Rationale Strategien zur Isolierung bakterieller Lassopeptide - Struktur, Biosynthese und Anwendungspotential

Bakterielle Lassopeptide sind ribosomal synthetisierte, bioaktive Peptide, die sich aus 16 bis 21 proteinogenen Aminosäuren zusammensetzen. Sie zeichnen sich durch eine verzweigt-zyklische Primärstruktur aus, die durch einen N-terminalen Makrolaktamring und einen linearen C-Terminus charakterisiert...

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Bibliographic Details
Main Author: Knappe, Thomas
Contributors: Marahiel, Mohamed A. (Prof. Dr.) (Thesis advisor)
Format: Dissertation
Published: Philipps-Universität Marburg 2011
Online Access:PDF Full Text
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Table of Contents: Bacterial lasso peptides are ribosomally assembled, bioactive peptides consisting of 16 - 21 proteinogenic amino acids. They share a branched cyclic primary structure characterized by an N-terminal macrolactam ring and a linear exocyclic C-terminus, which is threaded through the macrocycle and trapped either by steric hindrance of bulky side chains or covalently via disulfide bonds. The resulting lasso structure shows a remarkable stability towards proteolytic degradation, high temperatures and chemical denaturants and accounts in combination with the genetic encoding and the bacterial origin for the increasing interest in these unique structured peptides. In this study a genome mining based approach for the identification of novel lasso peptide biosynthetic gene clusters in bacteria was developed. Using this rational approach a cryptic gene cluster was identified in the genome of Burkholderia thailandensis E264 and a compound matching the predicted mass of the postulated lasso peptide could be detected in culture supernatants. Structural studies applying mass spectrometry and NMR spectroscopy confirmed the lasso structure of the isolated compound, which was named capistruin. The 19-aa lasso peptide showed antibacterial activity against members of the pathogenic Burkholderia cepacia complex and could be produced heterologously in Escherichia coli upon expression of the entire biosynthetic gene cluster. Subsequent mutational analysis of the ribosomal precursor protein led to the identification of four positions within the lasso sequence being critical for the maturation of the peptide. Inside the leader peptide a threonine residue at the P2 position of the protease cleavage site was found to be essential for the processing of the precursor. Stability studies of selected capistruin variants gave insights into structure stability relationships and identified Arg15 as the residue which is solely responsible for the trapping of the C-terminus within the 9-residue macrolactam ring. In addition, the capistruin R15A/F16A variant was the first temperature sensitive lasso peptide which could be thermally unfolded into a branched-cyclic structure in vitro. Furthermore, the branched-cyclic peptides BI-32169 and anantin were analyzed by mass spectrometry and NMR spectroscopy. The postulated lasso structure, which was assumed based on similarities of their primary structures with known lasso peptides, could be proven for both peptides. As the glucagon receptor antagonist BI-32169 differs from lasso peptides of class I and II, it can be considered as the first representative of the novel class III. The relaxed substrate specificity of the biosynthetic machinery of lasso peptides suggested their application as stable molecular scaffolds for the presentation of pharmacophors in order to combine their intrinsic stability with the function of bioactive epitopes. The conversion of the lasso peptide microcin J25 into a nanomolar inhibitor of avb3 and avb5 integrins with antiangiogenic activity by grafting of the integrin binding motif RGD onto the stable peptide framework demonstrates the so far unexplored pharmaceutical potential of lasso structured peptides. The results of this study provide significant insights into the structure, stability and biosynthesis of bacterial lasso peptides, which will improve genome mining for lasso structured peptides in the future and therefore contribute to the identification of novel, alternative peptide scaffolds for rational drug design.