Guillaume Gotthard

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 SsoPoxs active site is sub-optimal for phosphotriester binding, which depends not only upon shape but also on localised charge of the ligand.

Structural and Enzymatic characterization of the lactonase SisLac from Sulfolobus islandicus

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. Methodology/Principal Findings Here, 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/Significance We 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.

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.

Structural and Enzymatic Characterization of the Phosphotriesterase OPHC2 from Pseudomonas pseudoalcaligenes.

Background Organophosphates (OPs) are neurotoxic compounds for which current methods of elimination are unsatisfactory; thus bio-remediation is considered as a promising alternative. Here we provide the structural and enzymatic characterization of the recently identified enzyme isolated from Pseudomonas pseudoalcaligenes dubbed OPHC2. OPHC2 belongs to the metallo-β-lactamase superfamily and exhibits an unusual thermal resistance and some OP degrading abilities. Principal findings The X-ray structure of OPHC2 has been solved at 2.1 Å resolution. The enzyme is roughly globular exhibiting a αβ/βα topology typical of the metallo-β-lactamase superfamily. Several structural determinants, such as an extended dimerization surface and an intramolecular disulfide bridge, common features in thermostable enzymes, are consistent with its high Tm (97.8°C). Additionally, we provide the enzymatic characterization of OPHC2 against a wide range of OPs, esters and lactones. Significance OPHC2 possesses a broad substrate activity spectrum, since it hydrolyzes various phosphotriesters, esters, and a lactone. Because of its organophosphorus hydrolase activity, and given its intrinsic thermostability, OPHC2 is an interesting candidate for the development of an OPs bio-decontaminant. Its X-ray structure shed light on its active site, and provides key information for the understanding of the substrate binding mode and catalysis.

SacPox from the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius is a proficient lactonase

BackgroundSac 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.ResultsHere, 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.ConclusionKinetic 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.

Human-Phosphate-Binding-Protein inhibits HIV-1 gene transcription and replication

The Human Phosphate-Binding protein (HPBP) is a serendipitously discovered lipoprotein that binds phosphate with high affinity. HPBP belongs to the DING protein family, involved in various biological processes like cell cycle regulation. We report that HPBP inhibits HIV-1 gene transcription and replication in T cell line, primary peripherical blood lymphocytes and primary macrophages. We show that HPBP is efficient in naïve and HIV-1 AZT-resistant strains. Our results revealed HPBP as a new and potent anti HIV molecule that inhibits transcription of the virus, which has not yet been targeted by HAART and therefore opens new strategies in the treatment of HIV infection.

Crystallization and preliminary X-ray diffraction analysis of the lactonase VmoLac from Vulcanisaeta 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.

Crystallization and preliminary X-ray diffraction analysis of the organophosphorus hydrolase OPHC2 from Pseudomonas pseudoalcaligenes.

Enzymes that are capable of degrading neurotoxic organophosphorus compounds are of increasing interest because of the lack of efficient and clean methods for their removal. Recently, a novel organophosphorus hydrolase belonging to the metallo-β-lactamase superfamily was identified and isolated from the mesophilic bacterium Pseudomonas pseudoalcaligenes. This enzyme, named OPHC2, is endowed with significant thermal and pH stability, making it an appealing candidate for engineering studies to develop an efficient organophosphorus biodecontaminant. Combined with biochemical studies, structural information will help decipher the catalytic mechanism of organophosphorus hydrolysis by OPHC2 and identify the residues involved in its substrate specificity. Here, the expression, purification, crystallization and X-ray data collection at 2.1 Å resolution of OPHC2 are presented.

Ancestral mutations as a tool for solubilizing proteins: The case of a hydrophobic phosphate-binding protein.

Stable and soluble proteins are ideal candidates for functional and structural studies. Unfortunately, some proteins or enzymes can be difficult to isolate, being sometimes poorly expressed in heterologous systems, insoluble and/or unstable. Numerous methods have been developed to address these issues, from the screening of various expression systems to the modification of the target protein itself. Here we use a hydrophobic, aggregation‐prone, phosphate‐binding protein (HPBP) as a case study. We describe a simple and fast method that selectively uses ancestral mutations to generate a soluble, stable and functional variant of the target protein, here named sHPBP. This variant is highly expressed in Escherichia coli, is easily purified and its structure was solved at much higher resolution than its wild‐type progenitor (1.3 versus 1.9 Å, respectively).

The Level of DING Proteins Is Increased in HIV-Infected Patients: In Vitro and In Vivo Studies

DING proteins constitute an interesting family, owing to their intriguing and important activities. However, after a decade of research, little is known about these proteins. In humans, at least five different DING proteins have been identified, which were implicated in important biological processes and diseases, including HIV. Indeed, recent data from different research groups have highlighted the anti-HIV activity of some DING representatives. These proteins share the ability to inhibit the transcriptional step of HIV-1, a key step of the viral cycle that is not yet targeted by the current therapies. Since such proteins have been isolated from humans, we undertook a comprehensive study that focuses on the relationship between these proteins and HIV-infection in an infectious context. Hence, we developed a home-made ELISA for the quantification of the concentration of DING proteins in human serum. Using this method, we were able to determine the concentration of DING proteins in healthy and HIV-infected patients. Interestingly, we observed a significant increase of the concentration of DING proteins in non treated and treated HIV-infected patients compared to controls. In addition, cell cultures infected with HIV also show an increased expression of DING proteins, ruling out the possible role of antiretroviral treatment in the increase of the expression of DING proteins. In conclusion, results from this study show that the organism reacts to HIV-infection by an overexpression of DING proteins.