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Multi modular enzymes generate a variety of secondary metabolites with interesting pharmacological properties. For fermentative, semi-synthetic or chemo-enzymatic synthesis of these natural substances and their optimized derivatives, a detailed knowledge of their biosynthesis or of the enzymes involved is needed. In this work important aspects of the biosynthesis of the iterative non ribosomal products gramicidin S, a potent antibiotic, and thiocoraline, an anti-tumor agent, were clarified. Iterative non ribosomal products are characterized by repetitive units connected to cyclic structures and the incorporation of non-proteinogenic building blocks. Both the resulting rigid structure and the properties of the non-proteinogenic components are often crucial for the biological activity of these compounds.
In the first part of the work, the key step of an iterative non ribosomal peptide synthetase, the thioesterase catalyzed ligation of repetitive units and the subsequent cyclization, was investigated. On this, the recombinant gramicidin S thioesterase from Bacillus brevis was heterologously expressed in E. coli and characterized with a chemo-enzymatic approach. Using the native pentapeptidyl-thioester substrate, the thioesterase catalyzed the dimerization and subsequent cyclization of the decapeptide leading to gramicidin S. Interestingly, the detection of a linear decapeptidyl-N-acetylcysteamine-thioester as an enzyme dependent intermediate supports the iterative mechanism in vivo, in which the decapeptide is firstly bound to the peptidyl-carrier-protein and afterwards transferred to the adjacent thioesterase to be cyclized (backward mechanism). Furthermore, it was shown that the thioesterase can handle different substrates length, leading not only to dimerization, but also to trimerization and the formation of different ring sizes. The biocombinatorial potential of the thioesterase was tested by the implementation of substrate mixtures, which led to cycles composed of different substrates. Additionally, the ligation potential was investigated, which yield a heptadecapeptide as longest product.
In the second part of the work a key step of 3-hydroxy quinaldic acid biosynthesis was investigated. This aromatic acid is crucial for the DNA bisintercalative properties of the anti-tumor drug thiocoraline. By heterologous expression of the proteins TioI, TioG, TioF, TioK and the peptidyl-carrier-protein of TioK together with the in vitro conversion with possible substrates, key steps in the biosynthesis were clarified. The results showed that, contrary to the postulated biosynthesis, TioI catalyzes the β-hydroxylation of peptidyl-carrier-protein bound tryptophan and that after the release of the β-hydroxytryptophan from the synthetase TioK further biosynthetic steps were performed.