginosa orchestrates the expression of many genes in a cell density-dependent manner by using quorum sensing (QS). Two acyl-homoserine lactones (AHLs) are involved in QS circuits and contribute to the regulation of virulence factors production, biofilm formation, and antimicrobial sensitivity. Disrupting QS, a strategy referred to as quorum quenching (QQ) can be achieved using exogenous AHL-degrading Lactonases. However, the importance of enzyme specificity on quenching efficacy has been poorly investigated. Here, we used two Lactonases both targeting the signal molecules N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C-12 HSL) and butyryl-homoserine lactone (C-4 HSL) albeit with different efficacies on C-4 HSL. Interestingly, both Lactonases similarly decreased AHL concentrations and comparably impacted the expression of AHL-based QS genes. However, strong variations were observed in Pseudomonas Quinolone Signal (PQS) regulation depending on the Lactonase used. Both Lactonases were also found to decrease virulence factors production and biofilm formation in vitro, albeit with different efficiencies. Unexpectedly, only the Lactonase with lower efficacy on C-4 HSL was able to inhibit P. aeruginosa pathogenicity in vivo in an amoeba infection model. Similarly, proteomic analysis revealed large variations in protein levels involved in antibiotic resistance, biofilm formation, virulence and diverse cellular mechanisms depending on the chosen Lactonase. This global analysis provides evidences that QQ enzyme specificity has a significant impact on the modulation of QS-associated behavior in P. aeruginosa PA14.
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Lactonase specificity is key to quorum quenching in pseudomonas aeruginosa
Social Science Research Network, 2019Co-Authors: Benjamin Remy, Mikael Elias, Laure Plener, Philippe Decloquement, Nicholas Armstrong, David Daudé, Eric ChabriereAbstract:Background: Pseudomonas aeruginosa orchestrates the expression of many genes by using quorum sensing (QS) communication process. QS relies on an intricate molecular network including two acyl-homoserine lactones (AHL), involved in the modulation of virulence factors production, biofilm formation or antimicrobial sensitivity. Disrupting QS is a promising approach to modulate virulence while not challenging bacterial survival and can be achieved with AHL-degrading Lactonases. Methods: Here, we used two Lactonases both targeting the signal molecules N-(3-oxododecanoyl)-L-Homoserine lactone (3-oxo-C12 HSL) and butyryl-homoserine lactone (C4 HSL) albeit with different efficacy on C4 HSL. We used phenotypic analyses to characterize the specific impact of each Lactonases on P. aeruginosa virulence. We further used molecular approaches for finely determine the impact of Lactonases on gene regulation and protein expressions of P. aeruginosa. Findings: Although, both Lactonases similarly impacted AHL-based circuits, strong variations were however observed in Pseudomonas Quinolone Signal regulation yielding in different impacts on phenotypic traits related to virulence, biofilm, pathogenicity or antibiotic susceptibility. Proteomic analysis further allowed to identify the specific impacts of Lactonases on P. aeruginosa behavior. Interpretation: This global analysis provides the first evidence that QQ enzyme specificity is crucial to modulate QS-associated behavior in P. aeruginosa PA14 and will help to develop efficient quorum quenching strategies. Funding Statement: This work was supported by a project RAPID (LACTO-TEX) from Direction Generale de l'Armement (DGA, France), "Investissements d'avenir" program (Mediterranee Infection 10-IAHU-03) of the French Agence Nationale de la Recherche (ANR), "Emplois Jeunes Doctorants" program of Region Provence-Alpes-Cote d'Azur (PACA, France) and MnDrive initiative. Declaration of Interests: ME and EC have a patent WO2014167140 A1 licensed to Gene&GreenTK. LP, DD, BR, ME and EC report personal fees from Gene&GreenTK during the conduct of the study. The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Ethics Approval Statement: Not required.
