Methanogenesis is the biological production of methane and is utilized by methanogenic archaea (methanogens) to generate energy. This process is responsible for 70% of total atmospheric methane, a potent greenhouse gas and an important energy source (natural gas). In the future, reversing methanogenesis in an engineered methanogenic strain could be realized to efficiently convert natural gas into liquid fuels. Methyl coenzyme M reductase (Mcr) catalyzes the final reaction of methanogenesis in methanogens and the first reaction in the anaerobic oxidation of methane (AOM) carried out by the anaerobic methanotrophs (ANME). Cofactor F₄₃₀, a unique nickel-containing tetrapyrrole, serves as the prosthetic group and catalytic component of Mcr. Recently, multiple F₄₃₀ variants have been discovered in several methanogenic species, including Methanococcus maripaludis, Methanosarcina acetivorans, and Methanocaldococcus jannaschii. A novel variant reported here has an exact mass of 1008.3478, a similar absorption spectrum as unmodified F₄₃₀, and associates with purified Mcr from M. acetivorans. Based on the exact mass, this molecule is likely modified with a mercaptopropamide moiety. In some conditions, this modified F₄₃₀ comprises 30-50% of the total F₄₃₀ pool. We also report upon our work to identify the sulfur insertion enzyme required to produce methylthio-F₄₃₀ that functions with Mcr in ANME-1. We hypothesized that the insertion of the methylthio moiety is likely catalyzed by a methylthiotransferase (MTTase) homolog present in ANME. However, purified ANME MTTase does not appear to catalyze this reaction, and instead catalyzes the methylthiolation of N6-threonylcarbamoyladenosine (t6A) in tRNA. / Master of Science in Life Sciences / Methanogens are a unique but diverse group of microorganisms that produce methane to generate their energetic needs. The byproduct of their metabolism is methane gas, most of which escapes into the atmosphere. Methanogens produce 70% of Earth's atmospheric methane, which is a gas that has contributed to 20% of global warming since the start of the industrial era. However, methane, which makes up the majority of natural gas, is also an important source of energy, and natural gas generates 40% of the United States' electricity. An issue with natural gas is, as a gas, it readily leaks out in the extraction and transport process. A solution to this is to convert the gas into liquids, which do not display these negatives. It is possible, through a better understanding of how methanogens work, we could produce a methanogen strain that can efficiently convert methane into liquid fuels. The last methane-generating step in methanogenic metabolism uses a protein known as methyl-coenzyme M reductase (Mcr). To do this, Mcr uses a small molecule known as cofactor F₄₃₀. Recently, variants of the standard F₄₃₀ structure have been described, in both methanogens as well as another microbial group known as the anaerobic methanotrophs (ANME). ANME generate their energy through reversing methanogenic metabolism. The work here involves studying why and how methanogens and ANME make F₄₃₀ variants. The hope is this work will reveal either different functionalities of cofactor F₄₃₀ not previously known, or that they influence Mcr catalysis, potentially in the reverse (methane degradation) direction.

Wyosine derivatives are highly complex modified ribonucleic acid (RNA) bases found in archaea and eukarya. They are a modification of a genetically encoded guanosine found at position 37 of phenylalanine encoding transfer ribonucleic acid (tRNA). The second step in the biosynthesis of all wyosine derivatives, in both archaea and eukarya, is the transformation of N-methylguanosine to 4-demethylwyosine by the radical S-adenosyl-l-methionine enzyme TYW1. When these studies were initiated, the substrate of TYW1 was unknown. Four possible substrates; acetyl CoA, acetyl phosphate, phosphoenolpyruvate, and pyruvate; were tested for activity. Only incubation with pyruvate led to production of 4-demethylwyosine. As only two new carbons are incorporated into the RNA base at this step, ¹³C isotopologues were used to identify the carbons that are transferred into 4-demethylwyosine. These experiments revealed that C2 and C3 of pyruvate are incorporated into 4-demethylwyosine, with C1 lost as an unknown byproduct. Utilizing pyruvate containing deuteriums in place of protons on the C3 carbon, the regiochemistry of the addition was determined. It was found that C3 forms the methyl group of 4-demethylwyosine and C2 becomes the bridging carbon in the imidazoline ring. The site of hydrogen atom abstraction by 5'-deoxyadenosyl radical was identified as the N-methylguanosine methyl group through the use of tRNA containing a deuterated methyl group. The putative mechanism for this transformation involved the formation of an enzyme substrate Schiff base through a conserved lysine residue. Utilizing sodium cyanoborohydride a Schiff base was trapped between TYW1 and pyruvate. The mass of the trapped adduct responded as expected when different isotopologues of pyruvate were used, demonstrating that it is due to pyruvate. Moreover, the fragment of TYW1 that contained the trapped adduct contained two lysine residues, one of which was shown to be required for activity both in vivo and in vitro. It was initially proposed that TYW1 contained two iron-sulfur clusters, and then subsequently shown to have two 4Fe-4S clusters. Site directed mutagenesis, along with iron and sulfide analysis identified the cysteines; as C26, C39, and C52; coordinating the second 4Fe-4S cluster. This study identified pyruvate as the substrate of TYW1, and provided evidence for key steps in the transformation of N-methylguanosine to 4-demethylwyosine.

