77 research outputs found
The Demeter project. Eight millennia of agrobiodiversity changes in the northwest Mediterranean basin
The development of agricultural societies is closely entangled with that of domestic animals and plants. Local and traditional domestic breeds and varieties are the result of millennia of selection by farmers. DEMETER (2020-2025) is an international project which is aiming to characterize the changes in animal and plant agrobiodiversity (pigs, sheep, goats, and barley) in relation with environmental and socioeconomic factors in the northwestern Mediterranean basin since the beginnings of agriculture. The project is based on a combination of approaches including phenomics (through geometric morphometrics), databasing, zooarchaeology, archaeobotany, climate modeling, paleoproteins (ZooMs) and statistical analyses. Several hundreds of archaeological sites from the South of France and Catalonia will be studied, covering the maximum environmental, societal and cultural diversity of context over the course of the last eight millennia
Using RNA-seq to determine the transcriptional landscape and the hypoxic response of the pathogenic yeast Candida parapsilosis
<p>Abstract</p> <p>Background</p> <p><it>Candida parapsilosis </it>is one of the most common causes of <it>Candida </it>infection worldwide. However, the genome sequence annotation was made without experimental validation and little is known about the transcriptional landscape. The transcriptional response of <it>C. parapsilosis </it>to hypoxic (low oxygen) conditions, such as those encountered in the host, is also relatively unexplored.</p> <p>Results</p> <p>We used next generation sequencing (RNA-seq) to determine the transcriptional profile of <it>C. parapsilosis </it>growing in several conditions including different media, temperatures and oxygen concentrations. We identified 395 novel protein-coding sequences that had not previously been annotated. We removed > 300 unsupported gene models, and corrected approximately 900. We mapped the 5' and 3' UTR for thousands of genes. We also identified 422 introns, including two introns in the 3' UTR of one gene. This is the first report of 3' UTR introns in the Saccharomycotina. Comparing the introns in coding sequences with other species shows that small numbers have been gained and lost throughout evolution. Our analysis also identified a number of novel transcriptional active regions (nTARs). We used both RNA-seq and microarray analysis to determine the transcriptional profile of cells grown in normoxic and hypoxic conditions in rich media, and we showed that there was a high correlation between the approaches. We also generated a knockout of the <it>UPC2 </it>transcriptional regulator, and we found that similar to <it>C. albicans</it>, Upc2 is required for conferring resistance to azole drugs, and for regulation of expression of the ergosterol pathway in hypoxia.</p> <p>Conclusion</p> <p>We provide the first detailed annotation of the <it>C. parapsilosis </it>genome, based on gene predictions and transcriptional analysis. We identified a number of novel ORFs and other transcribed regions, and detected transcripts from approximately 90% of the annotated protein coding genes. We found that the transcription factor Upc2 role has a conserved role as a major regulator of the hypoxic response in <it>C. parapsilosis </it>and <it>C. albicans</it>.</p
Caractérisation structurale d'une méga-enzyme bactérienne participant à la biosynthÚse du carcinogÚne colibactine
