Publikationsserver der Universitätsbibliothek Marburg

Titel:Studien zur Biosynthese von Sactipeptiden: Charakterisierung der an der Thioetherbrückenbildung beteiligten Radical SAM Enzyme AlbA und SkfB
Autor:Flühe, Leif
Weitere Beteiligte: Marahiel, Mohamed A. (Prof. Dr.)
Veröffentlicht:2014
URI:https://archiv.ub.uni-marburg.de/diss/z2014/0072
URN: urn:nbn:de:hebis:04-z2014-00725
DOI: https://doi.org/10.17192/z2014.0072
DDC: Chemie
Titel(trans.):Biosynthesis of Sactipeptides: Characterization of the thioether bond forming Radical SAM enzymes AlbA und SkfB
Publikationsdatum:2014-08-07
Lizenz:https://rightsstatements.org/vocab/InC-NC/1.0/

Dokument

Schlagwörter:
Thioether, Radical based Reaction Mechanism, Peptide, Peptide Biosynthesis, Reaktionsmechanismus, Lanthipeptide, ribosomale Peptide, Sactipeptide, Naturstoff, Thioether, Radical SAM Enzyme, Biosynthese, Radical SAM Enzyme, Charakterisierung neuer Enzymklasse, Sactipeptides, Enzym

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.

Summary:
Sactipeptides are a new class of ribosomal assembled and posttranslational modified, mostly bioactive peptides, which are assembled of 26 to 35 proteinogenic amino acids. Their character-istic feature is a thioether bond linking the sulfur atom of a cysteine residue with the α-carbon of an acceptor amino acid. Until now the amino acids phenylalanine, threonine, methionine, aspar-agine, serine, alanine and tyrosine were identified as acceptors. All known sactipeptides pos-sess of an inflexible helical fold. Partial a cyclized peptide backbone is found. The sporulation killing factor (SKF) contains an additional disulfide bridge as posttranslational modification be-sides the sactipeptide like thioether linkage. In this thesis the biosynthesis of the thioether bond formation in the sactipeptides subtilosin A and SKF was investigated. It was shown that enzymes AlbA (subtilosin A) and SkfB (SKF) cata-lyze the generation of the thioether linkages. These proteins were identified as radical SAM en-zymes, as they were only able to catalyze the cleavage of S-adenosylmethionine into methio-nine and the 5’-deoxyadenosylradical under reductive condition and after the enzymes were fully loaded with iron- and sulfide-ions. Furthermore it was shown, that both enzymes contains two [4Fe-4S]-Clusters. Therefore the iron- and sulfur-content of the proteins and protein variants were measured. As well the proteins and protein variants were analyzed by several spectro-scopic techniques. The [4Fe-4S]-Cluster, which is coordinated by the characteristic radical SAM enzyme like CXXXCXXC motif is responsible for the cleavage of SAM, whereas the second [4Fe-4S]-Cluster plays a key role during thioether bond formation. By carrying out precursor peptide modification assays with and without the addition of iodoa-cetamide and subsequent HPLC-HRMS analysis, it was shown that AlbA and SkfB are able to catalyze the thioether linkage generation in the respective precursor peptide (SboA, SkfA). By assaying leaderpeptide less SboA and SkfA variants it was elucidated that the AlbA and SkfB catalyzed reactions are the first step during the biosynthesis of both sactipeptides. Precursor peptide modification assays with AlbA and SkfB variants, as well as interaction studies with Al-bA, AlbA variants and SboA showed an interaction between the second [4Fe-4S]-Cluster and the precursor peptide. Additional assays with SkfA variants, which should address issues con-cerning the substrate specificity of SkfB towards the donor and acceptor positions, were carried out. Within these assays it was e.g. shown that the enzyme class is not able to catalyze the formation of an ether linkage. Based on the obtained results a completely new radical based mechanism for thioether bond formation during sactipeptide biosynthesis is proposed and veri-fied.

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