Les centres FeS sont un des cofacteurs protéiques majeurs, ils se trouvent aussi bien chez les bactéries que chez les eucaryotes. Ils ont des rôles essentiels de transfert d'électron, liaison de substrat et son activation, régulation d'expression de gènes, donneur de soufre etc. Leur agencement est très varié, allant du centre [2Fe-2S] à l'agrégat plus complexe MoFe7S9X (X = C, N ou O) de la nitrogénase. L'assemblage de ces centres se fait par des machineries protéiques. Nous avons étudié le système ISC (Iron-Sulfur Cluster) chez les bactéries, qui fabrique des centres [2Fe-2S] et [4Fe-4S]. Il est composé des protéines IscS, IscU, IscA, HscA, HscB et d'une ferrédoxine. Deux de ces protéines, IscS, qui est une cystéine désulfurase et IscU, protéine dite échafaudage, sont le cœur de la machinerie puisque IscS apporte le soufre sur la protéine IscU, qui, avec le fer qu'elle aura obtenu d'une autre protéine (non clairement identifiée à ce jour), fabriquera le centre fer-soufre et le transfèrera à une apoprotéine. Nous avons isolé un complexe stable (IscS-D35A-IscU)2 contenant un centre [2Fe-2S] dans des conditions anaérobie. Différentes formes du complexe ont été obtenues et cristallisées afin d'obtenir leurs structures, résolues par remplacement moléculaire. Ces structures nous ont permis de proposer un mécanisme d'assemblage des centres [2Fe-2S] à l'échelle atomique et électronique. Nous avons d'autre part étudié la protéine HmdB probablement impliquée dans la maturation de l'hydrogénase à fer. HmdB fait partie de la superfamille des protéines à radical SAM. Des cristaux de l'apoprotéine ont été obtenus et sa structure a été résolue par remplacement moléculaire. Même si une partie de la structure n'est pas visible du fait de l'absence de centre [4Fe-4S], elle donne une première vue du site actif de la protéine. / FeS clusters are widely used protein cofactors, found both in bacteria and eukaryotes. They play key roles such as electron transfer, substrate binding and activation, regulation of gene expression, sulfur donor etc. They are really various, ranging from the [2Fe-2S] cluster to the more complex MoFe7S9X (X = C, N or O) agregate of nitrogenase. Clusters assembly is carried out by protein machineries. We studied the ISC (Iron-Sulfur Cluster) in bacteria, who assembles [2Fe-2S] and [4Fe-4S] clusters. It is composed of IscS, IscU, IscA, HscA, HscB proteins and a ferredoxin. Two of these proteins: the cysteine desulfurase IscS, and the scaffold protein IscU, represent the core of the machinery as IscS provides sulfur protein on IscU, which, with iron obtained from another protein (not clearly identified to date), assemble the iron-sulfur center. The latter transfers it to an apoprotein. We isolated under anaerobic conditions a stable (IscS-D35A-IscU)2 complex containing a [2Fe-2S] cluster. Different forms of the complex were obtained and their structures were solved by molecular replacement. These structures allowed us to propose a mechanism for the assembly of the [2Fe-2S] clusters at the atomic and electronic levels. We have also studied the HmdB protein, which is proposed to maturate the [Fe]-hydrogenase. HmdB is a member of the radical SAM proteins superfamily. Crystals of the apoprotein were obtained and its structure was solved by molecular replacement. Although part of the structure is not visible due to the absence of the [4Fe-4S] cluster, this structure gives a first view of the active site of the protein.

