The crucial part of the biosynthesis of lincosamide antibiotics lincomycin and celesticetin is the condensation of amino sugar and amino acid moieties. This reaction is catalysed by the oligomeric enzyme lincosamide synthetase (LS). One of the most important components of LS is adenylation domain recognizing and activating amino acid precursor. The substrate specificity of adenylation domain is determined by "nonribosomal code", 10 amino acids residues which side chains are in close contact with the activated substrate. The homologous adenylation domains LmbC from biosynthesis of lincomycin and CcbC from biosynthesis of celesticetin exhibit strong substrate specificity for their natural substrates (2S,4R)-4-propyl-L-proline (PPL) and L-proline, respectively. At first the effect of selected amino acid residues of LmbC nonribosomal code on the substrate specificity of the whole domain was tested. The amino acids residues, most important for preference of PPL substrate over L proline, were determined: G308, A207 and L246. Then the effect of double mutations in nonribosomal codes of both LmbC and CcbC on their substrate specificity was evaluated. The double mutants LmbC G308V + A207F and CcbC V306G + F205A were prepared and tested biochemically. The results brought new evidence of validity of homologous models...

Diese Arbeit befasst sich mit dem bisher noch nicht untersuchten Mechanismus der Cyanophycinbiosynthese. Hierzu wurden verschiedene kurze Cyanophycinmoleküle chemisch synthetisiert, die als definierte Primer in in vitro Experimenten verwendet wurden. Die Verwendung dieser Primer ermöglichte erstmals die Richtung der Verlängerung des Cyanophycinmoleküls aufzuklären. Die durchgeführten Experimente zeigten, dass der Einbau der konstituierenden Aminosäuren sukzessiv vom Carboxyterminus aus erfolgt. Weiterhin wurde gezeigt, dass auch die nicht proteinogenen Aminosäuren Ornithin und Citrullin vom Enzym eingebaut werden. Mittels ortsgerichteter Mutagenese wurde zudem eine Zuordnung unterschiedlicher Abschnitte der Cyanophycin-Synthetase zu den verschiedenen vom Enzym katalysierten Teilreaktionen versucht. Mutationen im N-terminalen Bereich der Cyanophycin-Synthetase aus Anabaena variabilis ATCC 29413 führten dazu, dass Aspartat nicht mehr in Cyanophycin eingebaut wurde, eine Mutation im C-terminalen Bereich bewirkte, dass Arginin nicht mehr mit Cyanophycin verknüpft werden konnte. Als Reaktionsmechanismus wird für die Bindung beider Aminosäuren jeweils eine Phosphorylierung des C-terminalen Aspartatrestes von Cyanophycin als Acylphosphat vorgeschlagen, wobei die Phosphorylierung der beta-Carboxylgruppe mittels gamma-[³²P]-ATP nachgewiesen werden konnte, die Phosphorylierung der alpha-Carboxylgruppe jedoch nicht. Durch Vergleiche mit Enzymen ähnlicher Aminosäuresequenz und bekannter Raumstruktur wird eine mögliche Begründung für diese unterschiedlichen Befunde gegeben. / This work is occupied with the till now uninvestigated mechanism of the biosynthesis of cyanophycin. Therefore different short cyanophycin molecules were synthesized chemically, which were employed as defined primers for in vitro experiments. The usage of these primers made it possible to clear up the direction of the elongation of the cyanophycin molecule. Experiments showed that the incorporation of the constituent amino acids happens successively starting from the carboxy-terminus. Further it was shown that the nonproteinogenic amino-acids ornithine and citrulline are incorporated by the enzyme. Using site-directed mutagenesis an assignment between segments of the cyanophycin synthetase to different parts of the reactions catalyzed by the enzyme was carried out. Mutations in the N-terminal part of cyanophycin synthetase of Anabaena variabilis ATCC 29413 lead to the finding, that aspartate was not incorporated into cyanophycin anymore. A mutation in the C-terminal part resulted in the disability of the enzyme to incorporate arginine into cyanophycin. As reaction mechanism for the attachment of both of the amino acids a phosphorylation of the C-terminal aspartate as an acylphosphate was proposed. The phosphorylation of the beta-carboxylic-group could be shown by using gamma-[³²P]-ATP, the phosphorylation of the alpha-carboxylic group could not be shown. By comparison with enzymes that share a similar amino acid sequence and have a solved crystal structure a possible explanation for this finding is given.

