La résistance bactérienne aux antibiotiques est un problème majeur de santé publique. Un moyen de la combattre est de viser des cibles non encore exploitées pour retarder l’apparition de la résistance. Dans ce contexte, nous avons entrepris la caractérisation sur les plans biochimique et structural de l’enzyme MraY, une protéine intégrale de membrane, membre d’une famille de transférases membranaires. MraY catalyse la première étape membranaire de la biosynthèse du peptidoglycane bactérien à savoir, le transfert du motif N-acétylmuramoyl-pentapeptide du précurseur cytoplasmique UDP-MurNAc-pentapeptide sur le transporteur membranaire, l’undécaprényl-phosphate aboutissant à la formation du lipide I. Aucune structure 3D de cette enzyme n’est disponible actuellement et aucun antibiotique en utilisation clinique ne la cible. D’une part nous avons entrepris la caractérisation structurale de cette enzyme par des approches de biophysique. Des essais de cristallisation 2D dans des systèmes membranaires ont permis d’observer au microscope électronique des dimères de MraY (taille de 70Å/50Å). Des expériences de diffusion des rayons X (SAXS) montrent un rayon de giration d’environ 42Å. Les résultats issus des expériences de SAXS ont été combinés à des approches de modélisation afin déterminer l’état d’oligomérisation de cette protéine en présence de détergents. Enfin, en vue de faciliter la cristallogenèse 3D, des chimères de MraY en fusion avec des protéines hydrosolubles de structure 3D résolues (mCherry et GFP) ont été construites. Des essais de cristallisation de la protéine seule et des chimères construites ont été effectués.D’autre part, nous avons élucidé le mécanisme catalytique de l’enzyme MraY et de son paralogue WecA. Au cours de ma thèse, j’ai pu montrer que cette famille de transférase membranaire présente un mécanisme catalytique commun qui procède en une seule étape par attaque directe d’un oxyanion du substrat lipidique, préalablement déprotoné par un résidu aspartate invariant, sur le phophate Beta du substrat nucléotidique. Cela conduit à la formation du produit lipidique et libération de l’UMP. / The growing emergence of multiresistance of pathogenic bacteria to currently used antibiotics is a major public health problem that requires the development of new therapeutic compounds and the identification and exploitation of novel targets. In this context, we undertook the biochemical and structural characterization of MraY enzyme, an integral membrane protein, member of the polyprenyl-phosphate N-acetylhexosamine 1-phosphate transferase superfamily. The MraY transferase catalyzes the first membrane step of bacterial cell wall peptidoglycan biosynthesis, namely the transfer of the N-acetylmuramoyl-pentapeptide moiety of the cytoplasmic precursor UDP-MurNAc-pentapeptide to the membrane transporter undecaprenyl phosphate, yielding C55-PP-MurNAc-pentapeptide (lipid I). To date, no crystal structure has been reported for this enzyme. On the one hand we have undertaken the structural characterization of this enzyme by differents biophysical approaches. Electron microscopic images after two-dimensional crystallization of the protein displayed a dimeric organisation of the MraY enzyme (size 70Å/50Å). Small X-ray scattering (SAXS) experiments have shown a radius of gyration of about 42A. The results of SAXS experiments were combined with modeling approaches to determine the oligomerization state of the protein in the presence of detergents. Finally, in order to facilitate 3D crystallisation of MraY, fusion proteins of MraY and mCherry/GFP were constructed. Crystallization trials of MraY alone and the constructed chimeras were made. On the other hand, we have elucidated the catalytic mechanism of the MraY transferase and its paralog WecA. In this study, we have shown that this family of membrane transferases has a common catalytic mechanism that proceeds by a single step displacement. During this “one-step” mechanism, the oxyanion of the poly-prenyl phosphate attacks the β phosphate of the nucleotide substrate, leading to the formation of lipid product and the liberation of UMP.