Some Escherichia coli strains from the human gut microbiota synthesize colibactin, a causative agent of colorectal cancer and a potential agent involved in other cancers. As is the case for many valuable secondary metabolites produced by micro-organisms, colibactin is synthesized by non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs). NRPSs/PKSs use diverse enzymatic domains that are organized into modules. A NRPS module incorporates an amino acid during metabolite assembly, while a PKS module adds an acyl chain. In addition to enzymatic domains, each module integrates a non-enzymatic domain, named carrier protein, that delivers the substrates and the growing metabolite to the enzymatic domains. In type I NRPSs/PKSs, several modules are commonly fused into a single polypeptide. Even if the structural characterization of these megaenzymes is very challenging due to their size and flexibility, major breakthroughs occurred recently both in the NRPS and the PKS fields. However, no fused hybrid NRPS/PKS, composed of a NRPS module associated to a PKS module, has been structurally characterized to date.The main goal of this thesis was the structural characterization of the hybrid PKS-NRPS ClbK, that adds into colibactin an aminomalonyl unit and a thiazole ring that could impart the genotoxic attributes of colibactin. This work demonstrated that ClbK is a 480 kDa dimer. The purified protein catalyzes the activation of cysteine, the precursor of the thiazole ring. Small angle X-ray scattering (SAXS) revealed that ClbK has an elongated shape with a maximal dimension of 30 nm. Ab initio models generated from SAXS data suggest that ClbK possesses catalytic chambers, a common feature of PKS proteins.In parallel, the structure of the PKS module of ClbK (85 kDa) was solved at 3 Ă
by X-ray crystallography. The structure revealed that the protein is dimeric and harbors several adaptations allowing its interactions with NRPS domains. The carrier protein domain is tethered at an unusual position, observed only once in PKS proteins. Characterization of the oligomeric state of several muti-domain fragments of ClbK suggest that the megaenzyme dimerizes owing to two domains located at its N-terminal and C-terminal extremities. Data obtained during this work associated with other structural data allowed us to propose the first architectural model of a hybrid NRPS/PKS protein.Certaines souches dâEscherichia coli rĂ©sidant dans notre microbiote intestinal produisent la colibactine, un carcinogĂšne dont le rĂŽle a Ă©tĂ© dĂ©montrĂ© dans le cancer colorectal et soupçonnĂ© dans dâautres cancers. La biosynthĂšse de la colibactine, comme celle de nombreux mĂ©tabolites secondaires microbiens dâintĂ©rĂȘt mĂ©dical, repose sur des mĂ©ga-enzymes appartenant Ă la famille des synthĂ©tases de peptides non ribosomaux (NRPS) et des synthases de polycĂ©tides (PKS). Les NRPS/PKS sont constituĂ©es de domaines enzymatiques variĂ©s, regroupĂ©s en modules. Un module NRPS incorpore un acide aminĂ© dans le mĂ©tabolite en construction alors quâun module PKS ajoute une chaĂźne acyle. En plus des domaines enzymatiques, chaque module intĂšgre un domaine non enzymatique nommĂ© « carrier protein », qui prĂ©sente les substrats et le mĂ©tabolite en construction aux autres domaines. Chez les NRPS/PKS de type I, plusieurs modules sont fusionnĂ©s au sein dâun mĂȘme polypeptide. La taille et la flexibilitĂ© de ces protĂ©ines les rend trĂšs difficiles Ă caractĂ©riser, mĂȘme si des avancĂ©es majeures ont eu lieu rĂ©cemment, Ă la fois pour les NRPS et les PKS. En revanche, aucune protĂ©ine hybride NRPS/PKS fusionnĂ©e, composĂ©e dâun module NRPS associĂ© Ă un module PKS, nâa Ă©tĂ© caractĂ©risĂ©e structuralement.Le but de cette thĂšse Ă©tait la caractĂ©risation structurale de lâhybride PKS-NRPS ClbK, responsable de lâincorporation dans la colibactine dâune unitĂ© aminomalonyle et dâun cycle thiazole potentiellement gĂ©notoxique. Ce travail a montrĂ© que ClbK forme un assemblage dimĂ©rique de 480 kDa. La protĂ©ine ClbK purifiĂ©e est capable dâactiver la cystĂ©ine, le prĂ©curseur du cycle thiazole. La diffusion des rayons X aux petits angles a rĂ©vĂ©lĂ© que ClbK possĂšde une forme allongĂ©e, de dimension maximale 30 nm et les modĂšles ab initio gĂ©nĂ©rĂ©s suggĂšrent la prĂ©sence de plusieurs chambres catalytiques, dĂ©jĂ identifiĂ©es chez les PKS.ParallĂšlement, la structure du module PKS de ClbK (85 kDa) a Ă©tĂ© rĂ©solue Ă 3 Ă