The biosynthetic pathway for the production of the dipeptide antibiotic bacilysin has been the subject of intense research over the past three decades. These studies revealed the role of multiple enzymes in the biosynthesis of this antibiotic. The identification of different enzymes was initially guided by genetic studies on different strains of Bacillus. The functional role of some these enzymes have been validated in vitro in the recent past. Despite this, the in vitro synthesis of bacilysin still remains elusive. The focus of this study was on two oxidoreductases - BacC and BacG. In the course of these studies, several variations to conventional oxidoreductase mechanisms were observed. These studies also provided us an opportunity to examine an oxidoreductase, BacC, at atomic resolution. This thesis describes these structural studies alongside efforts to achieve the biosynthesis of bacilysin in vitro. Chapter 1 provides an introduction to the broad goals of this thesis. First, the diversity of naturally occurring antibiotics is described. This is followed by a description of nonribosomal peptides and their preferred route for antibiotic synthesis. A summary of previous work in this area is provided to place this study in perspective. Earlier studies performed in this laboratory and others provided a framework for understanding the role of BacC and BacG. These studies have been described with an emphasis on the pivotal role of oxidoreductases in this process. In this context, known features of oxidoreductases, classification of the enzyme family, known reaction mechanisms, preferred substrates and cofactors of the enzyme have been summarized in this chapter. Chapter 2 describes the structural and biochemical characterization of B.subtilis BacG. The crystal structures of BacG determined in the apo form and ligand bound states could capture different conformational states of this enzyme. These structures revealed a basis to understand the ping-pong reaction mechanism. The catalytic residues Tyr-Ser-Lys-Asn involved in the proton relay were examined by mutational analysis. These biochemical studies could corroborate our observations derived from structural analysis. Put together, these studies suggest synchronized conformational changes in BacG that can rationalize cofactor specificity and catalytic action on di hydroxyphenyl pyruvate to form tetra hydroxyphenyl pyruvate en route to anticapsin biosynthesis. Bacillus subtilis BacC could be structurally characterized at 1.19Å resolution. The atomic resolution structure formed the basis for the analysis reported in this chapter. The structure revealed aspects of non-covalent interactions that could be unambiguously determined due to the high resolution diffraction data. The atomic resolution structure also enabled us to conduct charge density analysis on this protein. Atomic displacement parameters were used as a tool to explore paths of non-covalent interactions. A commercially available substrate, 3-Quinuclidinone, was used to characterize enzymatic activity. We note that this enzyme follows a rapid equilibrium random mechanism. Furthermore, the kinetic profiles were conclusive to draw inferences on allosteric interactions. A comparison between the NADH-complex and the apo enzyme structure suggests aspects of nuanced atomic displacement that governs the intra structural signal transduction. Taken together, this study provided a template to analyze the role of non-covalent interactions in regulating enzymatic activity. Chapter 4 is based on a survey of oxidoreductases that have been previously described in literature. During this study, we collated the extensive structural and biochemical data in this family of enzymes. However, we noted that the data remains disperse thereby limiting efforts to understand the reaction mechanism from a structural perspective. Here we collate information of known sequences, structures, cofactors, ligand preferences, reaction mechanisms and their influence on higher order association and catalytic activity in this class of enzymes. Chapter 5 summarizes the findings on two oxidoreductases (BacC, BacG). These studies on two closely related oxidoreductases BacC and BacG performing different roles in the same biosynthetic pathway revealed aspects biosynthesis that are often poorly recognized in protein engineering. The role of the reaction mechanism and their influence on the cofactor specificity could be inferred from the studies on these two enzymes. These studies also suggest the feasibility of evaluating aspects of enzyme activity and regulation provided the wealth of a priori information that is currently available. Put together, these studies provide a data-set for protein engineering efforts on oxidoreductases with general inferences for other enzymes in the short-chain dehydrogenases/ reductases (SDR) family. Appendix 1 provides a schematic representation of our efforts to biosynthetically obtain bacilysin in vitro. The identification, mass spectrometry of the products and substrates en route to bacilysin biosynthesis are compiled in this section. Appendix 2 describes the preliminary characterization of B.subtilis BacF. This part of the work describes the cloning, expression and purification of BacF and attempts to obtain suitable diffracting crystals for structural analysis.

