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Mise en place d’un workflow d’identification de microorganismes et de leurs métabolites secondaires d’origine non ribosomique par spectrométrie de masse / Establishment of a workflow for the identification of microorganisms and their secondary metabolites of non-ribosomal origin by mass spectrometryChevalier, Mickaël 18 December 2018 (has links)
Nous avons choisi de réaliser un Workflow permettant l’incrimination, la discrimination et l’identification de peptides non ribosomiques (NRPs) connus ou inconnus dans une philosophie tournée vers le criblage de nouveaux composés actifs. Cette combinaison de méthodes analytiques commence par l’identification du microorganisme par une méthode de profilage phénotypique par spectrométrie de masse (MS). Puis, nous avons choisi de combiner pour la première fois une méthode de calcul itérative et les informations contenues dans la base de données, Norine, dédiée aux NRPs. Cette combinaison nous a permis de déterminer la composition élémentaire de peptide non ribosomique à partir de données HRMS combinées à un maillage vectoriel reliant les différents NRPs de Norine et les formules chimiques. Nous illustrons égalementque d’une part cette méthode démontrée à partir des données MS peut-être extrapolée aux données de fragmentation MS/MS et que d’autre part elle présente un intérêt pour la déréplication des NRPs mais aussi la caractérisation structurale de nouveaux composés actifs pour des applications en particulier dans les secteurs de la santé et phytosanitaires. La performance du workflow sera illustrée par l’identification de lipopeptides produit par des souches de Pseudomonas. Ces lipopeptides sont particulièrement intéressants car se sont des composés ayant des applications potentielles en biocontrôle. / We have chosen to carry out a Workflow allowing the incrimination, the discrimination and the identification of known or unknown nonribosomal peptides (NRPs) in a philosophy oriented towards the screening of new active compounds. This combination of analytical methods begins with the identification of the microorganism by a phenotypic profiling method by mass spectrometry (MS). Then, we chose to combine for the first time an iterative calculation method and the information contained in the database, Norine, dedicated to NRPs. This combination allowed us to determine the nonribosomal peptide elemental composition from HRMS data combined with a vector mesh linking the different Norine NRPs and the chemical formulas. We also illustrate that on the one hand this method demonstrated from the MS data can be extrapolated to the MS / MS fragmentation data and that on the other hand it is of interest for the dereplication of NRPs but also the structural characterization of new compounds for applications especially in the health and plant health sectors. The workflow performance will be illustrated by the identification of lipopeptides produced by Pseudomonas strains. These lipopeptides are particularly interesting because they are compounds with potential applications in biocontrol.
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Biosynthesis and Incorporation of Nonproteinogenic Amino Acids into Non-ribosomal Peptide Natural ProductsWidboom, Paul Fredrick January 2008 (has links)
Thesis advisor: Steven D. Bruner / Complex and unique enzymology is often behind the biosynthesis of natural products. This thesis is focused on how non-proteinogenic amino acids are biosynthesized and then incorporated into natural products. Chapters two, three and four deal with a unique dioxygenase found in vancomycin biosynthesis. Chapter five elaborates on the biochemical characterization along with efforts toward structural characterization of a terminal non-ribosomal peptide synthetase module. The vancomycin biosynthetic enzyme DpgC belongs to a small class of oxygenation enzymes that are not dependent on an accessory cofactor or metal ion. The detailed mechanism of cofactor-independent oxygenases has not been established. We have solved the first structure of an enzyme of this oxygenase class complexed with a bound substrate mimic. The use of a designed, synthetic substrate analog allows unique insights into the chemistry of oxygen activation. The structure confirms the absence of cofactors, and electron density consistent with molecular oxygen is present adjacent to the site of oxidation on the substrate. Molecular oxygen is bound in a small hydrophobic pocket and the substrate provides the reducing power to activate oxygen for downstream chemical steps. Our results resolve the unique and complex chemistry of DpgC, a key enzyme in the biosynthetic pathway of an important class of antibiotics. Mechanistic parallels exist between DpgC and cofactor-dependent flavoenzymes, providing information regarding the general mechanism of enzymatic oxygen activation. / Thesis (PhD) — Boston College, 2008. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.
