Dokument

Zusammenfassung:
Sactipeptide sind eine neue Klasse ribosomal synthetisierter, meist bioaktiver Peptide, die aus 26 bis 35 proteinogenen Aminosäuren bestehen. Ihr charakteristisches Merkmal ist eine Thio-etherbindung, die das Schwefelatom einer Cysteingruppe mit dem α-Kohlenstoffatom einer Ak-zeptoraminosäure verknüpft. Als Akzeptoren wurden bis zum jetzigen Zeitpunkt die Aminosäu-ren Phenylalanin, Threonin, Methionin, Asparagin, Serin, Alanin und Tyrosin identifiziert. Alle bekannten Sactipeptide verfügen über eine relativ starre helikale Struktur. Teilweise wird auch ein zyklisches Peptidrückgrat gefunden. Der Sporulation Killing Factor (SKF) verfügt neben der Thioetherbrücke über eine Disulfidbindung zwischen zwei Cysteinen. Im Rahmen dieser Arbeit wurde die Biosynthese der Thioetherbildung in den Sactipeptide Subti-losin A und SKF untersucht. Es wurde gezeigt, dass die Bildung der Thioetherbrücken von den Enzymen AlbA (Subtilosin A) und SkfB (SKF) katalysiert wird. Diese beiden Proteine wurden als Radical SAM Enzyme identifiziert, da sie nur in der mit Eisen und Schwefel beladenen Form und nur unter reduktiven Bedingungen in der Lage sind S-Adenosylmethionin (SAM) in Methio-nin und das 5’-Deoxyadenosylradikal zu spalten. Des Weiteren wurde durch die Bestimmung des Eisengehaltes von-Varianten der beiden Enzyme und auch durch spektroskopische Unter-suchungen dieser Proteinvarianten gezeigt, dass AlbA wie auch SkfB zwei [4Fe-4S]-Cluster binden. Der über ein für Radical SAM Enzyme charakteristisches CXXXCXXC-Motiv gebundene [4Fe-4S]-Cluster ist für die Spaltung von SAM verantwortlich wohin gegen der andere [4Fe-4S]-Cluster eine wichtige Rolle in der Synthese des Thioethers spielt. Durch Vorläuferpeptidmodifikationsassays mit und ohne Zugabe von Iodacetamid, die anschlie-ßend per HPLC-HRMS analysiert wurden, wurde gezeigt, dass die Enzyme jeweils für die Syn-these der Thioetherbrücken in dem dazugehörigen Vorläuferpeptid (SboA, SkfA) verantwortlich sind. Durch Leaderpeptidlose SboA und SkfA-Varianten wurde gezeigt, dass die Reaktion den ersten Schritt in der jeweiligen Biosynthese bildet. Durch AlbA und SkfB-Varianten wie auch Interaktionsstudien mit AlbA, AlbA-Varianten und SboA wurde eine Wechselwirkung des zwei-ten gebundenen [4Fe-4S]-Clusters und dem Vorläuferpeptid nachgewiesen. Des Weiteren wur-den SkfA-Varianten hergestellt die Fragen zur Substratspezifität im Bezug auf Donor- und Ak-zeptorposition in der SkfB katalysierten Reaktion beantworten sollten. Dabei wurde u.a. gezeigt, dass die Enzymklasse nicht in der Lage ist die Synthese von Etherbindungen zu katalysieren. Aufbauend auf den dabei erhaltenen Ergebnissen wurde ein vollständig neuer, über radikale Zwischenstufen verlaufender Mechanismus für die Synthese der Thioetherbindungen vorge-schlagen und verifziert.

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