Un des problèmes majeurs en enzymologie est la fonctionnalisation de liaisons C-H peu réactives. Ces réactions nécessitent des cofacteurs spécialisés tels que l'hème, des Fer-oxo ou encore la vitamine B12. En 2001, il a été montré que des centres Fe-S particuliers liant la S-Adénosyle méthionine (SAM) pouvaient activer des liaisons C-H non réactives. Les enzymes utilisant ce cofacteur constituent une super-famille appelée « Radical SAM ». Les thiométhyltransférases (MTTases) sont des enzymes « Radical SAM » qui catalysent l'insertion d'un groupe thiométhyle (-SCH3) dans des liaisons C-H non réactives. Par des expériences in vivo et in vitro, nous avons montré qu'on pouvait les regrouper en trois classes. La première classe (RimO) catalyse la formation du β-thiométhylaspartate 89 sur la protéine ribosomale S12 (β-ms-D89-S12) alors que les deux dernières (MiaB et MtaB/eMtaB) catalysent respectivement la thiométhylation des nucléosides 2-methylthio-N6-isopentenyladenosine 37 (ms2i6A-37) et 2-methylthio-N6-threoninecarbamoyl adenosine 37 (ms2t6A-37) de certains ARNts. L'étude in vitro du mécanisme de ces enzymes a permis de démontrer que les MTTases catalysent l'insertion d'un groupement -SCH3 de façon catalytique invalidant l'hypothèse généralement retenue dans la littérature que le soufre inséré dérive de la destruction d'un centre Fe-S. / One of the chemically most challenging problems in enzymology is the functionalization of non-reactive C–H bonds. Such reactions require specialized cofactors as heme, Fe-µ-oxo or vitamin B12. In 2001, special Fe-S centers able to bind S-Adenosylmethionine (SAM) have been shown to activate non-reactive C-H bonds toward subsequent functionalization. Enzymes containing this new cofactor constitute a super-family called “Radical SAM”. Methylthiotransferases (MTTases) belong to the “Radical SAM” super-family and catalyse the insertion of a methylthio group (-SCH3) in C-H bonds. By doing in vitro and in vivo experiments, we were able to classify them into three groups. The first class, (RimO) catalyses the formation of β-methylthio-aspartate 89 on the ribosomal protein S12 (β-ms-D89-S12) whereas the two others (MiaB and MtaB/eMtaB) respectively catalyse the thiomethylation of the nucleosides 2-methylthio-N6-isopentenyladenosine 37 (ms2i6A-37) and 2-methylthio-N6-threoninecarbamoyl adenosine 37 (ms2t6A-37) present in some tRNA. Our studies have shown that, in vitro, MTTases catalyse the insertion of a SCH3 group in a catalytic way suggesting entirely new radical sulfur insertion mechanisms.

