Rationales Enzymdesign zur Optimierung dimodularer Peptidsynthetasen

Table of Contents: Many microorganisms use multimodular nonribosomal peptide synthetases (NRPS) for the production of structurally diverse and pharmacological interesting compounds. Each module within these multifunctional NRPSs represents a functional unit of catalytic domains, which is responsible for recognition, activation and incorporation of the substrate into the growing peptide product. Due to the modular arrangement NRPSs are especially suitable for the construction of artificial enzymes. In this work different strategies of domain and module fusions were used to develop dimodular synthetases for the production of the dipeptide alpha-aspartyl-phenylalanine (Asp-Phe), which is used as a precursor in the production of the high intensity sweetener aspartame. Comparison of six Asp-Phe-Synthetases exposed the influence of artificial fusions on the activity of the hybrid enzymes. Varying turnover rates as well as turnover-dependent formation of the by-product beta-Asp-Phe were observed. The knowledge to design hybrid peptide synthetases is enlarged by this comparison of different fusion strategies. Structural diversity of nonribosomally synthesized peptides is mainly insured by catalysis of modifying domains. The remarkable heterocyclization-(Cy)-domain catalyzes the peptide bond-formation associated with the cyclization of the side chains of cysteine, serine or threonine. This reaction results in the formation of thiazoline and oxazoline rings respectively. Based on the first two modules of the bacitracin synthetase A, a model system was constructed that produces the heterocyclic isoleucinylcystein. Starting from this model system dimodular hybrid synthetases were assembled, which were able to form new heterocyclic products. The activities of these constructs demonstrate the biocombinatorial potential of the Cy-domain. Additionally the reaction sequence of peptide bond-formation and cyclization was clarified by mutation of conserved residues within the Cy-domain of the model system. On the basis of mutated synthetases that catalyzes the peptide bond-formation but not the cyclization, the independence of condensation and heterocyclization was demonstrated.