La colistine, antibiotique appartenant à la famille des polymyxines, est un polypeptide cyclique, cationique, ciblant les membranes bactériennes. Elle est produite par Paenibacillus polymyxa via des complexes multi-enzymatiques appelés Non-Ribosomal Peptides Synthétases (NRPS). Dans le cas de la mucoviscidose, et malgré des effets secondaires importants, la colistine est utilisée comme ultime recours pour lutter contre les bactéries Gram-négatives multirésistantes responsables d’infections pulmonaires dont Pseudomonas aeruginosa. Jusqu’ici les systèmes génétiques à l’origine de la production de la colistine étaient peu connus. Au cours de cette étude, nous avons caractérisé par LC-MS haute résolution des molécules antimicrobiennes, dont des colistines, produites par un nouveau Paenibacillus. Afin d’identifier et de cloner le cluster de gène responsable de la production de ces antibiotiques, une banque d’ADN génomique a été construite et criblée par homologie de séquence avec des systèmes de production déjà connus. Ce criblage a permis de sélectionner quatre clones d’intérêt. L’étude in silico de leurs séquences a permis d’identifier les différents modules d’un nouveau cluster NRPS qui serait à l’origine de la synthèse de variants de la colistine. À terme, cette découverte pourrait permettre de mieux contrôler la production de la colistine et d’obtenir des composés plus actifs et/ou présentant des effets secondaires amoindris. En parallèle à ce premier travail, nous avons également recherché la présence de nouvelles NRPS chez une centaine de micro-organismes issus d’une station d’étude environnementale du laboratoire (vasière intertidale). Ce travail a permis de découvrir des nouvelles séquences et d’isoler un nouveau micro-organisme producteur d’antibiotique(s). / Colistin is a cationic cyclic polypeptide antibiotic belonging to the polymyxin family and targeting bacterial membranes. It is produced by Paenibacillus polymyxa through a Nonribosomal Peptide Synthetase (NRPS) mechanism. In the context of cystic fibrosis (CF), colistin is used for the treatment of lung infections caused by multiresistant Gram-negative bacteria including Pseudomonas aeruginosa. Unfortunately, this molecule is also known for its strong side effects. So far, genetic systems controlling the production of polymyxins were little known. In this study we characterized by High-resolution LC-MS the antimicrobial molecules, including colistins, of a new Paenibacillus. A genomic library of this strain was constructed and screened to identify genes involved in the production of these antibiotics. A degenerated PCR screening was performed and allowed to select four clones in the genomic library. In silico study allowed to identify a new NRPS gene cluster responsible for the biosynthesis of colistin variants. In the future, this work might allow the harnessing of the production of colistin derived structures, more active and/or showing fewer side effects. In parallel, a second investigation was performed in order to find new NRPS genes in a collection of one hundred intertidal mudflat bacterial isolates. This work has allowed the identification of new sequences and the characterization of a new antimicrobial producing strain.

Modulare Polyketidsynthasen (PKS) sind Multienzymsysteme des bakteriellen Sekundärstoffwechsels. An ihnen läuft eine schrittweise Biosynthese vielfältiger Kohlenstoff-Gerüste ab, die von einfachen Carbonsäure-Einheiten ausgeht. Polyketid-Verbindungen zeigen eine große Bandbreite pharmazeutisch interessanter Aktivitäten. In dieser Arbeit wurde eine Reihe von Evolutionsstudien durchgeführt. Zunächst wurden die phylogenetischen Beziehungen zwischen modularen PKS und anderen PKS-Systemen sowie Fettsäuresynthasen untersucht, wodurch ihre zentrale Stellung innerhalb eines langen Evolutionsprozesses gezeigt werden konnte. Eine detaillierte Analyse der Phylogenien von Domänen bakterieller modularer PKS ergab, dass das Ausmaß an Genduplikationen, Genverlusten und Ereignissen horizontalen Gentransfers zwischen den verschiedenen Bakteriengruppen beträchtlich variiert. Aus der Genomsequenz des Actinobakteriums Streptomyces avermitilis wurden die Phylogenien aller Domänentypen rekonstruiert. Der Vergleich dieser Einzelphylogenien ermöglichte es, eine Reihe von homologen Rekombinationsereignissen aufzufinden. Homologe Rekombination scheint der Hauptmechanismus zu sein, auf dem die Strukturvielfalt der Polyketide in Bakterien beruht. Mit Hilfe eines „genome mining“-Ansatzes konnte im Genom des Cyanobakteriums Nostoc punctiforme eine Reihe von Biosynthese-Clustern, die zu den PKS und nichtribosomalen Peptidsynthetasen gehören, identifiziert werden. Durch chromatographische und massenspektrometrische Analysen von Zellextrakten und Kulturüberständen konnten einige der Biosynthese-Cluster bestimmten Metaboliten zugeordnet werden. Eines der Cluster wurde hinsichtlich des produzierten Metaboliten und der Regulationsstruktur eingehender charakterisiert. Die Folgerungen aus den gewonnen Ergebnissen werden im allgemeinen Zusammenhang der Evolution metabolischer Diversität ausführlich diskutiert. / Modular polyketide synthases (PKS) are multienzym systems of bacterial secondary metabolism. They perform a stepwise biosynthesis of diverse carbon skeletons from simple carboxylic acid units. Polyketide compounds possess a wide range of pharmaceutically interesting activities. In this study, a series of evolutionary analyses was performed. Initially, the phylogenetic relationships between modular PKS and other PKS systems as well as fatty acid synthases were investigated revealing their central position within a long evolutionary process. In detail reconstruction of the phylogenies of bacterial modular PKS domains demonstrated that the extent of gene duplications, gene losses and horizontal gene transfer events varies considerably between different bacterial groups. Using the genome sequence of the actinobacterium Streptomyces avermitilis the phylogenies of all domain types were reconstructed. Comparison of these phylogenies allowed for detecting numerous events of homologous recombination, which appears to be the main mechanism underlying polyketide structural diversity in bacteria. A genome mining approach revealed a number of biosynthesis clusters of the PKS and nonribosomal peptide synthetase type in the genome of the cyanobacterium Nostoc punctiforme. Cell extracts and culture supernatants were analysed by means of liquid chromatography and mass spectrometry and some of the biosynthesis clusters could be assigned to specific metabolites. One of the clusters was characterised in greater detail regarding the produced metabolite and the cluster’s regulatory structure. The implications of the results are extensively discussed within the general context of the evolution of metabolic diversity.

