Chemoenzymatische Synthese von Bacitracin-Derivaten und Untersuchungen zur Optimierung der in vitro Zyklisierung des Surfactins

Nichtribosomale Peptidsynthetasen (NRPS) sind modular aufgebaute Multienzymkomplexe, die in der Natur für die Synthese einer Vielzahl strukturell diverser Peptide mit bedeutenden pharmakologischen Wirkungsprofilen verantwortlich sind. Ein wichtiges Merkmal vieler dieser Peptide stellen zyklische Str...

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Bibliographic Details
Main Author: Wagner, Björn
Contributors: Marahiel, A. Mohamed (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Language:German
Published: Philipps-Universität Marburg 2006
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Nonribosomal peptide synthetases (NRPS) are large multienzyme complexes, which are responsible for the synthesis of structural diverse peptide products that feature distinguished pharmacological profiles. One important structural aspect of many nonribosomal peptides is the introduction of heterocyclic elements into the peptide by the attack of an amino acid side-chain nucleophile onto the peptide backbone. To constrain the peptide into its bioactive conformation, additional macrocyclization can be observed, which is catalyzed in the last step of the biosynthesis by cyclase activity. This work presents a method to selectively alter the heterocyclic metal binding subunit of Bacitracin A by the synthesis of heterocyclic building blocks that have been successfully coupled to the linear decapeptide and subsequently cyclized by the excised bacitracin cyclase. Utilization of this cyclase allowed the first generation of branched cyclic bacitracin derivatives containing thiazole, oxazole, and Methyloxazole moieties. The generated bacitracin derivatives showed bactericidal activity against a representative gram-positive indicator strain, indicating the possibility to alter the biological important heterocyclic subunit and to overcome existing limitations for the application of bacitracin. Additional investigations of this work concern the activation of the surfactin cyclase (Srf-TE) in organic solvents or ionic liquids. By this solvent engineering approach it was demonstrated that the catalytic activity of Srf-TE in DMF provides a quantitative method for the in vitro cyclization of predominantly hydrophobic surfactin-substrates and eliminates side-product formation caused by hydrolysis. This chemoenzymatic cyclization in DMF was shown for the first time to be a selective method for the cyclization of authentic surfactin carrying a long chain C14-fatty acid. The surfactin cyclase inactive mutant (P26G) was restored to full cyclization activity in DMF. This finding led to the identification of an oxyanion-hole consensus motive (P-x), conserved in NRPS cyclases. Mutational studies involving the consensus-motive of the nucleophilic elbow revealed, that substitution of a single amino acid residue (Ala81) had tremendous impact on reaction catalysis. Introduction of an additional hydrogen bond donor (Ser81) into this position of the primary sequence provided evidence of the molecular recognition of a -amino-nucleophile and was applied for the production of a cyclic surfactin macrolactam (surfactam).