Biochemische und strukturelle Untersuchungen der Biosynthese unnatürlicher Aminosäuren als Bausteine nicht-ribosomaler Peptide

Structural diversity of non-ribosomally synthesized peptides is achieved by non-proteinogenic building blocks. These are often essential for the bioactivity of the natural product, but the biosynthesis of numerous building blocks is not understood so far. The presented work deals with three chemically demanding modification reactions of free amino acids that are catalyzed by so called tailoring enzymes. The unusual, modified amino acids are used as building blocks for non-ribosomal peptide biosynthesis. The first part of the work presents the in vitro investigation of the biosynthesis pathway of Nδ hydroxylated and Nδ-formylated ornithine residues. These building blocks are needed for the biosynthesis of the tris-hydroxamate siderophore coelichelin since they are responsible for Fe(III) coordination. The flavin-dependent monooxygenase CchB and the formyltransferase CchA were recombinantly produced and biochemically characterized whereas CchA showed no activity in vitro. Contrary, CchB hydroxylates exclusively free L ornithine and therefore catalyzes the first step in the biosynthesis of coelichelin. The second part of the work deals with a stereospecific Cβ-hydroxylation reaction of L arginine that is catalyzed by the non-heme Fe(II)- and α-ketoglutarate-dependent monooxygenase VioC from Streptomyces vinaceus. The resulting building block erythro-β-hydroxy-arginine is modified again and then incorporated into the peptide scaffold of viomycin. Interestingly, a large number of non-heme Fe(II)-/α-ketoglutarate-dependent monooxygenases catalyze the formation of threo diastereomers. The biochemical and structural characterization of the monooxygenase VioC was conducted in this work and the unexpected substrate tolerance and unusual erythro stereospecificity could be explained. In the third part of the work another Cβ-functionalization of a free substrate is examined. The non-proteinogenic precursor (2S,3S)-β-methyl-phenylalanine is incorporated into the glycopeptide-antibiotic mannopeptimycin. The activity of the S-adenosylmethionine-dependent methyltransferase MppJ was tested in vitro to investigate the biosynthetic pathway of β-methyl-phenylalanine in Streptomyces hygroscopicus. Unexpectedly, the α-keto acid phenylpyruvate is methylated by MppJ instead of phenylalanine. A following transamination reaction catalyzed by the pyridoxal-5’-phosphate-dependent aminotransferase IlvE from the primary metabolism of Streptomyces coelicolor yielded the two diastereomers (2S,3S)- and (2S,3R)-β-methyl-phenylalanine. It could not be shown whether the methylation reaction is stereospecific or not but new findings concerning the Cβ-functionalization of phenylalanine respectively phenylpyruvate were gained during this work.