par cristallographie aux rayons X. Ce fragment forme un assemblage dimĂ©rique et possĂšde plusieurs adaptations facilitant ses interactions avec les domaines NRPS en amont et en aval. Le domaine « carrier protein » est dĂ©tectĂ© Ă une position inhabituelle, observĂ©e une seule fois chez les PKS. La caractĂ©risation de lâĂ©tat oligomĂ©rique en solution de plusieurs fragments multi-domaines de ClbK suggĂšre que ClbK dimĂ©rise grĂące Ă deux domaines enzymatiques situĂ©s Ă ses extrĂ©mitĂ©s N- et C-terminales. Lâensemble des donnĂ©es collectĂ©es au cours de cette thĂšse et les donnĂ©es structurales disponibles nous ont permis de proposer le premier modĂšle architectural dâune protĂ©ine hybride NRPS/PKS
Structural characterization of a bacterial megaenzyme involved in the biosynthesis of the colibactin carcinogen
Certaines souches dâEscherichia coli rĂ©sidant dans notre microbiote intestinal produisent la colibactine, un carcinogĂšne dont le rĂŽle a Ă©tĂ© dĂ©montrĂ© dans le cancer colorectal et soupçonnĂ© dans dâautres cancers. La biosynthĂšse de la colibactine, comme celle de nombreux mĂ©tabolites secondaires microbiens dâintĂ©rĂȘt mĂ©dical, repose sur des mĂ©ga-enzymes appartenant Ă la famille des synthĂ©tases de peptides non ribosomaux (NRPS) et des synthases de polycĂ©tides (PKS). Les NRPS/PKS sont constituĂ©es de domaines enzymatiques variĂ©s, regroupĂ©s en modules. Un module NRPS incorpore un acide aminĂ© dans le mĂ©tabolite en construction alors quâun module PKS ajoute une chaĂźne acyle. En plus des domaines enzymatiques, chaque module intĂšgre un domaine non enzymatique nommĂ© « carrier protein », qui prĂ©sente les substrats et le mĂ©tabolite en construction aux autres domaines. Chez les NRPS/PKS de type I, plusieurs modules sont fusionnĂ©s au sein dâun mĂȘme polypeptide. La taille et la flexibilitĂ© de ces protĂ©ines les rend trĂšs difficiles Ă caractĂ©riser, mĂȘme si des avancĂ©es majeures ont eu lieu rĂ©cemment, Ă la fois pour les NRPS et les PKS. En revanche, aucune protĂ©ine hybride NRPS/PKS fusionnĂ©e, composĂ©e dâun module NRPS associĂ© Ă un module PKS, nâa Ă©tĂ© caractĂ©risĂ©e structuralement.Le but de cette thĂšse Ă©tait la caractĂ©risation structurale de lâhybride PKS-NRPS ClbK, responsable de lâincorporation dans la colibactine dâune unitĂ© aminomalonyle et dâun cycle thiazole potentiellement gĂ©notoxique. Ce travail a montrĂ© que ClbK forme un assemblage dimĂ©rique de 480 kDa. La protĂ©ine ClbK purifiĂ©e est capable dâactiver la cystĂ©ine, le prĂ©curseur du cycle thiazole. La diffusion des rayons X aux petits angles a rĂ©vĂ©lĂ© que ClbK possĂšde une forme allongĂ©e, de dimension maximale 30 nm et les modĂšles ab initio gĂ©nĂ©rĂ©s suggĂšrent la prĂ©sence de plusieurs chambres catalytiques, dĂ©jĂ identifiĂ©es chez les PKS.ParallĂšlement, la structure du module PKS de ClbK (85 kDa) a Ă©tĂ© rĂ©solue Ă 3 Ă
par cristallographie aux rayons X. Ce fragment forme un assemblage dimĂ©rique et possĂšde plusieurs adaptations facilitant ses interactions avec les domaines NRPS en amont et en aval. Le domaine « carrier protein » est dĂ©tectĂ© Ă une position inhabituelle, observĂ©e une seule fois chez les PKS. La caractĂ©risation de lâĂ©tat oligomĂ©rique en solution de plusieurs fragments multi-domaines de ClbK suggĂšre que ClbK dimĂ©rise grĂące Ă deux domaines enzymatiques situĂ©s Ă ses extrĂ©mitĂ©s N- et C-terminales. Lâensemble des donnĂ©es collectĂ©es au cours de cette thĂšse et les donnĂ©es structurales disponibles nous ont permis de proposer le premier modĂšle architectural dâune protĂ©ine hybride NRPS/PKS.Some Escherichia coli strains from the human gut microbiota synthesize colibactin, a causative agent of colorectal cancer and a potential agent involved in other cancers. As is the case for many valuable secondary metabolites produced by micro-organisms, colibactin is synthesized by non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs). NRPSs/PKSs use diverse enzymatic domains that are organized into modules. A NRPS module incorporates an amino acid during metabolite assembly, while a PKS module adds an acyl chain. In addition to enzymatic domains, each module integrates a non-enzymatic domain, named carrier protein, that delivers the substrates and the growing metabolite to the enzymatic domains. In type I NRPSs/PKSs, several modules are commonly fused into a single polypeptide. Even if the structural characterization of these megaenzymes is very challenging due to their size and flexibility, major breakthroughs