Le microbiote humain est constitué de milliers d’espèces bactériennes qui synthétisent de nombreux métabolites secondaires. Cependant, notre connaissance des produits naturels dérivés du microbiome est encore limitée. Parmi eux, les RiPPs (Ribosomally Synthesized and Post-translationally modified Peptides) apparaissent comme une famille majeure de produits naturels possédant diverses structures et fonctions biologiques dont des propriétés antibiotiques, en faisant une famille de molécules d’intérêt majeur pour la santé publique. La biosynthèse des RiPPs commence par la traduction d’un peptide précurseur, qui est ensuite maturé par l’action d’une ou plusieurs enzymes avant l’excision d’une séquence signal et l’export du produit naturel actif. La diversité structurale et fonctionnelle des RiPPs démontre la nécessité de la compréhension des voies de biosynthèse de ces produits naturels, de l’étude systématique des mécanismes de modification et de la caractérisation des maturases associées. En particulier, une famille de métallo-enzymes, les enzymes à radical S-adénosyl-L-méthionine (SAM), a récemment été impliquée dans la biosynthèse de nombreux RiPPs. Ces enzymes catalysent un large éventail de réaction, via un mécanisme de chimie radicalaire, aboutissant à une grande variété de modifications post-traductionnelles. Néanmoins, les voies de biosynthèse de nombreux RiPP restent mal comprises.En 2011, il a été montré que Ruminococcus gnavus, un membre important du microbiote humain, produisait un peptide actif contre Clostridium perfringens, la Ruminococcin C (RumC). Le séquençage de l’opéron de biosynthèse de RumC montre la présence de cinq gènes codants des peptides précurseurs (RumC1-5) et deux gènes codant des enzymes (RumMC1 et RumMC2).L’objectif de ma thèse est de mieux comprendre les voies de biosynthèse des produits naturels au sein du microbiome humain. Nous avons démontré l’appartenance des protéines RumMC1 et RumMC2 à la famille des enzymes à radical SAM, ainsi que leurs implications dans la formation de quatre modifications post-traductionnelles (ponts α-thioether) essentielles à l’activité antibiotique de RumC1 et RumC2. Ces études nous ont permis de proposer un mécanisme catalytique pour la maturation de la Rummonicoccin C et ainsi de mieux documenter cette famille d’enzymes émergentes. / The human microbiota consists of thousands bacterial species which synthesize numerous secondary metabolites. However, our knowledge of microbiome-derived natural products is still limited. Among them, RiPPs (Ribosomally synthesized and Post-translationally modified Peptides) are emerging as a major family of natural products possessing diverse structures and biological functions including antibiotic properties, making them a major family of molecules of interest for public health. The biosynthesis of RiPPs occurred by the translation of a precursor peptide, which is then matured via the action of one or more enzymes before the excision of a signal sequence and the export of the active natural product. The structural and functional diversity of RiPPs demonstrates the need for understanding the biosynthetic pathways of these natural products, the systematic study of the modification mechanisms and the characterization of associated maturases. In particular, a family of metallo-enzymes, the S-adenosyl-L-methionine radical (SAM) enzymes, has recently been implicated in the biosynthesis of many RiPPs. These enzymes catalyze a wide range of reactions, via a mechanism of radical chemistry, resulting in a wide variety of post-translational modifications. Nevertheless, the biosynthetic pathways of many RiPPs remain poorly understood.In 2011, it was shown that Ruminococcus gnavus, a major member of the human microbiota, produced an active peptide against Clostridium perfringens, Ruminococcin C (RumC). Sequencing of the RumC biosynthesis operon shows the presence of five genes encoding precursor peptides (RumC1-5) and two genes encoding enzymes (RumMC1 and RumMC2).The aim of my thesis is to understand the biosynthetic pathways of natural products within the human microbiome. We have demonstrated that the RumMC1 and RumMC2 proteins belong to the radial SAM enzyme family, as well as their involvement in the formation of four post-translational modifications (α-thiother bridges) essential for the antibiotic activity of RumC1 and RumC2. These studies allowed us to propose a catalytic mechanism for the maturation of Rummonicoccin and thus to better document this family of emerging enzymes.