Le phénomène de résistance aux antibiotiques est un grave problème de santé publique. Afin de lutter contre l’émergence continue de résistances, le développement de nouveaux agents antibactériens s’avère nécessaire. La translocase bactérienne MraY, enzyme transmembranaire impliquée dans la biosynthèse du peptidoglycane, est essentielle à la survie de la bactérie. Sans équivalent chez les eucaryotes, elle n’est actuellement la cible d’aucun médicament et constitue donc une cible de choix pour le développement de nouveaux antibiotiques. Des molécules naturelles de structure complexe telles que les muraymycines ou les liposidomycines, inhibent cette enzyme avec, de plus, une bonne activité anti-bactérienne.L’objectif de ce travail est de développer la synthèse d’analogues simplifiés de ces inhibiteurs naturels, de structure originale. Un squelette carboné de type aminoribosyluridine, motif central rencontré dans la plupart des inhibiteurs naturels, a été retenu comme châssis moléculaire. L’objectif a été de fonctionnaliser le squelette sur le carbone 5’ de l’uridine par un motif éthynyle permettant l’accès à de nombreuses molécules, grâce à la réaction de cycloaddition azoture-alcyne catalysée par le cuivre. L’étape clé envisagée dans l’approche de synthèse proposée est une réaction de glycosylation entre deux intermédiaires clefs, préparés à l’échelle de plusieurs grammes au cours de cette thèse : - un dérivé du D-ribose fluoré en position anomérique. - un dérivé alkylé en position 5’ de l’uridine, dont l’obtention stéréocontrôlée a été optimisée au cours de l’étude. L’étape clé entre les deux intermédiaires précédents, conduisant au pharmacophore visé sous sa forme protégée, a été réalisée à l’échelle de la millimole. La réaction de cycloaddition a été testée et a permis l’accès à une première famille de molécules. De manière complémentaire, la synthèse de deux autres familles de composés comportant un groupement méthylène supplémentaire entre le triazole et le squelette aminoribosyluridine a été réalisée à partir d'un même époxyde clé. L'activité biologique de toutes les molécules a été évaluée sur l'enzyme MraY purifiée et sur différentes souches bactériennes Gram (+) et Gram (-). / The antibiotic resistance phenomenon is a serious public health problem. To fight against the continuous emergence of resistant strains, the development of new antibacterial agents is of critical importance. The bacterial translocase MraY, a transmembrane enzyme involved in the peptidoglycan biosynthesis, is essential to the survival of the bacteria. Unparalleled in eukaryote cells, this enzyme is currently untargeted by any medication and is therefore a druggable target for the development of future new antibiotics. Natural structuraly complex molecules such as liposidomycins or muraymycines inhibit this enzyme and exhibit also a good anti-bacterial activity. This work aimed to develop the synthesis of simplified analogs of these natural inhibitors. A key aminoribosyluridine scaffold, motif encountered in the most of natural inhibitors, has been selected as molecular frame. The objective was to functionalize this backbone on the 5 'carbon of uridine by a terminal alkyne goup, allowing access to many molecules through the reaction of copper catalyzed azide-alkyne cycloaddition. The critical step envisaged in the proposed synthetic approach is a glycosylation reaction between two key intermediates prepared at the multi-gram scale during this thesis : - A D-ribose derivative, fluorinated in anomeric position. - A 5'-alkylated uridine derivative. The key step between these two intermediates, leading directly to the refered pharmacophore in its protected form, was conducted at the millimole scale. The cycloaddition reaction has been then tested and has provided access to a first family of molecules. In a complementary manner, two others small library of compounds, bearing an additional methylene group between the triazole and the aminoribosyluridine core, were prepared from the same key epoxide. The biological activity of all molecules was evaluated on purified MraY and on different bacterial strains Gram (+) and Gram (-).