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Studies on Redesign and Solution Structure Determination of Nonribosomal Peptide Synthetases and Redox Regulation of PhosphatasesChen, Cheng-Yu January 2013 (has links)
<p>We present a computational structure-based redesign of the phenylalanine adenylation domain of the non-ribosomal peptide synthetase (NRPS) enzyme gramicidin S synthetase A (GrsA-PheA) for a set of non-cognate substrates for which the wild-type enzyme has little or virtually no specificity. Experimental validation of a set of top-ranked computationally-predicted enzyme mutants shows significant improvement in the specificity for the target substrates. We further present enhancements to the methodology for computational enzyme redesign that are experimentally shown to result in significant additional improvements in the target substrate specificity. The mutant with the highest activity for a non-cognate substrate exhibits 1/6 of the wild-type enzyme/wild-type substrate activity, further confirming the feasibility of our computational approach. Our results suggest that structure-based protein design can identify active mutants different from those selected by evolution.</p><p>Knowledge about the structures of individual domains and domain interactions can further our redesign of the NRPS enzymes for new bioactive nature product. So far, little structure information has been available for the auxiliary domains such as the epimerization domains and how they interact with the NRPS modules. Solution structure studies by nuclear magnetic resonance (NMR) provide advantages for understanding the dynamics of the domains and reveal active conformations that sometimes are not represented by the crystal structures. However, the large size of the NRPS proteins present challenges for structure studies in solution. In chapter 3, we study the solution structure of the 56 kDa epimerization domain of GrsA (GrsA-PheE) by NMR. We use multidimensional backbone resonance experiments as well as specific labeling strategy to assign the backbone resonances of GrsA-PheE. Secondary structures are determined by sets of residual dipolar couplings (RDCs) measured in multiple alignment media. To determine the global fold of the protein, we obtain long-range distance restraints by measuring the paramagnetic relaxation enhancements (PREs) from 15 site-directed spin labeling samples. </p><p>In chapter 4, we investigate the redox regulation of phosphatases. The activity levels of protein tyrosine phosphatases (PTPs) in cells are highly regulated in various ways including by phosphorylation, localization and protein-protein interaction. Additionally, redox-dependent modification has emerged as a critical part in attenuating PTPs activity in response to cellular stimuli. The tandem Src homology 2 domain-containing PTPs (SHPs) belong to the family of nonreceptor PTPs. The activity level of SHPs is highly regulated by interaction of SH2 domain, phosphorylation level of C-terminal tail and by reversible oxidation. In vivo evidence has shown the reversible oxidation of catalytic cysteine inhibits SHPs activity transiently as a result, affecting the phosphorylation level of its target proteins. In this chapter, we investigate in vitro the reversible oxidation of full-length and catalytic domain of SHP-1 and SHP-2 by using kinetic measurements and mass spectrometry. We have confirmed the susceptibility of the active site cysteines of SHPs to oxidative inactivation, with rate constants for oxidation similar to other PTPs (2-10 M-1s-1). Both SHP-1 and SHP-2 can be reduced and reactivated with the reductants DTT and gluthathione, whereas only the catalytic domain of SHP-2 is subject to reactivation by thioredoxin. Unlike PTPs whose oxidation contains a catalytic cysteine disulfide bonding to a backdoor cysteine or forms a sulfenylamide bonding to nearby backbone nitrogen, we have found that in the reversibly oxidized SHPs, the catalytic cysteines is re-reduced while two conserved backdoor cysteines form a disulfide linkage. Knocking out either of the backdoor cysteine preserves the reversibility of the oxidized SHPs with a disulfide formation between the catalytic cysteine and the remaining backdoor cysteine. However, removal of both backdoor cysteines leads to irreversible oxidative inactivation, demonstrating that these two cysteines are necessary and sufficient for ensuring reversible oxidation of the SHPs. Our results extend the mechanisms by which redox regulation of PTPs is used to modulate intracellular signaling pathways.</p> / Dissertation
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Investigação do potencial antifúngico e envolvimento de genes biossintéticos em actinobactérias isoladas da CaatingaVASCONCELOS, Nataliane Marques de 26 February 2016 (has links)