Le cofacteur F420 est un coenzyme d’oxydoréduction essentiel pour la méthanogenèse chez les archées, un processus qui influence fortement les interactions métaboliques au sein du microbiote intestinal ; en outre, il joue un rôle important dans la pathogénicité de la bactérie Mycobacterium tuberculosis. L’étude de sa biosynthèse présente donc un intérêt majeur en Biologie.La formation du chromophore du F420 est catalysée par la F0-synthase, qui contient, de façon unique, deux domaines caractéristiques des enzymes à radical SAM (rSAM). Ces enzymes catalysent le clivage de la S-adénosylméthionine (SAM) pour former un radical 5′ déoxyadénosyle, capable d’initier un grand nombre de réactions radicalaires.Nous avons réussi à identifier les substrats de la F0-synthase et à reconstituer la synthèse du F0 in vitro. Nous avons également démontré que cette enzyme contient deux centre [4Fe-4S] 2+/1+ rSAM fonctionnels et caractérisé les étapes de la synthèse du F0. Ceci nous a permis de proposer un mécanisme réactionnel pour la F0 synthase. Nous avons ensuite entrepris la comparaison de la F0 synthase avec les deux autres enzymes rSAM tyrosine lyases connues à ce jour : ThiH, impliquée dans la biosynthèse de la vitamine B1, et HydG, impliquée dans la biosynthèse du cofacteur métallique de l’hydrogénase à fer-fer. Nous avons ainsi découvert de nouveaux aspects de la réaction de clivage de la tyrosine par ces enzymes, permettant une meilleure compréhension de ce groupe émergent au sein de la superfamille des enzymes rSAM. / Cofactor F420 is a redox coenzyme crucial for methanogenesis in Archaea, a process which plays a major role in metabolic interactions in the gut microbiota ; It also constitutes a key pathogenicity factor for Mycobacterium tuberculosis. Understanding the biosynthesis of this cofactor is thus of major interest.The biosynthesis of the chromophore of F420 is catalyzed by F0 synthase, which comprises, in a unique manner, two radical SAM (rSAM) domains. These enzymes catalyze the cleavage of S adenosylmethionine (SAM) to produce a 5′-deoxyadenosyl radical, which can initiate a broad range of radical reactions.We succeeded to identify the substrates of F0-synthase and to perform the biosynthesis of F0 in vitro. We ascertained that F0-synthase contains two functional [4Fe-4S]2+/1+ rSAM clusters, and characterized the steps of the reaction of F0 synthesis. Based on these date, we proposed a mechanism for the F0-synthase reaction. Furthermore, we compared F0 synthase with the two other radical SAM tyrosine lyases identified to date: ThiH, which is involved in vitamin B1 biosynthesis, and HydG, which is involved in the biosynthesis of the metal cofactor of iron-iron hydrogenases. We obtained novel insights of the reaction of tyrosine cleavage catalyzed by these enzymes, providing a better understanding of this emerging group in the rSAM enzyme superfamily.

De nombreuses protéines possèdent des cofacteurs inorganiques contenant des métaux de transition. Les propriétés physico-chimiques de ces métaux permettent aux enzymes qui les portent de catalyser des réactions impossibles par l'utilisation des seules potentialités chimiques des vingt-deux acides aminés. Cependant, ces métaux sont toxiques pour la cellule lorsqu'ils sont libres. La synthèse et l'incorportion de ces cofacteurs dans les enzymes nécessitent alors des machineries protéiques complexes d'assemblage. Au cours de cette thèse, les mécanismes de synthèse des centres FeS par les machineries ISC (Iron-Sulfur Cluster) et Hyd (Hydrogenase) ont été étudiés. Le système ISC correspond à la machinerie primaire d'assemblage des centres FeS chez les bactéries, et un système équivalent existe chez les eucaryotes au niveau de la mitochondrie. Le système Hyd est la machinerie de maturation de l'hydrogénase à FeFe chez plusieurs eucaryotes inférieurs (algues et protistes) et dans une grande variété de bactéries. Dans un premier temps, nous nous sommes intéressés à la machinerie ISC d'Archaeoglobus fulgidus dont le coeur est composé de la cystéine désulfurase IscS et de la protéine échafaudage IscU ; IscS apportant le soufre nécessaire à l'assemblage du centre FeS sur IscU. Au cours de cette étude, il est apparu que IscS d'Archaeoglobus fulgidus ne possède pas d'activité cystéine désulfurase, mais qu'elle joue tout de même un rôle fondamental dans la synthèse du centre FeS sur le complexe IscSU en fournissant sa cystéine active en tant que ligand de l'agrégat. Dans un second temps, nous avons étudié la protéine à radical S-adénosyl-L-méthionine HydG, responsable de la synthèse des ligands CN- et CO du sous-agrégat à 2 Fe des hydrogénases à FeFe, qui était la seule maturase du système Hyd dont la structure n'était pas connue. Nos résultats structuraux et fonctionnels suggèrent que HydG synthétise successivement le ligand CN- dans un site actif basique, puis le ligand CO sur le cinquième Fe de son agrégat [5Fe-4S] C-terminal. Ce dernier pourrait être stabilisé par un ligand cystéine ou homocystéine. / Many proteins have inorganic cofactors containing transition metals. The physicochemical properties of these metals allow the enzymes, which carry them to catalyze reactions not possible when only using the chemical properties of the twenty-two amino acids. However, these metals are toxic to the cell when they are free. Consequently, the synthesis and incorporation of these cofactors into enzymes requires complex protein assembles. In this thesis, the FeS clusters synthesis mechanisms by the ISC (Iron-Sulfur Cluster) and Hyd (Hydrogenase) machineries were studied. The ISC system corresponds to the primary FeS clusters assembly machinery in bacteria, and a homologous system exists in mitochondria. The Hyd system is FeFe-hydrogenase active site maturation machinery found in several lower eukaryotes (algae and protists) and in a wide variety of bacteria. Initially, we studied the ISC machinery from Archaeoglobus fulgidus whose core is composed of the cysteine desulfurase IscS and the scaffold protein IscU; IscS delivers the sulfur needed for the FeS assembly to IscU. From this study we conclude that IscS from Archaeoglobus fulgidus has no cysteine desulfurase activity, but it still plays a fundamental role in FeS cluster synthesis by IscSU complex by providing a cysteine ligand to the nascent cluster. Secondly, we studied the radical S-adenosyl-L-methionine HydG, responsible for the synthesis of CN- and CO ligand of the active site [FeFe] subcluster, which was the only Hyd system maturase for which the structure was unknown. Our structural and functional results suggest that HydG successively synthesizes the CN- ligand at a basic site, and then the CO ligand at the unique fifth Fe ion of its C-terminal [5Fe-4S] cluster. The latter could be stabilized by either a cysteine or a homocysteine ligand.

Les antibiotiques sont des médicaments qui ont changé notre manière d’aborder les infections bactériennes et sont devenus l’un des symboles de la médecine moderne. Cependant leur utilisation massive a conduit à l'émergence de souches bactériennes multirésistantes. Ce problème est sans aucun doute un des grands défis que la médecine actuelle doit relever. Sachant que les bactéries évoluent à un rythme plus rapide que la production de nouveaux antibiotiques, il est urgent de trouver des approches alternatives. Il a été mis en évidence que ces mêmes bactéries sont capables de sécréter différents peptides antimicrobiens, ou bactériocines. Ces macromolécules présentent une grande diversité structurale et sont très efficaces pour combattre un grand nombre de souches pathogènes de façon spécifique. Les bactériocines ont un immense potentiel dans les domaines agroalimentaire et pharmaceutique. Notre projet s’intéresse aux bactériocines RumCs produites par une souche dérivée de Ruminococcus gnavus, une bactérie anaérobie stricte, membre dominant du microbiote intestinal humain. Le travail présenté dans ce manuscrit concerne la mise au point d’un système d’expression et de maturation hétérologue chez E. coli de la bactériocine RumC1. La caractérisation biochimique du peptide RumC1 montre que les bactériocines RumCs appartiennent à la famille des sactipeptides pour laquelle l’étape de biosynthèse fait intervenir une enzyme radical-SAM. Les sactipeptides présentent dans leurs séquences peptidiques un ou plusieurs ponts thioéther entre une cystéine et le carbone alpha d’un acide aminé partenaire. RumC1 renferme 4 ponts thioéther ce qui lui confère une structure originale en double épingle à cheveux. L’activité biologique de RumC1 montre que ce peptide est efficace contre un large spectre de bactéries à Gram positif incluant des pathogènes résistants tels que S.aureus et E. faecalis. Dans ces études nous n’avons pas noté de toxicité significative de RumC1 sur différentes lignées cellulaires humaine ni observé de phénomène de résistance. Les travaux en cours visent notamment à définir le mode d’action de RumC1 et à évaluer l’activité biologique de RumC1 dans un contexte d’infection in vivo chez la souris. / Antibiotics are drugs that have changed the way we approach bacterial infections and have become one of the symbols of modern medicine. However, their widespread use has led to the emergence of multiresistant bacterial strains. This problem is undoubtedly one of the major challenges facing today's medicine. Knowing that bacteria evolve at a faster rate than the discovery of new antibiotics, it is urgent to find alternative approaches. It has been shown that these same bacteria are capable of secreting antimicrobial peptides, the bacteriocins. These macromolecules have a high structural diversity and are very effective in combating a large number of pathogenic strains in a specific way. Bacteriocins have immense potential in the agro-food and pharmaceutical sectors. Our project focuses on the bacteriocins RumCs produced by a strain derived from Ruminococcus gnavus, a strict anaerobic bacterium of the human intestinal microbiota. The work presented in this manuscript concerns the development of a heterologous expression and maturation system in E. coli of the bacteriocin RumC1. The biochemical characterization of the RumC1 peptide shows that the RumCs bacteriocins belong to the family of sactipeptides for which the biosynthesis step involves a radical-SAM enzyme. The sactipeptides have in their peptide sequences one or more thioether bridges between a cysteine and the alpha carbon of a partner amino acid. RumC1 contains 4 thioether bridges which gives it an original structure in double hairpin. The biological activity of RumC1 shows that this peptide is effective against a broad spectrum of Gram-positive bacteria including resistant pathogens such as S.aureus and E. faecalis. In these studies, we did not note any significant toxicity of RumC1 on different human cell lines nor observed resistance phenomena. Current work aims to define the mode of action of RumC1 and to evaluate the biological activity of RumC1 in an in vivo context of infection in mice.

