Mechanismen der C-Domänen-vermittelten Lipoinitiation in der nichtribosomalen Peptidsynthese
Nichtribosomal synthetisierte Lipopeptide aus mikrobiellen Organismen weisen ein breites Spektrum an biologischen Aktivitäten auf, zu denen beispielsweise antibiotische, antitumorale aber auch biotensidische Eigenschaften zählen. Daher sind diese Naturstoffe von pharmazeutischem sowie biotechnologis...
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Table of Contents: Nonribosomally synthesized lipopeptides of microbial origin contain a fatty acyl moiety and exhibit diverse biological activities including antibiotic, anticancer or biosurfactant properties. Thus, these natural products are subject of intense research and present a potent class of new agents for pharmaceutical and biotechnological applications. The biosynthesis of lipopeptides usually starts by N-acylation of the peptide backbone and is referred to as lipoinitiation. This study adresses the exploration and characterization of different mechanisms of lipoinitiation consisting of activation and transfer of the lipid moiety. These reactions were elucidated during the biosynthesis of the lipoheptapeptide surfactin from Bacillus subtilis and of the acidic lipodepsipeptide CDA (calcium-dependent antibiotic) from Streptomyces coelicolor A3(2) displaying two novel strategies for the incorporation of a fatty acid into a peptidic natural product. The involved domains were dissected from their nonribosomal peptide synthetases (NRPSs) by genetic techniques and were heterologously produced as singular proteins. Commercially not available substrates were chemically synthesized. Biochemical studies revealed in both systems that the starter C domains from the initiation modules of the corresponding NRPSs are essential for the transfer of the fatty acid acting as acyltransferases. The catalytic motif HHxxxDG identified in other C domains was shown to be indispensable in this reaction which was investigated by mutational studies pointing towards a common mechanism of C domain catalyzed reactions. The investigated lipoinitiation reactions during surfactin and CDA biosynthesis differred in the mode of fatty acid activation. For surfactin production, enzymes from the primary metabolic pathways activate the fatty acid by coupling it to coenzyme A resulting in a reactive thioester that directly serves as a substrate for the N-acylation of the peptidyl-carrier protein(PCP)-bound amino acid. However, the starter C domain from the CDA synthetase needs an acyl-carrier protein(ACP)-bound fatty acid for the transfer reaction. Both starter C domains exhibit high selectivities for their substrates except for the PCP domain, which could be arbitrarily substituted with other PCP domains. The second part of the thesis aimed at the characterization of a cyclization domain of the bacitracin synthetase BacA-Cy2 which is closely related to C domains. It catalyzes the formation of a thiazoline in the antibiotic bacitracin from Bacillus licheniformis and was dissected from the native synthetase resulting in a singular protein to allow detailed characterization and the determination of substrate specificity.