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Previous issue date: 2016-02-26 / CNPq / A resistência microbiológica aos antibióticos constitui uma série problemas de saúde pública por dificultar o tratamento das infecções. As actinobactérias são fontes importantes para a descobertas de novas moléculas com atividades biológicas. A este grupo, o gênero Streptomyces possuem dentre outros, dois grupos de enzimas multimoduladoras conhecidas como policetídeo sintase (PKS) e peptídeo sintase não ribossomal (NRPS), genes relacionados com a produção de metabólitos secundários. O presente trabalho teve como objetivo investigar o potencial in vitro de metabólitos bioativos produzidos por Actinobactérias isoladas do bioma Caatinga com atividade contra diferentes isolados clínicos de Candida spp. Após ensaio primário das 45 actinobactérias apenas a linhagem PR- 32 apresentou atividade contra Candida spp, com halos de até 20 mm no meio ISP2. Posteriormente, essa linhagem foi cultivada em seis diferentes meios de cultura sendo observada melhor produção do metabólito secundário no meio 400 em 48 horas (h) de fermentação. A determinação da concentração mínima inibitória (CMI) foi determinada a partir do extrato etanólico da biomassa de PR- 32 em pH 7.0 e foi evidenciada uma CMI entre 31,25 μg/mL a 3,9 μg/mL para as leveduras testadas. A cinética de morte reforçou o resultado da CMI e mostrou que no período de 4-8 h o extrato inibiu as cepas de Candida spp. A caracterização da actinobactéria foi identificada por metodologias clássicas e pela pesquisa do gene 16S rRNA como Streptomyces sp. PR- 32. Os resultados desta caracterização sugerem uma possível espécie nova, contudo outras análises ainda precisam ser realizadas. Foi evidenciada a presença do gene nrps com aproximadamente 750 kb. Diante destes resultados podemos concluir que Streptomyces sp. PR- 32 é um isolado promissor para produção de compostos antifúngicos, sendo possível sugerir que a atividade biológica deste metabólito secundário é regulado por peptídeo sintase não ribossomal (NRPS). / The microbial resistance to antibiotics is a series of public health problems for hindering the treatment of infections. The actinomycetes are important sources for new molecules with biological activities discovered. In this group, the genus Streptomyces have among others, multimoduladoras two groups of enzymes known as polyketide synthase (PKS) and non-ribosomal peptide synthase (NRPS), genes involved in production of secondary metabolites. This study aimed to investigate the potential in vitro bioactive metabolites produced by isolated Actinobacteria biome Caatinga with activity against different clinical isolates of Candida spp. After screening of actinomycetes in 45 primary test only the PR- 32 strain showed activity against Candida spp, with halos of up to 20 mm in the middle ISP2. Subsequently, this strain was grown in six different culture media is best seen in secondary metabolite production means 400 at 48 h of fermentation. The determination of the minimum inhibitory concentration (MIC) was determined from the ethanolic extract of the biomass of PR- 32 at pH 7.0 and one MIC was observed between 31.25 mg / mL 3.9 mg / mL for yeast tested. The kinetics of death reinforced the result of CMI and showed that in the 4-8 hour period the extract inhibited the strains of Candida spp. The characterization of actinobacteria was identified by classical methods and research 16S rRNA gene as Streptomyces sp. PR- 32. The results of this characterization suggests a possible new species, but other tests that must be performed. the presence of the NRPS gene of approximately 750 kb was observed. From these results we conclude that Streptomyces sp. PR- 32 is a promising isolated to produce antifungal compounds, it is possible to suggest that the biological activity of this secondary metabolite is regulated by peptide synthase not ribosomal (NRPS).
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Cílené vyhledávání genů sekundárního metabolismu ve streptomycetách. / The directed search of genes for secondary metabolites in streptomycetes.Bakal, Tomáš January 2011 (has links)
Discoveries of new natural antibiotics are now relatively rare, therefore the construction of strains producing hybrid substances seems to be a very promising opportunity to gain new interesting biologically active compounds. This work is part of a larger project focused on the preparation of new biologically active substances derived from the antibiotic lincomycin. Lincomycin is composed of saccharide (MTL) and amino acid (propylhygric acid) moieties condensed by amide bond. Various modifications of amino acid moiety, especially of the side alkyl chain, are known to improve the antibiotic properties of final molecule. The bottleneck of biosynthesis of such modified compounds is the condensing enzyme NDL-synthetase, and especially its A-domain, which, similarly to nonribosomal peptide synthetases (NRPS), specifically recognizes and activates the amino acid precursor. In this work a set of degenerate primers for PCR searching of NRPS A-domains was proposed and the conditions of PCR reaction were optimized. In the first step a collection approximately 800 isolates of soil actinomycetes will serve as a source of genetic information for search of interesting NRPS A-domains, applicable for the construction of hybrid biosynthetic clusters. The isolates of this collection have been also characterized taxonomically...
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Bioinformatics and Biological Databases: 1) Sigma-54 Promoter Database – A Database of Sigma-54 Promoters Covering a Wide Range of Bacterial Genomes 2) ClusterMine360 – A Database of PKS/NRPS BiosynthesisConway, Kyle 14 January 2013 (has links)
The Sigma-54 Promoter Database contains computationally predicted sigma-54 promoters from
over 60 prokaryotic species. Organisms from all major phyla were analysed and results were
made available online at http://www.sigma54.ca. This database is particularly unique due to its
inclusion of intragenic regions, grouping of data by COG and COG category, and the ability to
summarize results either by phylum or database-wide.