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the structural determinants accounting for the broad substrate specificity of the quorum quenching Lactonase gcl
ChemBioChem, 2019Co-Authors: Celine Bergonzi, Michael Schwab, Tanushree Naik, Mikael EliasAbstract:Quorum quenching Lactonases are enzymes capable of hydrolyzing lactones, including N-acyl homoserine lactones (AHLs). AHLs are molecules known as signals in bacterial communication dubbed quorum sensing. Bacterial signal disruption by Lactonases was previously reported to inhibit behavior regulated by quorum sensing, such as the expression of virulence factors and the formation of biofilms. Herein, we report the enzymatic and structural characterization of a novel Lactonase representative from the metallo-β-lactamase superfamily, dubbed GcL. GcL is a broad spectrum and highly proficient Lactonase, with kcat /KM values in the range of 104 to 106 m-1 s-1 . Analysis of free GcL structures and in complex with AHL substrates of different acyl chain length, namely, C4-AHL and 3-oxo-C12-AHL, allowed their respective binding modes to be elucidated. Structures reveal three subsites in the binding crevice: 1) the small subsite where chemistry is performed on the lactone ring; 2) a hydrophobic ring that accommodates the amide group of AHLs and small acyl chains; and 3) the outer, hydrophilic subsite that extends to the protein surface. Unexpectedly, the absence of structural accommodation for long substrate acyl chains seems to relate to the broad substrate specificity of the enzyme.
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Evaluation of biological and enzymatic quorum quencher coating additives to reduce biocorrosion of steel
2019Co-Authors: Siqia Huang, Mikael Elias, Michael Schwab, Celine Ergonzi, Randall E. HicksAbstract:Microbial colonization can be detrimental to the integrity of metal surfaces and lead to microbiologically influenced corrosion (MIC). Biocorrosion is a serious problem for aquatic and marine industries in the world. In Minnesota (USA), where this study was conducted, biocorrosion severely affects the maritime transportation industry. The anticorrosion activity of a variety of compounds, including chemical (magnesium peroxide) and biological (surfactin, capsaicin, and gramicidin) molecules were investigated as coating additives. We also evaluated a previously engineered, extremely stable, non-biocidal enzyme known to interfere in bacterial signaling, SsoPox (a quorum quenching Lactonase). Experimental steel coupons were submerged in water from the Duluth Superior Harbor (DSH) for 8 weeks in the laboratory. Biocorrosion was evaluated by counting the number and the coverage of corrosion tubercles on coupons and also by ESEM imaging of the coupon surface. Three experimental coating additives significantly reduced the formation of corrosion tubercles: surfactin, magnesium peroxide and the quorum quenching Lactonase by 31%, 36% and 50%, respectively. DNA sequence analysis of the V4 region of the bacterial 16S rRNA gene revealed that these decreases in corrosion were associated with significant changes in the composition of bacterial communities on the steel surfaces. These results demonstrate the potential of highly stable quorum quenching Lactonases to provide a reliable, cost-effective method to treat steel structures and prevent biocorrosion.
Dan S Tawfik - One of the best experts on this subject based on the ideXlab platform.
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the evolutionary origins of detoxifying enzymes the mammalian serum paraoxonases pons relate to bacterial homoserine Lactonases
Journal of Biological Chemistry, 2013Co-Authors: Hagi Arrogovsky, Adria Hugenmatte, Dan S TawfikAbstract:Serum paraoxonases (PONs) are detoxifying Lactonases that were first identified in mammals. Three mammalian families are known, PON1, 2, and 3 that reside primarily in the liver. They catalyze essentially the same reaction, lactone hydrolysis, but differ in their substrate specificity. Although some members are highly specific, others have a broad specificity profile. The evolutionary origins and substrate specificities of PONs therefore remain poorly understood. Here, we report a newly identified family of bacterial PONs, and the reconstruction of the ancestor of the three families of mammalian PONs. Both the mammalian ancestor and the characterized bacterial PONX_OCCAL were found to efficiently hydrolyze N-acyl homoserine lactones that mediate quorum sensing in many bacteria, including pathogenic ones. The mammalian PONs may therefore relate to a newly identified family of bacterial, PON-like “quorum-quenching” Lactonases. The appearance of PONs in metazoa is likely to relate to innate immunity rather than detoxification. Unlike the bacterial PON, the mammalian ancestor also hydrolyzes, with low efficiency, lactones other than homoserine lactones, thus preceding the detoxifying functions that diverged later in two of the three mammalian families. The bifunctionality of the mammalian ancestor and the trade-off between the quorum-quenching and detoxifying Lactonase activities explain the broad and overlapping specificities of some mammalian PONs versus the singular specificity of others.
