Table of Contents:
Sactipeptides are a new class of ribosomal assembled and posttranslational modified, mostly bioactive peptides, which are assembled of 26 to 35 proteinogenic amino acids. Their character-istic feature is a thioether bond linking the sulfur atom of a cysteine residue with the α-carbon of an acceptor amino acid. Until now the amino acids phenylalanine, threonine, methionine, aspar-agine, serine, alanine and tyrosine were identified as acceptors. All known sactipeptides pos-sess of an inflexible helical fold. Partial a cyclized peptide backbone is found. The sporulation killing factor (SKF) contains an additional disulfide bridge as posttranslational modification be-sides the sactipeptide like thioether linkage. In this thesis the biosynthesis of the thioether bond formation in the sactipeptides subtilosin A and SKF was investigated. It was shown that enzymes AlbA (subtilosin A) and SkfB (SKF) cata-lyze the generation of the thioether linkages. These proteins were identified as radical SAM en-zymes, as they were only able to catalyze the cleavage of S-adenosylmethionine into methio-nine and the 5’-deoxyadenosylradical under reductive condition and after the enzymes were fully loaded with iron- and sulfide-ions. Furthermore it was shown, that both enzymes contains two [4Fe-4S]-Clusters. Therefore the iron- and sulfur-content of the proteins and protein variants were measured. As well the proteins and protein variants were analyzed by several spectro-scopic techniques. The [4Fe-4S]-Cluster, which is coordinated by the characteristic radical SAM enzyme like CXXXCXXC motif is responsible for the cleavage of SAM, whereas the second [4Fe-4S]-Cluster plays a key role during thioether bond formation. By carrying out precursor peptide modification assays with and without the addition of iodoa-cetamide and subsequent HPLC-HRMS analysis, it was shown that AlbA and SkfB are able to catalyze the thioether linkage generation in the respective precursor peptide (SboA, SkfA). By assaying leaderpeptide less SboA and SkfA variants it was elucidated that the AlbA and SkfB catalyzed reactions are the first step during the biosynthesis of both sactipeptides. Precursor peptide modification assays with AlbA and SkfB variants, as well as interaction studies with Al-bA, AlbA variants and SboA showed an interaction between the second [4Fe-4S]-Cluster and the precursor peptide. Additional assays with SkfA variants, which should address issues con-cerning the substrate specificity of SkfB towards the donor and acceptor positions, were carried out. Within these assays it was e.g. shown that the enzyme class is not able to catalyze the formation of an ether linkage. Based on the obtained results a completely new radical based mechanism for thioether bond formation during sactipeptide biosynthesis is proposed and veri-fied.