<p>Phospho-MurNAc-pentapeptide translocase (MraY) is a conserved membrane-spanning enzyme involved in the biosynthesis of bacterial cell walls. MraY generates lipid I by transferring the phospho-MurNAc-pentapeptide to the lipid carrier undecaprenyl-phosphate. MraY is a primary target for antibiotic development because it is essential in peptidoglycan synthesis and targeted by 5 classes of natural product antibiotics. The structure of this enzyme will provide insight into the catalytic mechanism and a platform for future antibiotic development. MraY genes from 19 bacteria were cloned, expressed, purified and assayed for biochemical stability. After initial crystallization screening, I found that MraY from Aquifex aeolicus (MraYAA) produced diffracting crystals. Recombinant MraYAA is functional and shows inhibition by the natural inhibitor capuramycin. After extensive optimization of crystallization conditions, we extended the resolution limit of the crystal to 3.3 Å. The crystal structure, the first structure of the polyprenyl-phosphate N-acetyl hexosamine 1-phosphate transferase (PNPT) superfamily, reveals the architecture of MraYAA and together with functional studies, allow us to identify the location of Mg2+ at the active site and the putative binding sites of both substrates. My crystallographic studies provide insights into the mechanism of how MraY attaches a building block of peptidoglycan to the carrier lipid.</p><p>Voltage-gated Na+ (NaV) channels initiate action potentials in neurons and cardiac myocytes. NaV channels are composed of a transmembrane domain responsible for voltage-dependent Na+ conduction and a cytosolic C-terminal domain (CTD) that regulates channel function through interactions with many auxiliary proteins including members of the fibroblast growth factor homologous factor (FHF) family and calmodulin (CaM). Through the collaboration between our lab and Geoffrey Pitt's lab, we report the first crystal structure of the ternary complex of the human NaV1.5 CTD, FGF13, and Ca2+-free CaM at 2.2 Å. Combined with functional experiments based on structural insights, we present a platform to understand roles of these auxiliary proteins in NaV channel regulation and the molecular basis of mutations that lead to neuronal and cardiac diseases. Furthermore, we identify a critical interaction that contributes to the specificity between individual NaV CTD isoforms and distinctive FHFs.</p> / Dissertation

This thesis focuses upon pacidamycin, a member of the uridyl peptide antibiotics, a family of antibiotics which exhibit an, as yet, clinically unexploited mode of action, against MraY. The Goss group has previously demonstrated the ease of accessing N and C-termini analogues of pacidamycin utilizing precursor directed biosynthesis. The central diamino acid is key to pacidamycin's activity, yet little work has been carried out, to date, to investigate the SAR around this moiety. Particularly this thesis describes work toward generating pacidamycin analogues using the complementary tools of organic synthesis and biosynthesis. Chapter 1 introduces natural compounds and their importance in clinical use, provides a brief overview of the history of antibiotics and focuses on the urgent need for new antibiotics displaying new chemical architectures and possessing novel modes of action. This chapter also introduces uridyl peptide antibiotics and overviews the SAR studies around these unusual peptides, focusing on pacidamycin in particular. Diaminobutyric acid is central to these structures and a discussion of a selection of published methods to synthesis α, β-diaminobutyric acid (DABA) is also presented. Chapter 2 describes the synthesis of DABA and two analogues, in which the C-methyl moiety has been substituted by an ethyl or a cyclopropyl group. The mutasynthesis approach utilised in the attempt to generate novel pacidamycins and discussion around the results observes is also described. Chapter 3 demonstrates a three step one-pot reaction to access 1,3-disubstituted urea molecules. The chapter starts with a brief overview of previously established methods in the literature to access these useful molecules, and then moves towards a discussion about the reaction optimisation. The chapter also describes a family of analogues generated utilising this novel approach; and exploring the use of these analogues in the mutasynthesis of pacidamycin. In order to access the desired pacidamycin analogues with the modified diamino acid residue, it was determined that it is currently not possible to use a mutasynthesis approach, instead an approach of total synthesis needed to be employed. Chapter 4 describes this total synthesis. The C- terminal urea motif was generated using a novel 1-pot phosphine free route developed during this study. To access the central native (2S, 3S)- DABA, a variation of the route of Merino et al's via Garner's aldehyde was initially utilised. Subsequently, a shorter and more flexible approach from Soloshonok et al via a Ni (II) Schiff base complex of glycine was adopted. Unpublished results from the Goss group have shown that the 2',3'dihydroxy uridine analogues in pacidamycin conferred broader spectra of activity. Work towards the synthesis of these analogues has been conducted. The order of assembly of the peptide and the nucleoside fragments was in alignment with Boojamra et al's approach. If the de-protection chemistry had worked according to plan, this would have resulted with a synthesis that is at least 6 steps shorter and higher yielding then Boojamra's. The introduction in this chapter reports the various methods previously reported in the literature for the total synthesis of pacidamycin. A discussion about the current progress in the total synthesis highlighting the difficulties faced is also shown. Chapter 5 demonstrates utilising semi-synthesis as a useful tool to generate novel pacidamycins by applying a Pictet-Spengler reaction on pacidamycin 4. This chapter starts with an overview of this phosphate mediated Pictet-Spengler reaction. In addition, a discussion about the large-scale fermentation of Streptomyces coeruleorubidus, the wild type producer of pacidamycin, and the generation of pacidamycin analogues utilising a semi-synthesis approach is also presented. Chapter 6 describes the future work following on from this study building upon each of the above chapters.