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reconstructing a missing link in the evolution of a recently diverged phosphotriesterase by active site loop remodeling
Biochemistry, 2012Co-Authors: Livnat Afriatjurnou, Colin J Jackson, Dan S TawfikAbstract:Only decades after the introduction of organophosphate pesticides, bacterial phosphotriesterases (PTEs) have evolved to catalyze their degradation with remarkable efficiency. Their closest known relatives, Lactonases, with promiscuous phosphotriasterase activity, dubbed PTE-like Lactonases (PLLs), share only 30% sequence identity and also differ in the configuration of their active-site loops. PTE was therefore presumed to have evolved from a yet unknown PLL whose primary activity was the hydrolysis of quorum sensing homoserine lactones (HSLs) (Afriat et al. (2006) Biochemistry 45, 13677-13686). However, how PTEs diverged from this presumed PLL remains a mystery. In this study we investigated loop remodeling as a means of reconstructing a homoserine Lactonase ancestor that relates to PTE by few mutational steps. Although, in nature, loop remodeling is a common mechanism of divergence of enzymatic functions, reproducing this process in the laboratory is a challenge. Structural and phylogenetic analyses enabled us to remodel one of PTE's active-site loops into a PLL-like configuration. A deletion in loop 7, combined with an adjacent, highly epistatic, point mutation led to the emergence of an HSLase activity that is undetectable in PTE (k(cat)/K(M) values of up to 2 × 10(4)). The appearance of the HSLase activity was accompanied by only a minor decrease in PTE's paraoxonase activity. This specificity change demonstrates the potential role of bifunctional intermediates in the divergence of new enzymatic functions and highlights the critical contribution of loop remodeling to the rapid divergence of new enzyme functions.
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apoe induces serum paraoxonase pon1 activity and stability similar to apoa i
Biochemistry, 2010Co-Authors: Leonid Gaidukov, Michael Aviram, Mira Rosenblat, Viji R, Shiri Yacobson, Dan S TawfikAbstract:Serum paraoxonase (PON1) is an anti-atherogenic interfacially activated lipo-Lactonase that was shown to selectively bind high-density lipoprotein (HDL) carrying apolipoprotein A-I (apoA-I). ApoA-I binding occurs with nanomolar affinity and induces a dramatic increase in enzyme stability and Lactonase activity. This study examined the association of PON1 with reconstituted HDL (rHDL) carrying apolipoprotein E, and its consequences on the stability and enzymatic activity of PON1, and on its anti-atherogenic potential. The results indicate that reconstituted HDL particles prepared with two most common isoforms of apoE (apoE3 and apoE4) associate with rePON1 in a manner and affinity similar to those of apoA-I. Binding to apoE-HDL stimulates the Lactonase activity and stabilizes the enzyme, although the latter occurs to a >10-fold lesser extent compared to apoA-I-HDL particles. The anti-atherogenic potential of PON1, measured by inhibition of LDL oxidation and stimulation of macrophage cholesterol efflux, was...
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the latent promiscuity of newly identified microbial Lactonases is linked to a recently diverged phosphotriesterase
Biochemistry, 2006Co-Authors: Livnat Afriat, Giuseppe Manco, Cintia Roodveldt, Dan S TawfikAbstract: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.
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the histidine 115 histidine 134 dyad mediates the Lactonase activity of mammalian serum paraoxonases
Journal of Biological Chemistry, 2006Co-Authors: Olga Khersonsky, Dan S TawfikAbstract:Serum paraoxonases (PONs) are calcium-dependent Lactonases that catalyze the hydrolysis and formation of a variety of lactones, with a clear preference for lipophilic lactones. However, the Lactonase mechanism of mammalian PON1, a high density lipoprotein-associated enzyme that is the most studied family member, remains unclear, and other family members have not been examined at all. We present a kinetic and site-directed mutagenesis study aimed at deciphering the Lactonase mechanism of PON1 and PON3. The pH-rate profile determined for the Lactonase activity of PON1 indicated an apparent pKa of ∼7.4. We thus explored the role of all amino acids in the PON1 active site that are not directly ligated to the catalytic calcium and that possess an imidazolyl or carboxyl side chain (His115, His134, His184, His285, Asp183, and Asp269). Extensive site-directed mutagenesis studies in which each amino acid candidate was replaced with all other 19 amino acids were conducted to identify the residue(s) that mediate the Lactonase activity of PONs. The results indicate that the Lactonase activity of PON1 and PON3 and the esterase activity of PON1 are mediated by the His115-His134 dyad. Notably, the phosphotriesterase activity of PON1, which is a promiscuous activity of this enzyme, is mediated by other residues. To our knowledge, this is one of few examples of a histidine dyad in enzyme active sites and the first example of a hydrolytic enzyme with such a dyad.