ClusterMine360 (http://www.clustermine360.ca/) is a database of microbial polyketide and nonribosomal peptide gene clusters. It takes advantage of crowd-sourcing by allowing members of the community to make contributions while automation is used to help achieve high data
consistency and quality. The database currently has over 200 gene clusters from over 185
compound families. It also features a unique sequence repository containing over 10,000
PKS/NRPS domains. The sequences are filterable and downloadable as individual or multiple
sequence FASTA files. This database will be a useful resource for members of the PKS/NRPS
research community enabling them to keep up with the growing number of sequenced gene
clusters and rapidly mine these clusters for functional information.
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Bioinformatics and Biological Databases: 1) Sigma-54 Promoter Database – A Database of Sigma-54 Promoters Covering a Wide Range of Bacterial Genomes 2) ClusterMine360 – A Database of PKS/NRPS BiosynthesisConway, Kyle 14 January 2013 (has links)
The Sigma-54 Promoter Database contains computationally predicted sigma-54 promoters from
over 60 prokaryotic species. Organisms from all major phyla were analysed and results were
made available online at http://www.sigma54.ca. This database is particularly unique due to its
inclusion of intragenic regions, grouping of data by COG and COG category, and the ability to
summarize results either by phylum or database-wide.
ClusterMine360 (http://www.clustermine360.ca/) is a database of microbial polyketide and nonribosomal peptide gene clusters. It takes advantage of crowd-sourcing by allowing members of the community to make contributions while automation is used to help achieve high data
consistency and quality. The database currently has over 200 gene clusters from over 185
compound families. It also features a unique sequence repository containing over 10,000
PKS/NRPS domains. The sequences are filterable and downloadable as individual or multiple
sequence FASTA files. This database will be a useful resource for members of the PKS/NRPS
research community enabling them to keep up with the growing number of sequenced gene
clusters and rapidly mine these clusters for functional information.
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Bioinformatics and Biological Databases: 1) Sigma-54 Promoter Database – A Database of Sigma-54 Promoters Covering a Wide Range of Bacterial Genomes 2) ClusterMine360 – A Database of PKS/NRPS BiosynthesisConway, Kyle January 2013 (has links)
The Sigma-54 Promoter Database contains computationally predicted sigma-54 promoters from
over 60 prokaryotic species. Organisms from all major phyla were analysed and results were
made available online at http://www.sigma54.ca. This database is particularly unique due to its
inclusion of intragenic regions, grouping of data by COG and COG category, and the ability to
summarize results either by phylum or database-wide.
ClusterMine360 (http://www.clustermine360.ca/) is a database of microbial polyketide and nonribosomal peptide gene clusters. It takes advantage of crowd-sourcing by allowing members of the community to make contributions while automation is used to help achieve high data
consistency and quality. The database currently has over 200 gene clusters from over 185
compound families. It also features a unique sequence repository containing over 10,000
PKS/NRPS domains. The sequences are filterable and downloadable as individual or multiple
sequence FASTA files. This database will be a useful resource for members of the PKS/NRPS
research community enabling them to keep up with the growing number of sequenced gene
clusters and rapidly mine these clusters for functional information.