occurred recently both in the NRPS and the PKS fields. However, no fused hybrid NRPS/PKS, composed of a NRPS module associated to a PKS module, has been structurally characterized to date.The main goal of this thesis was the structural characterization of the hybrid PKS-NRPS ClbK, that adds into colibactin an aminomalonyl unit and a thiazole ring that could impart the genotoxic attributes of colibactin. This work demonstrated that ClbK is a 480 kDa dimer. The purified protein catalyzes the activation of cysteine, the precursor of the thiazole ring. Small angle X-ray scattering (SAXS) revealed that ClbK has an elongated shape with a maximal dimension of 30 nm. Ab initio models generated from SAXS data suggest that ClbK possesses catalytic chambers, a common feature of PKS proteins.In parallel, the structure of the PKS module of ClbK (85 kDa) was solved at 3 Ă
by X-ray crystallography. The structure revealed that the protein is dimeric and harbors several adaptations allowing its interactions with NRPS domains. The carrier protein domain is tethered at an unusual position, observed only once in PKS proteins. Characterization of the oligomeric state of several muti-domain fragments of ClbK suggest that the megaenzyme dimerizes owing to two domains located at its N-terminal and C-terminal extremities. Data obtained during this work associated with other structural data allowed us to propose the first architectural model of a hybrid NRPS/PKS protein
Caractérisation structurale d'une méga-enzyme bactérienne participant à la biosynthÚse du carcinogÚne colibactine
Some Escherichia coli strains from the human gut microbiota synthesize colibactin, a causative agent of colorectal cancer and a potential agent involved in other cancers. As is the case for many valuable secondary metabolites produced by micro-organisms, colibactin is synthesized by non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs). NRPSs/PKSs use diverse enzymatic domains that are organized into modules. A NRPS module incorporates an amino acid during metabolite assembly, while a PKS module adds an acyl chain. In addition to enzymatic domains, each module integrates a non-enzymatic domain, named carrier protein, that delivers the substrates and the growing metabolite to the enzymatic domains. In type I NRPSs/PKSs, several modules are commonly fused into a single polypeptide. Even if the structural characterization of these megaenzymes is very challenging due to their size and flexibility, major breakthroughs occurred recently both in the NRPS and the PKS fields. However, no fused hybrid NRPS/PKS, composed of a NRPS module associated to a PKS module, has been structurally characterized to date.The main goal of this thesis was the structural characterization of the hybrid PKS-NRPS ClbK, that adds into colibactin an aminomalonyl unit and a thiazole ring that could impart the genotoxic attributes of colibactin. This work demonstrated that ClbK is a 480 kDa dimer. The purified protein catalyzes the activation of cysteine, the precursor of the thiazole ring. Small angle X-ray scattering (SAXS) revealed that ClbK has an elongated shape with a maximal dimension of 30 nm. Ab initio models generated from SAXS data suggest that ClbK possesses catalytic chambers, a common feature of PKS proteins.In parallel, the structure of the PKS module of ClbK (85 kDa) was solved at 3 Ă
by X-ray crystallography. The structure revealed that the protein is dimeric and harbors several adaptations allowing its interactions with NRPS domains. The carrier protein domain is tethered at an unusual position, observed only once in PKS proteins. Characterization of the oligomeric state of several muti-domain fragments of ClbK suggest that the megaenzyme dimerizes owing to two domains located at its N-terminal and C-terminal extremities. Data obtained during this work associated with other structural data allowed us to propose the first architectural model of a hybrid NRPS/PKS protein.Certaines souches dâEscherichia coli rĂ©sidant dans notre microbiote intestinal produisent la colibactine, un carcinogĂšne dont le rĂŽle a Ă©tĂ© dĂ©montrĂ© dans le cancer colorectal