Eric Chabriere - One of the best experts on this subject based on the ideXlab platform.
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Lactonase Specificity Is Key to Quorum Quenching in Pseudomonas aeruginosa
FRONTIERS IN MICROBIOLOGY, 2020Co-Authors: Benjamin Remy, Mikael Elias, Laure Plener, Philippe Decloquement, Nicholas Armstrong, David Daudé, Eric ChabriereAbstract:The human opportunistic pathogen Pseudomonas aeruginosa orchestrates the expression of many genes in a cell density-dependent manner by using quorum sensing (QS). Two acyl-homoserine lactones (AHLs) are involved in QS circuits and contribute to the regulation of virulence factors production, biofilm formation, and antimicrobial sensitivity. Disrupting QS, a strategy referred to as quorum quenching (QQ) can be achieved using exogenous AHL-degrading Lactonases. However, the importance of enzyme specificity on quenching efficacy has been poorly investigated. Here, we used two Lactonases both targeting the signal molecules N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C-12 HSL) and butyryl-homoserine lactone (C-4 HSL) albeit with different efficacies on C-4 HSL. Interestingly, both Lactonases similarly decreased AHL concentrations and comparably impacted the expression of AHL-based QS genes. However, strong variations were observed in Pseudomonas Quinolone Signal (PQS) regulation depending on the Lactonase used. Both Lactonases were also found to decrease virulence factors production and biofilm formation in vitro, albeit with different efficiencies. Unexpectedly, only the Lactonase with lower efficacy on C-4 HSL was able to inhibit P. aeruginosa pathogenicity in vivo in an amoeba infection model. Similarly, proteomic analysis revealed large variations in protein levels involved in antibiotic resistance, biofilm formation, virulence and diverse cellular mechanisms depending on the chosen Lactonase. This global analysis provides evidences that QQ enzyme specificity has a significant impact on the modulation of QS-associated behavior in P. aeruginosa PA14.
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Lactonase specificity is key to quorum quenching in pseudomonas aeruginosa
Social Science Research Network, 2019Co-Authors: Benjamin Remy, Mikael Elias, Laure Plener, Philippe Decloquement, Nicholas Armstrong, David Daudé, Eric ChabriereAbstract:Background: Pseudomonas aeruginosa orchestrates the expression of many genes by using quorum sensing (QS) communication process. QS relies on an intricate molecular network including two acyl-homoserine lactones (AHL), involved in the modulation of virulence factors production, biofilm formation or antimicrobial sensitivity. Disrupting QS is a promising approach to modulate virulence while not challenging bacterial survival and can be achieved with AHL-degrading Lactonases. Methods: Here, we used two Lactonases both targeting the signal molecules N-(3-oxododecanoyl)-L-Homoserine lactone (3-oxo-C12 HSL) and butyryl-homoserine lactone (C4 HSL) albeit with different efficacy on C4 HSL. We used phenotypic analyses to characterize the specific impact of each Lactonases on P. aeruginosa virulence. We further used molecular approaches for finely determine the impact of Lactonases on gene regulation and protein expressions of P. aeruginosa. Findings: Although, both Lactonases similarly impacted AHL-based circuits, strong variations were however observed in Pseudomonas Quinolone Signal regulation yielding in different impacts on phenotypic traits related to virulence, biofilm, pathogenicity or antibiotic susceptibility. Proteomic analysis further allowed to identify the specific impacts of Lactonases on P. aeruginosa behavior. Interpretation: This global analysis provides the first evidence that QQ enzyme specificity is crucial to modulate QS-associated behavior in P. aeruginosa PA14 and will help to develop efficient quorum quenching strategies. Funding Statement: This work was supported by a project RAPID (LACTO-TEX) from Direction Generale de l'Armement (DGA, France), "Investissements d'avenir" program (Mediterranee Infection 10-IAHU-03) of the French Agence Nationale de la Recherche (ANR), "Emplois Jeunes Doctorants" program of Region Provence-Alpes-Cote d'Azur (PACA, France) and MnDrive initiative. Declaration of Interests: ME and EC have a patent WO2014167140 A1 licensed to Gene&GreenTK. LP, DD, BR, ME and EC report personal fees from Gene&GreenTK during the conduct of the study. The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Ethics Approval Statement: Not required.