Les antibiotiques sont des molécules incontournables dans le traitement des infections bactériennes. L’émergence et la dissémination de la résistance aux antibiotiques chez la pathogène opportuniste Pseudomonas aeruginosa, ont amené l’Organisation Mondiale de la Santé à déclarer indispensable le développement de nouvelles approches thérapeutiques pour lutter contre cette bactérie. Bien que certaines alternatives aient été envisagées, la préservation de l’activité d’antibiotiques majeurs tels que les aminosides et la colistine est primordiale. La caractérisation des mécanismes de résistance à ces médicaments est nécessaire pour la mise au point de nouvelles molécules et mieux prendre en charge les patients. Dans ce contexte, nous montrons que des mutations dans le gène fusA1 (codant le facteur d’élongation EF-G1A) et dans l’opéron pmrAB (système à deux composants PmrAB) entrainent une augmentation de la résistance aux aminosides chez des mutants isolés au laboratoire et des souches issues de patients, atteints ou non, de mucoviscidose. Certaines substitutions d’acide aminé dans EF-G1A accroissent les niveaux de résistance de 2 à 16 fois aux quatre sous-classes d’aminosides. Par ailleurs, des changements d’acide aminé dans le système à deux composants PmrAB activent l’expression des gènes PA4773-PA4774-PA4775, et la production de norspermidine et de spermidine. La synthèse de ces polyamines va de pair avec une baisse de 4 à 16 fois de la sensibilité aux aminosides à noyan 2-désoxystreptamine bisubstitué en 4,6 (gentamicine, amikacine et tobramycine). De plus, il apparaît que la résistance des mutants pmrB à la colistine est en partie dépendante de la pompe d’efflux MexXY(OprM), un système impliqué dans la résistance naturelle, adaptative ou acquise aux aminosides. Enfin, nous montrons que les mutants pmrB surproduisent des alcaloïdes contenant un motif azétidine, par une voie de synthèse non-ribosomale et dépendante du quorum sensing. Ces alcaloïdes diminuent la virulence de P. aeruginosa dans le modèle Galleria mellonella. / Antibiotics are invaluable drugs to combat bacterial infections. Emergence and spread of antibiotic resistance in the opportunistic pathogen Pseudomonas aeruginosa have led the World Health Organization to consider as a crucial priority the development of new therapeutic approaches to fight this bacterium. In addition to other alternatives, preservation of activity of major antibiotics such as aminoglycosides and colistin is primordial. Consequently, characterization of the resistance mechanisms to these drugs is a prerequisite to design novel molecules, and improve patient care. In this context, we show that mutations in gene fusA1 (encoding elongation factor EF-G1A) and in operon pmrAB (two-component system PmrAB) lead to an increased resistance to aminoglycosides in in vitro-selected mutants and strains isolated from cystic fibrosis (CF) and non-CF patients. Certain amino acid substitutions in EF-G1A confer a 2- to 16-fold increased resistance to the four aminoglycoside subclasses. On the other hand, amino acid variations in two-component system PmrAB activate the expression of genes PA4773-PA4774-PA4775, and production of norspermidine and spermidine. This upregulated polyamine biosynthesis is associated with a 4- to 16-fold decreased susceptibility to 4,6-di-substituted deoxystreptamine aminoglycosides (gentamicin, amikacin and tobramycin). Moreover, our work reveals that the acquired resistance of pmrB mutants to colistin partially depends upon pump MexXY(OprM), a system that otherwise mediates intrinsic, adaptive and acquired resistance to aminoglycosides. Finally, we show that pmrB mutants overproduce azetidine-containing alkaloids by a quorum-sensing-regulated, nonribosomal peptide synthetase pathway. These alkaloids impair the virulence of P. aeruginosa in a Galleria mellonella infection model.