Streptogramin B-Antibiotika und azide Lipopeptide: Chemoenzymatische Derivatisierung der Makrozyklen und Biosynthese nichtproteinogener Bausteine

Nonribosomal peptides are characterized by a high structural diversity that results from the presence of various nonproteinogenic amino acids in their peptide backbones. In many cases, these building blocks are important or even essential for the bioactivity of the natural products. Furthermore, the bioactivity is ensured by the conformational rigidity of the peptide backbone that is guaranteed by its macrocyclic structure. The macrocyclization of the peptide is catalyzed during the last step of nonribosomal peptide synthesis by so called thioesterase (TE)-domains. The here presented work addresses the development of a chemoenzymatic strategy for the synthesis and the modification of Streptogramin (SB)-antibiotics as well as the elucidation and characterization of the biosynthesis of the nonproteinogenic 3-methyl-glutamate (3-MeGlu) building block of the acidic lipopeptides. The first part of this work describes a TE-based chemoenzymatic approach for the rapid and convenient SB-synthesis in which diversity is generated by standard solid phase protocols and stereoselective enzymatic cyclization. For this approach, the recombinant thioesterase domain SnbDE TE of the pristinamycin I nonribosomal peptide synthetase from Streptomyces pristinaespiralis was cloned, overproduced, and biochemically characterized. This cyclase catalyses the macrolactonization of linear peptide thioester analogues of pristinamycin I with high regio- and stereoselectivity. The stereoselective cyclization out of complex in situ racemizing substrate mixtures enables an elegant SB-synthesis via a dynamic kinetic resolution assay. A remarkable substrate tolerance was detected for the enzymatic cyclization including all seven positions of the peptide backbone. An N-methylated peptide bond between positions 4 and 5 is mandatory for a high substrate turnover. The second part of this work is dedicated to in vitro reconstitution of the biosynthetic strategy of 3-methyl-glutamate (3-MeGlu), which is a building block important for the bioactivity of the acidic lipopeptides CDA, daptomycin and A54145. The investigations show that GlmT from Streptomyces coelicolor, DptI from S. roseosporus, and LptI from S. fradiae are S-adenosyl-methionine (SAM)-dependent α-ketoglutarate (αKG) methyltransferases catalyzing the stereospecific methylation of αKG resulting in the formation of (3R)-3-methyl-2-oxoglutarate. Furthermore, the branched chain amino acid transaminase IlvE (SCO5523) from S. coelicolor was characterized as an efficient catalyst for the following transamination leading to (2S,3R)-3-MeGlu. This stereospecific two-step conversion of αKG to (2S,3R)-3-MeGlu completes our understanding of the biosynthesis and incorporation of β-methylated amino acids into nonribosomal peptides and expanses our knowledge of the mechanisms generating structural diversity in natural products.