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the quorum quenching Lactonase from alicyclobacter acidoterrestris purification kinetic characterization crystallization and crystallographic analysis
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2017Co-Authors: Celine Bergonzi, Eric Chabriere, Michael Schwab, Mikael EliasAbstract:Lactonases comprise a class of enzymes that hydrolyze lactones, including acyl-homoserine lactones (AHLs); the latter are used as chemical signaling molecules by numerous Gram-negative bacteria. Lactonases have therefore been demonstrated to quench AHL-based bacterial communication. In particular, Lactonases are capable of inhibiting bacterial behaviors that depend on these chemicals, such as the formation of biofilms or the expression of virulence factors. A novel representative from the metallo-β-lactamase superfamily, named AaL, was isolated from the thermoacidophilic bacterium Alicyclobacter acidoterrestris. Kinetic characterization proves AaL to be a proficient Lactonase, with catalytic efficiencies (kcat/Km) against AHLs in the region of 105 M-1 s-1. AaL exhibits a very broad substrate specificity. Its structure is expected to reveal the molecular determinants for its substrate binding and specificity, as well as to provide grounds for future protein-engineering projects. Here, the expression, purification, characterization, crystallization and X-ray diffraction data collection of AaL at 1.65 A resolution are reported.
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Crystal structure of VmoLac, a tentative quorum quenching Lactonase from the extremophilic crenarchaeon Vulcanisaeta moutnovskia
Scientific Reports, 2015Co-Authors: Julien Hiblot, Janek Bzdrenga, Charlotte Champion, Eric Chabriere, Mikael EliasAbstract:A new representative of the Phosphotriesterase-Like Lactonases (PLLs) family from the hyperthermophilic crenarchaeon Vulcanisaeta moutnovskia has been characterized and crystallized. VmoLac is a native, proficient Lactonase with promiscuous, low phosphotriesterase activity. VmoLac therefore represents an interesting candidate for engineering studies, with the aim of developing an efficient bacterial quorum-quenching agent. Here, we provide an extensive biochemical and kinetic characterization of VmoLac and describe the X-ray structures of the enzyme bound to a fatty acid and to its cognate substrate 3-oxo-C10 AHL (Acyl-Homoserine Lactone). The structures highlight possible structural determinants that may be involved in its extreme thermal stability (Tm = 128 degrees C). Moreover, the structure reveals that the substrate binding mode of VmoLac significantly differs from those of its close homologues, possibly explaining the substrate specificity of the enzyme. Finally, we describe the specific interactions between the enzyme and its substrate, and discuss the possible lactone hydrolysis mechanism of VmoLac.
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SacPox from the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius is a proficient Lactonase
BMC research notes, 2014Co-Authors: Janek Bzdrenga, Mikael Elias, Julien Hiblot, Guillaume Gotthard, Charlotte Champion, Eric ChabriereAbstract:Sac Pox, an enzyme from the extremophilic crenarchaeal Sulfolobus acidocaldarius (Sac), was isolated by virtue of its phosphotriesterase (or paraoxonase; Pox) activity, i.e. its ability to hydrolyze the neurotoxic organophosphorus insecticides. Later on, Sac Pox was shown to belong to the Phosphotriesterase-Like Lactonase family that comprises natural Lactonases, possibly involved in quorum sensing, and endowed with promiscuous, phosphotriesterase activity. Here, we present a comprehensive and broad enzymatic characterization of the natural Lactonase and promiscuous organophosphorus hydrolase activities of Sac Pox, as well as a structural analysis using a model. Kinetic experiments show that Sac Pox is a proficient Lactonase, including at room temperature. Moreover, we discuss the observed differences in substrate specificity between Sac Pox and its closest homologues Sso Pox and Sis Lac together with the possible structural causes for these observations.
Walter Fast - One of the best experts on this subject based on the ideXlab platform.