et soupçonnĂ© dans dâautres cancers. La biosynthĂšse de la colibactine, comme celle de nombreux mĂ©tabolites secondaires microbiens dâintĂ©rĂȘt mĂ©dical, repose sur des mĂ©ga-enzymes appartenant Ă la famille des synthĂ©tases de peptides non ribosomaux (NRPS) et des synthases de polycĂ©tides (PKS). Les NRPS/PKS sont constituĂ©es de domaines enzymatiques variĂ©s, regroupĂ©s en modules. Un module NRPS incorpore un acide aminĂ© dans le mĂ©tabolite en construction alors quâun module PKS ajoute une chaĂźne acyle. En plus des domaines enzymatiques, chaque module intĂšgre un domaine non enzymatique nommĂ© « carrier protein », qui prĂ©sente les substrats et le mĂ©tabolite en construction aux autres domaines. Chez les NRPS/PKS de type I, plusieurs modules sont fusionnĂ©s au sein dâun mĂȘme polypeptide. La taille et la flexibilitĂ© de ces protĂ©ines les rend trĂšs difficiles Ă caractĂ©riser, mĂȘme si des avancĂ©es majeures ont eu lieu rĂ©cemment, Ă la fois pour les NRPS et les PKS. En revanche, aucune protĂ©ine hybride NRPS/PKS fusionnĂ©e, composĂ©e dâun module NRPS associĂ© Ă un module PKS, nâa Ă©tĂ© caractĂ©risĂ©e structuralement.Le but de cette thĂšse Ă©tait la caractĂ©risation structurale de lâhybride PKS-NRPS ClbK, responsable de lâincorporation dans la colibactine dâune unitĂ© aminomalonyle et dâun cycle thiazole potentiellement gĂ©notoxique. Ce travail a montrĂ© que ClbK forme un assemblage dimĂ©rique de 480 kDa. La protĂ©ine ClbK purifiĂ©e est capable dâactiver la cystĂ©ine, le prĂ©curseur du cycle thiazole. La diffusion des rayons X aux petits angles a rĂ©vĂ©lĂ© que ClbK possĂšde une forme allongĂ©e, de dimension maximale 30 nm et les modĂšles ab initio gĂ©nĂ©rĂ©s suggĂšrent la prĂ©sence de plusieurs chambres catalytiques, dĂ©jĂ identifiĂ©es chez les PKS.ParallĂšlement, la structure du module PKS de ClbK (85 kDa) a Ă©tĂ© rĂ©solue Ă 3 Ă
par cristallographie aux rayons X. Ce fragment forme un assemblage dimĂ©rique et possĂšde plusieurs adaptations facilitant ses interactions avec les domaines NRPS en amont et en aval. Le domaine « carrier protein » est dĂ©tectĂ© Ă une position inhabituelle, observĂ©e une seule fois chez les PKS. La caractĂ©risation de lâĂ©tat oligomĂ©rique en solution de plusieurs fragments multi-domaines de ClbK suggĂšre que ClbK dimĂ©rise grĂące Ă deux domaines enzymatiques situĂ©s Ă ses extrĂ©mitĂ©s N- et C-terminales. Lâensemble des donnĂ©es collectĂ©es au cours de cette thĂšse et les donnĂ©es structurales disponibles nous ont permis de proposer le premier modĂšle architectural dâune protĂ©ine hybride NRPS/PKS
Caractérisation structurale d'une méga-enzyme bactérienne participant à la biosynthÚse du carcinogÚne colibactine
Some Escherichia coli strains from the human gut microbiota synthesize colibactin, a causative agent of colorectal cancer and a potential agent involved in other cancers. As is the case for many valuable secondary metabolites produced by micro-organisms, colibactin is synthesized by non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs). NRPSs/PKSs use diverse enzymatic domains that are organized into modules. A NRPS module incorporates an amino acid during metabolite assembly, while a PKS module adds an acyl chain. In addition to enzymatic domains, each module integrates a non-enzymatic domain, named carrier protein, that delivers the substrates and the growing metabolite to the enzymatic domains. In type I NRPSs/PKSs, several modules are commonly fused into a single polypeptide. Even if the structural characterization of these megaenzymes is very challenging due to their size and flexibility, major breakthroughs occurred recently both in the NRPS and the PKS fields. However, no fused hybrid NRPS/PKS, composed of a NRPS module associated to a PKS module, has been structurally characterized to date.The main goal of this thesis was the structural characterization of the hybrid PKS-NRPS ClbK, that adds into colibactin an aminomalonyl unit and a thiazole ring that could impart the genotoxic attributes of colibactin. This work demonstrated that ClbK is a 480 kDa dimer. The purified protein catalyzes the activation of cysteine, the precursor of the thiazole ring. Small angle X-ray scattering (SAXS) revealed that ClbK has an elongated shape with a maximal dimension of 30 nm. Ab initio models generated from SAXS data suggest that ClbK possesses catalytic chambers, a common feature of PKS proteins.In parallel, the structure of the PKS module of ClbK (85 kDa) was solved at 3 Ă