<p>The molybdenum cofactor (Moco) is an essential enzyme cofactor found in all kingdoms of life. Moco plays central roles in many vital biological processes, and must be biosynthesized de novo. During its biosynthesis, the characteristic pyranopterin ring of Moco is constructed by a complex rearrangement of guanosine 5'-­triphosphate (GTP) into cyclic pyranopterin (cPMP) through the action of two enzymes, MoaA and MoaC. However, the mechanisms and the functions of the two enzymes are under significant debate. To elucidate their physiological roles, I took a multidisciplinary approach to functionally characterize MoaA and MoaC in vivo and in vitro. In this dissertation, I report the first isolation and characterization of the physiological MoaC substrate, 3',8-­ cyclo-­7,8-­dihydro-­guanosine 5'-triphosphate (3',8-cH2GTP). I also report the first X-­ray crystal structures of MoaC in complex with this highly air sensitive substrate, and its product cPMP. These studies, combined with in vitro experiments using substrate analogs, catalytically impaired mutants, and synthetic peptides, have enabled me to delineate the functions of the Moco biosynthetic enzymes, MoaA and MoaC, and proposed mechanistic models for their roles in the formation of cPMP.</p> / Dissertation

Structural biology uncovers life's secrets by studying protein structures via techniques like X-ray crystallography. This knowledge drives advancements in protein engineering for the improvement of human lives. Yet, obtaining high-quality crystals in X-ray crystallography is challenging. To overcome this, we used Translocation ETS Leukemia protein Sterile Alpha Motif domain (TELSAM), a promising polymer-forming crystallization chaperone (PFCC), to enhance protein crystallization. Human thirty-eight-negative kinase-1 (TNK1), a key player in cancer progression, possess a ubiquitin association (UBA) domain that binds polyubiquitin and regulates TNK1 activity and stability. Although sequence analysis hints at an unconventional TNK1 UBA domain architecture, its molecular structure lacks experimental validation. To gain insight into TNK1 regulation, we fused the UBA domain to the 1TEL crystallization chaperone and obtained crystals diffracting as far as 1.53 Ã…. 1TEL enabled solution of the X-ray phases. GG and GSGG linkers allowed the UBA to reproducibly find a productive binding mode against its 1TEL polymer and to crystallize at protein concentrations as low as 0.1 mg/mL. Our findings support a TELSAM fusion crystallization mechanism, highlighting fewer crystal contacts compared to traditional crystals. Both modeling and experimental validation indicate that the UBA domain exhibits selectivity towards polyubiquitin chain length and linkages. Radical S-adenosylmethionine (SAM) enzymes catalyze various radical-mediated substrate transformations. Despite the growing interest of computational enzyme design in industrial small molecule synthesis, radical SAM enzymes remain relatively unexplored. We used PyRosetta to leverage hydrogen bonding design (hbDes) and hydrophobic interaction design (hpDes) to enable a radical cyclization reaction on our selected substrate. Although the purified enzymes demonstrated activation potential with a reducing agent, enzymatic assays failed to exhibit activity against the reactant. To obtain successful results, addressing additional questions and issues is required, which may involve the implementation of machine learning.