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structural and biochemical characterization of aidc a quorum quenching Lactonase with atypical selectivity
Biochemistry, 2015Co-Authors: Romila Mascarenhas, Pei W Thomas, Dali Liu, Quyen Q Hoang, Boguslaw Nocek, Walter FastAbstract:Quorum-quenching catalysts are of interest for potential application as biochemical tools for interrogating interbacterial communication pathways, as antibiofouling agents, and as anti-infective agents in plants and animals. Herein, the structure and function of AidC, an N-acyl-l-homoserine lactone (AHL) Lactonase from Chryseobacterium, is characterized. Steady-state kinetics show that zinc-supplemented AidC is the most efficient wild-type quorum-quenching enzymes characterized to date, with a kcat/KM value of approximately 2 × 106 M–1 s–1 for N-heptanoyl-l-homoserine lactone. The enzyme has stricter substrate selectivity and significantly lower KM values (ca. 50 μM for preferred substrates) compared to those of typical AHL Lactonases (ca. >1 mM). X-ray crystal structures of AidC alone and with the product N-hexanoyl-l-homoserine were determined at resolutions of 1.09 and 1.67 A, respectively. Each structure displays as a dimer, and dimeric oligiomerization was also observed in solution by size-exclusion ...
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mechanism of the quorum quenching Lactonase aiia from bacillus thuringiensis 1 product bound structures
Biochemistry, 2008Co-Authors: Dali Liu, Pei W Thomas, Walter Fast, Jessica Momb, Gregory A Petsko, Aaron Moulin, Dagmar RingeAbstract:Enzymes capable of hydrolyzing N-acyl-l-homoserine lactones (AHLs) used in some bacterial quorum-sensing pathways are of considerable interest for their ability to block undesirable phenotypes. Most known AHL hydrolases that catalyze ring opening (AHL Lactonases) are members of the metallo-β-lactamase enzyme superfamily and rely on a dinuclear zinc site for catalysis and stability. Here we report the three-dimensional structures of three product complexes formed with the AHL Lactonase from Bacillus thuringiensis. Structures of the Lactonase bound with two different concentrations of the ring-opened product of N-hexanoyl-l-homoserine lactone are determined at 0.95 and 1.4 A resolution and exhibit different product configurations. A structure of the ring-opened product of the non-natural N-hexanoyl-l-homocysteine thiolactone at 1.3 A resolution is also determined. On the basis of these product-bound structures, a substrate-binding model is presented that differs from previous proposals. Additionally, the pr...
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mechanism of the quorum quenching Lactonase aiia from bacillus thuringiensis 2 substrate modeling and active site mutations
Biochemistry, 2008Co-Authors: Jessica Momb, Pei W Thomas, Dali Liu, Gregory A Petsko, Dagmar Ringe, Canhui Wang, Hua Guo, Walter FastAbstract:Proteins in the metallo-β-lactamase superfamily span all three domains of life and are quite diverse, encompassing noncatalytic proteins as well as a diverse set of enzymes (1). At least 17 different activities have been reported within this superfamily, including nitric oxide and oxygen reduction as well as cleavage of C−O, C−N, C−S, S−O, P−O, and possibly P−N bonds (2). Many of these proteins are important for understanding clinically important processes, including anticancer drug detoxification pathways, mRNA processing, and antibacterial resistance mechanisms (2). The metal centers at the active sites of these enzymes are considerably diverse, with examples of mononuclear proteins, mononuclear proteins with the metal ion bound at a different site, dinuclear proteins with both metals bound in the proximity of each other in a cocatalytic site, and two-metal proteins where the second metal-binding site is distant but allosterically linked to the active site (3,4). Both zinc(II) and iron(II) can confer activity to various superfamily members, and some enzymes appear to incorporate (and display activity with) a wide variety of different divalent metal ions found in their host organism’s growth medium (5–7). Even within a single family of enzymes, there is considerable diversity in metal stoichiometry, metal placement, and catalytic mechanism (3). The rich functionality and high diversity of this superfamily, as well as the clinical importance of some members, make it an attractive area for studying the divergence and evolution of enzyme activities. Recognition of a signature sequence motif, HxHxDH∼H∼D∼H, in the quorum-quenching N-acyl-l-homoserine lactone hydrolases (AHL1 Lactonases) led to the adoption of this family of enzymes into the metallo-β-lactamase superfamily (8). Although initial reports suggested that the AHL Lactonases were not metal-dependent, subsequent work on at least two different isoforms has shown that they are in fact dinuclear metalloproteins with two zinc(II) ions bound in the proximity of each other at the active site, and that these metal ions are essential for catalytic activity (9–13). The first reported structure of AHL Lactonase did not have a bound substrate or inhibitor, but a subsequent structure was reported containing l-homoserine lactone, a competitive inhibitor that lacks the N-acyl substitution usually found in substrates (11,12). On the basis of the structure of this complex, a catalytic mechanism was proposed using a substrate binding orientation similar to that of the l-homoserine lactone inhibitor, but the opposite of that observed in enzyme−substrate and enzyme−product complex structures of related metallo-β-lactamase superfamily members (3,12,14,15). This hypothesis sparked us to further investigate the catalytic mechanism of AHL Lactonases. The preceding paper in this series describes structural determination of three product-bound complexes and their implications for both substrate orientation and the ring-opening mechanism (16). Here, we use computational modeling, site-directed mutagenesis, synthesis of alternative substrates, and steady-state kinetics to test the proposed substrate binding model and the importance of several active site residues. In conclusion, a detailed catalytic mechanism is proposed for the AHL Lactonase from Bacillus thuringiensis.