by X-ray crystallography. The structure revealed that the protein is dimeric and harbors several adaptations allowing its interactions with NRPS domains. The carrier protein domain is tethered at an unusual position, observed only once in PKS proteins. Characterization of the oligomeric state of several muti-domain fragments of ClbK suggest that the megaenzyme dimerizes owing to two domains located at its N-terminal and C-terminal extremities. Data obtained during this work associated with other structural data allowed us to propose the first architectural model of a hybrid NRPS/PKS protein.Certaines souches dâEscherichia coli rĂ©sidant dans notre microbiote intestinal produisent la colibactine, un carcinogĂšne dont le rĂŽle a Ă©tĂ© dĂ©montrĂ© dans le cancer colorectal et soupçonnĂ© dans dâautres cancers. La biosynthĂšse de la colibactine, comme celle de nombreux mĂ©tabolites secondaires microbiens dâintĂ©rĂȘt mĂ©dical, repose sur des mĂ©ga-enzymes appartenant Ă la famille des synthĂ©tases de peptides non ribosomaux (NRPS) et des synthases de polycĂ©tides (PKS). Les NRPS/PKS sont constituĂ©es de domaines enzymatiques variĂ©s, regroupĂ©s en modules. Un module NRPS incorpore un acide aminĂ© dans le mĂ©tabolite en construction alors quâun module PKS ajoute une chaĂźne acyle. En plus des domaines enzymatiques, chaque module intĂšgre un domaine non enzymatique nommĂ© « carrier protein », qui prĂ©sente les substrats et le mĂ©tabolite en construction aux autres domaines. Chez les NRPS/PKS de type I, plusieurs modules sont fusionnĂ©s au sein dâun mĂȘme polypeptide. La taille et la flexibilitĂ© de ces protĂ©ines les rend trĂšs difficiles Ă caractĂ©riser, mĂȘme si des avancĂ©es majeures ont eu lieu rĂ©cemment, Ă la fois pour les NRPS et les PKS. En revanche, aucune protĂ©ine hybride NRPS/PKS fusionnĂ©e, composĂ©e dâun module NRPS associĂ© Ă un module PKS, nâa Ă©tĂ© caractĂ©risĂ©e structuralement.Le but de cette thĂšse Ă©tait la caractĂ©risation structurale de lâhybride PKS-NRPS ClbK, responsable de lâincorporation dans la colibactine dâune unitĂ© aminomalonyle et dâun cycle thiazole potentiellement gĂ©notoxique. Ce travail a montrĂ© que ClbK forme un assemblage dimĂ©rique de 480 kDa. La protĂ©ine ClbK purifiĂ©e est capable dâactiver la cystĂ©ine, le prĂ©curseur du cycle thiazole. La diffusion des rayons X aux petits angles a rĂ©vĂ©lĂ© que ClbK possĂšde une forme allongĂ©e, de dimension maximale 30 nm et les modĂšles ab initio gĂ©nĂ©rĂ©s suggĂšrent la prĂ©sence de plusieurs chambres catalytiques, dĂ©jĂ identifiĂ©es chez les PKS.ParallĂšlement, la structure du module PKS de ClbK (85 kDa) a Ă©tĂ© rĂ©solue Ă 3 Ă
par cristallographie aux rayons X. Ce fragment forme un assemblage dimĂ©rique et possĂšde plusieurs adaptations facilitant ses interactions avec les domaines NRPS en amont et en aval. Le domaine « carrier protein » est dĂ©tectĂ© Ă une position inhabituelle, observĂ©e une seule fois chez les PKS. La caractĂ©risation de lâĂ©tat oligomĂ©rique en solution de plusieurs fragments multi-domaines de ClbK suggĂšre que ClbK dimĂ©rise grĂące Ă deux domaines enzymatiques situĂ©s Ă ses extrĂ©mitĂ©s N- et C-terminales. Lâensemble des donnĂ©es collectĂ©es au cours de cette thĂšse et les donnĂ©es structurales disponibles nous ont permis de proposer le premier modĂšle architectural dâune protĂ©ine hybride NRPS/PKS
Sclérodermie systémique et facteurs environnementaux
PARIS5-BU MĂ©d.Cochin (751142101) / SudocPARIS-BIUM (751062103) / SudocCentre Technique Livre Ens. Sup. (774682301) / SudocSudocFranceF
The inherent flexibility of type I non-ribosomal peptide synthetase multienzymes drives their catalytic activities