New antibiotics with novel targets or mechanisms of action are needed to counter the steady emergence of bacterial pathogens that are resistant to antibiotics used in the clinic. MraY, a promising novel target for antibiotic development, initiates the lipid cycle for the biosynthesis of peptidoglycan cell wall, which is essential for the survival of most, if-not-all, bacteria. MraY is an enzyme that catalyzes the transfer and attachment of phospho-MurNAc-pentapeptide to a lipid carrier, undecaprenylphosphate. Muraymycins are recently discovered lipopeptidyl nucleoside antibiotics that exhibit remarkable antibiotic activity against Gram-positive as well as Gram-negative bacteria by inhibiting MraY. We conducted a thorough examination of the metabolic profile of Streptomyces sp. strain NRRL 30473, a known producer of muraymycins. Eight muraymycins were isolated and characterized by a suite of spectroscopic methods, including three new members of muraymycin family named B8, B9 and C5. Muraymycins B8 and B9, which differ from other muraymycins by having an elongated fatty acid side chain, showed potent antibacterial activity against Escherichia coli ∆tolC mutant and pM IC50 against Staphylococcus aureus MraY. Muraymycin C5, which is characterized by an N-acetyl modification of the disaccharide’s primary amine, greatly reduced its antibacterial activity, which possibly indicates this modification is used for self-resistance. In addition to the discovery of new muraymycins, eleven enzymes from the biosynthetic pathway were functionally assigned and characterized in vitro. Six enzymes involved in the biosynthesis of amino ribofuranosylated uronic acid moiety of muraymycin were characterized: Mur16, a non-heme, Fe(II)-dependent α-ketoglutarate: UMP dioxygenase; Mur17, an L-threonine: uridine-5′-aldehyde transaldolase; Mur20, an L-methionine:1-aminotransferase; Mur26, a low specificity pyrimidine nucleoside phosphorylase; Mur18, a primary amine-requiring nucleotidylyltransferase; Mur19, a 5-amino-5-deoxyribosyltransferase. A one-pot enzyme reaction was utilized to produce this disaccharide moiety and its 2′′-deoxy analogue. Two muraymycin-modifying enzymes that confer self-resistance were functionally assigned and characterized: Mur28, a TmrB-like ATP-dependent muraymycin phosphotransferase, and Mur29, a muraymycin nucleotidyltransferase. Notably, Mur28 preferentially phosphorylates the intermediate, aminoribofuranosylated uronic acid, in the muraymycin biosynthetic pathway to produce a cryptic phosphorylated-dissacharide intermediate. Mur23 and Mur24 were assigned as two enzymes that modify the cryptic phosphorylated intermediate by attachment of an aminopropyl group. Mur24 catalyzes the incorporation of butyric acid into the phosphorylated-disaccharide. Following the incorporation, Mur23 catalyzes a PLP-dependent decarboxylation. Finally, Mur15, which belongs to the cupin family, is functionally assigned as a non-heme, Fe(II)-dependent α-ketoglutarate dioxygenase that catalyzes the β-hydroxylation of a leucine moiety in muraymycin D1 to form muraymycin C1. Mur15 can also hydroxylate the γ-position of leucine moiety to muraymycins with fatty acid chain in β-position.