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structure and specificity of a quorum quenching Lactonase aiib from agrobacterium tumefaciens
Biochemistry, 2007Co-Authors: Dali Liu, Pei W Thomas, Jessica Momb, Quyen Q Hoang, Gregory A Petsko, Dagmar Ringe, Walter FastAbstract:N-Acyl-l-homoserine lactone (AHL) mediated quorum-sensing regulates virulence factor production in a variety of Gram-negative bacteria. Proteins capable of degrading these autoinducers have been called “quorum-quenching” enzymes, can block many quorum-sensing dependent phenotypes, and represent potentially useful reagents for clinical, agricultural, and industrial applications. The most characterized quorum-quenching enzymes to date are the AHL Lactonases, which are metalloproteins that belong to the metallo-beta-lactamase superfamily. Here, we report the cloning, heterologous expression, purification, metal content, substrate specificity, and three-dimensional structure of AiiB, an AHL Lactonase from Agrobacterium tumefaciens. Much like a homologous AHL Lactonase from Bacillus thuringiensis, AiiB appears to be a metal-dependent AHL Lactonase with broad specificity. A phosphate dianion is bound to the dinuclear zinc site and the active-site structure suggests specific mechanistic roles for an active site ...
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three dimensional structure of the quorum quenching n acyl homoserine lactone hydrolase from bacillus thuringiensis
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Dali Liu, Pei W Thomas, Everett Stone, Walter Fast, Gregory A Petsko, Bryan W Lepore, Dagmar RingeAbstract:The three-dimensional structure of the N-acyl-l-homoserine lactone hydrolase (AHL Lactonase) from Bacillus thuringiensis has been determined, by using single-wavelength anomalous dispersion (SAD) phasing, to 1.6-A resolution. AHLs are produced by many Gram-negative bacteria as signaling molecules used in quorum-sensing pathways that indirectly sense cell density and regulate communal behavior. Because of their importance in pathogenicity, quorum-sensing pathways have been suggested as potential targets for the development of novel therapeutics. Quorum-sensing can be disrupted by enzymes evolved to degrade these lactones, such as AHL Lactonases. These enzymes are members of the metallo-β-lactamase superfamily and contain two zinc ions in their active sites. The zinc ions are coordinated to a number of ligands, including a single oxygen of a bridging carboxylate and a bridging water/hydroxide ion, thought to be the nucleophile that hydrolyzes the AHLs to ring-opened products, which can no longer act as quorum signals.
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Crystal structure of VmoLac, a tentative quorum quenching Lactonase from the extremophilic crenarchaeon Vulcanisaeta moutnovskia
Scientific Reports, 2015Co-Authors: Julien Hiblot, Janek Bzdrenga, Charlotte Champion, Eric Chabriere, Mikael EliasAbstract:A new representative of the Phosphotriesterase-Like Lactonases (PLLs) family from the hyperthermophilic crenarchaeon Vulcanisaeta moutnovskia has been characterized and crystallized. VmoLac is a native, proficient Lactonase with promiscuous, low phosphotriesterase activity. VmoLac therefore represents an interesting candidate for engineering studies, with the aim of developing an efficient bacterial quorum-quenching agent. Here, we provide an extensive biochemical and kinetic characterization of VmoLac and describe the X-ray structures of the enzyme bound to a fatty acid and to its cognate substrate 3-oxo-C10 AHL (Acyl-Homoserine Lactone). The structures highlight possible structural determinants that may be involved in its extreme thermal stability (Tm = 128 degrees C). Moreover, the structure reveals that the substrate binding mode of VmoLac significantly differs from those of its close homologues, possibly explaining the substrate specificity of the enzyme. Finally, we describe the specific interactions between the enzyme and its substrate, and discuss the possible lactone hydrolysis mechanism of VmoLac.