International audienceNon-ribosomal peptide synthetases (NRPSs) are multienzymes that produce complex natural metabolites with many applications in medicine and agriculture. They are composed of numerous catalytic domains that elongate and chemically modify amino acid substrates or derivatives and of non-catalytic carrier protein domains that can tether and shuttle the growing products to the different catalytic domains. The intrinsic flexibility of NRPSs permits conformational rearrangements that are required to allow interactions between catalytic and carrier protein domains. Their large size coupled to this flexibility renders these multi-domain proteins very challenging for structural characterization. Here, we summarize recent studies that offer structural views of multi-domain NRPSs in various catalytically relevant conformations, thus providing an increased comprehension of their catalytic cycle. A better structural understanding of these multienzymes provides novel perspectives for their re-engineering to synthesize new bioactive metabolites
Experimental waterlogging of grape seeds, impact on seed shape and geometrical reversing for morphometric inference
International audienceWhile the impact of charring on grape seed morphology is well documented, waterlogged pips have up to now been considered in morphometric studies to undergo very limited changes and to be directly comparable to reference collections of fresh seeds. In this study we investigate the impact of waterlogging using different chemicals (H2O2, HNO3, NaOH, KOH) on pips of various cultivars and wild grapevines. Pips are soaked individually in a given chemical solution for increasing periods of time, photographed at each stage, and their dorsal and lateral outlines analysed using Elliptic Fourier Transforms to quantify shape changes. We observe that the different products have comparable effects. The seeds start to swell and then return to their original shapes. Deformation is limited and primarily affects the beak which is slightly blunt and somewhat more pointed than on the original pips. These changes are reminiscent of the deformations observed in waterlogged archaeological seeds. Our results show that they are not strong enough to affect significantly the identification of wild and domesticated morphotypes by seed outline analyses. The effect is greater on the identifications at cultivars level but the accuracy can still be considered acceptable. We experiment the application of an average reverse vector of shape changes to compensate for some of the deformation of artificially waterlogged pips. This correction appears to have a positive effect on the accuracy of identifications for some cultivars. Our conclusion is that outline analysis can be confidently performed on archaeological waterlogged grape pips to identify wild or domesticated status and that it is more appropriate to aim at identifying groups of varieties than individual cultivars. Considering that deformations due to charring and waterlogging have differentiated effects on the identifications it is recommended to take into account both waterlogged and charred seed assemblages
Architecture of a PKS-NRPS hybrid megaenzyme involved in the biosynthesis of the genotoxin colibactin
International audienceThe genotoxin colibactin produced by Escherichia coli is involved in the development of colorectal cancers. This secondary metabolite is synthesized by a multi-protein machinery, mainly composed of non-ribosomal peptide synthetase (NRPS)/polyketide synthase (PKS) enzymes. In order to decipher the function of a PKS-NRPS hybrid enzyme implicated in a key step of colibactin biosynthesis, we conducted an extensive structural characterization of the ClbK megaenzyme. Here we present the crystal structure of the complete trans-AT PKS module of ClbK showing structural specificities of hybrid enzymes. In addition, we report the SAXS solution structure of the full-length ClbK hybrid that reveals a dimeric organization as well as several catalytic chambers. These results provide a structural framework for the transfer of a colibactin precursor through a PKS-NRPS hybrid enzyme and can pave the way for re-engineering PKS-NRPS hybrid megaenzymes to generate diverse metabolites with many applications
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