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Rôle du système de sécrétion de type III SPI-1 et des mégaenzymes NRPS dans le cycle de vie de Xanthomonas albilineans, lagent causal de l'échaudure des feuilles de la canne à sucre / Role of the SPI-1 type III secretion system and of the NRPS megaenzymes in the life cycle of Xanthomonas albilineans, the causal agent of sugarcane leaf scaldMarguerettaz, Mélanie 07 December 2010 (has links)
Xanthomonas albilineans est l'agent causal de l'échaudure des feuilles de la canne à sucre. Peu d'éléments sont connus sur les bases moléculaires de l'invasion du xylème de la canne à sucre par cette bactérie. Nous manquons également d'informations sur l'écologie de X. albilineans qui, dans certaines zones de culture et dans des conditions climatiques particulières, est capable d'infecter la canne à sucre par voie aérienne. La découverte récente dans le génome de X. albilineans de clusters de gènes spécifiques à cette espèce ouvre de nouvelles perspectives. Bien qu'aucune association de X. albilineans avec un hôte animal n'ait été décrite à ce jour, cette bactérie possède un système de sécrétion de type III SPI-1 (Salmonella Pathogenicity Island-1). Nous avons montré que ce système n'est pas indispensable à la multiplication de X. albilineans in planta. Des analyses de phylogénie, de recombinaison et de sélection de ce système au sein de souches représentatives de la diversité de X. albilineans permettent de proposer de nouvelles hypothèses sur l'écologie de cette bactérie. X. albilineans possède aussi plusieurs clusters de gènes NRPS (NonRibosomal Peptide Synthetase), dont l'un est impliqué dans la biosynthèse de l'albicidine, une phytotoxine responsable de l'apparition de symptômes foliaires. Des analyses in silico ont permis de montrer que les autres clusters de gènes NRPS de X. albilineans sont impliqués dans la biosynthèse et la sécrétion de nouvelles petites molécules. Le rôle de ces molécules reste inconnu mais, d'après l'analyse fonctionnelle des NRPS de X. albilineans, au moins une de ces molécules influence la multiplication de la bactérie in planta. / Xanthomonas albilineans is a systemic, xylem-invading pathogen that causes leaf scald disease of sugarcane. Very little is currently known about the molecular bases of the invasion of the sugarcane xylem by this bacterium. Information is also lacking on the ecology of X. albilineans which is able, in certain geographical locations and under certain climate conditions, to infect sugarcane after aerial transmission. The recent discovery in the genome of X. albilineans of gene clusters specific to this species offers new research perspectives. X. albilineans, which is not known to be animal-associated, possesses a type III secretion system (T3SS) belonging to the SPI-1 (Salmonella Pathogenicity Island-1) injectisome family. Functional analyses confirmed that this system is not required by X. albilineans to spread within xylem vessels and to cause disease symptoms. Based on phylogenetic, recombination and selection analyses of T3SS SPI-1 sequences from strains spanning the genetic diversity of X. albilineans, new hypotheses were proposed regarding the ecology of this bacterium. X. albilineans possesses also several NRPS (NonRibosomal Peptide Synthetase) gene clusters, including a gene cluster which was previously shown to be involved in the biosynthesis of albicidin, a phytotoxin causing foliar symptoms. In silico analyses indicated that other NRPS gene clusters of X. albilineans are involved in biosynthesis and secretion of new small molecules with unknown function. However, functional analyses of NRPSs showed that at least one these new small molecules influence the spreading of X. albilineans in planta.
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Etude biosynthétique des dérivés polykétides PKS-NRPS de type pyrrocidine chez Acremonium zeae / Biosynthetic study of pyrrocidine related compounds, polyketides PKS-NRPS in Acremonium zeaeEar, Alexandre 13 October 2014 (has links)
Les composés de type pyrrocidine sont des polykétides PKS-NRPS possédant des activités biologiques intéressantes comme antifongique ou antibiotique. La synthèse totale de ces composés est un réél challenge comme il est constitué de 10 centres chiraux et d'un macrocycle complexe. L'étude de leur biosynthèse pourrait être d'une aide importante afin de comprendre le mécanisme de formation de cette structure spéciale, et en particulier l'étape de la cyclisation complexe.Le précurseur linéaire de ces polykétides étant composé par une chaine nonakétide (partie PKS) et d'une L-tyrosine (partie NRPS), des hypothèses sur leur biosynthèse ont été émises dans cette thèse. Des expériences d'incorporation de précurseurs marqués ou non vont être réalisées dans différents milieux de culture en vue d'obtenir des informations sur cette biosynthèse, et plus précisément le passage du précurseur linéaire vers la structure polycyclique complexe. En parallèle, des supplémentations des cultures d'A. zeae avec des dérivés de la tyrosine seront faites dans le but d'obtenir des analogues pouvant avoir des activités biologiques nouvelles ou meilleures que les pyrrocidines. / Pyrrocidine and its related compounds are PKS-NRPS polyketides having biological interests such as antifungal or antibiotic activities. The total synthesis of these entities has been challenging since the family of hirsutellone is composed by 10 chiral centers and a complex macrocycle. Studying the biosynthesis of these compounds can be an asset for the comprehension of this special molecular structure, especially for the complex cyclization step. Knowing that the linear precursor of these molecules is constituted by a nonaketide chain (PKS part) and by an L-tyrosine (NRPS part), hypotheses about the biosynthesis of hirsutellone-related compounds have been developed in this thesis. Some incorporation experiments of labeled or unlabeled compounds has been done in different culture media in order to have more information about this biosynthesis, in particular the conversion of the linear precursor into this complex polycyclic structure. In parallel, the supplementation of L-tyrosine derivatives will help us to get some analogs of pyrrocidine which can have new or better activities than natural products.
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