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Inhaled Lactonase Reduces Pseudomonas aeruginosa Quorum Sensing and Mortality in Rat Pneumonia
PLoS ONE, 2014Co-Authors: Sami Hraiech, Mikael Elias, Julien Hiblot, John Lafleur, Hubert Lepidi, Laurent Papazian, Jean-marc Rolain, Didier Raoult, Mark W. Silby, Janek BzdrengaAbstract:Rationale: The effectiveness of antibiotic molecules in treating Pseudomonas aeruginosa pneumonia is reduced as a result of the dissemination of bacterial resistance. The existence of bacterial communication systems, such as quorum sensing, has provided new opportunities of treatment. Lactonases efficiently quench acyl-homoserine lactone-based bacterial quorum sensing, implicating these enzymes as potential new anti-Pseudomonas drugs that might be evaluated in pneumonia. Objectives: The aim of the present study was to evaluate the ability of a Lactonase called SsoPox-I to reduce the mortality of a rat P. aeruginosa pneumonia. Methods: To assess SsoPox-I-mediated quorum quenching, we first measured the activity of the virulence gene lasB, the synthesis of pyocianin, the proteolytic activity of a bacterial suspension and the formation of biofilm of a PAO1 strain grown in the presence of Lactonase. In an acute lethal model of P. aeruginosa pneumonia in rats, we evaluated the effects of an early or deferred intra-tracheal treatment with SsoPox-I on the mortality, lung bacterial count and lung damage. Measurements and Primary Results: SsoPox-I decreased PAO1 lasB virulence gene activity, pyocianin synthesis, proteolytic activity and biofilm formation. The early use of SsoPox-I reduced the mortality of rats with acute pneumonia from 75% to 20%. Histological lung damage was significantly reduced but the lung bacterial count was not modified by the treatment. A delayed treatment was associated with a non-significant reduction of mortality. Conclusion: These results demonstrate the protective effects of Lactonase SsoPox-I in P. aeruginosa pneumonia and open the way for a future therapeutic use.
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SacPox from the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius is a proficient Lactonase
BMC research notes, 2014Co-Authors: Janek Bzdrenga, Mikael Elias, Julien Hiblot, Guillaume Gotthard, Charlotte Champion, Eric ChabriereAbstract:Sac Pox, an enzyme from the extremophilic crenarchaeal Sulfolobus acidocaldarius (Sac), was isolated by virtue of its phosphotriesterase (or paraoxonase; Pox) activity, i.e. its ability to hydrolyze the neurotoxic organophosphorus insecticides. Later on, Sac Pox was shown to belong to the Phosphotriesterase-Like Lactonase family that comprises natural Lactonases, possibly involved in quorum sensing, and endowed with promiscuous, phosphotriesterase activity. Here, we present a comprehensive and broad enzymatic characterization of the natural Lactonase and promiscuous organophosphorus hydrolase activities of Sac Pox, as well as a structural analysis using a model. Kinetic experiments show that Sac Pox is a proficient Lactonase, including at room temperature. Moreover, we discuss the observed differences in substrate specificity between Sac Pox and its closest homologues Sso Pox and Sis Lac together with the possible structural causes for these observations.
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differential active site loop conformations mediate promiscuous activities in the Lactonase ssopox
PLOS ONE, 2013Co-Authors: Julien Hiblot, Mikael Elias, Guillaume Gotthard, Eric ChabriereAbstract: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.
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structural and enzymatic characterization of the Lactonase sislac from sulfolobus islandicus
PLOS ONE, 2012Co-Authors: Julien Hiblot, Guillaume Gotthard, Eric Chabriere, Mikael EliasAbstract:Background A 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.
