Investigation of RiPPs originating from two-domain precursors Jethro Hemmann 2 and Gerald Lackner 1 1 Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute, Germany, 2 Leibniz Institute for Natural Product Research and Infection Biology, HKI, Germany Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a structurally diverse class of secondary metabolites with a wide range of bioactivities. They all share a ribosomal origin, as the peptide sequence is encoded in a so-called precursor gene. The precursor peptide usually consists of a leader sequence followed by a core sequence. Different enzymes recognize the leader and install post-translational modifications on the core peptide, which, after proteolytic cleavage, is released as the mature natural product. Often, precursor peptides are short and lack clear structural features. As an exception, the family of the “nitrile hydratase leader peptides” (NHLP) are characterized by unusually long leader sequences that show similarity to the enzyme nitrile hydratase [1]. In certain species of the order Burkholderiales, these NHLP precursors also appear as tandem genes, i.e., two copies of the precursor are present in a row. Intriguingly, in a few strains, these genes are fused into a single two-domain precursor, resulting in a ~270 amino acid precursor protein. The function of the two leader domains as well as the nature of the resulting RiPP(s) is currently unclear. Here, we investigated these so-far uncharacterized RiPP clusters in order to identify the produced metabolite(s), characterize the biosynthetic enzymes, and explore the role of the two leader domains in the precursors. Following a bottom-up approach, we heterologously expressed the biosynthetic gene cluster containing the fused two-domain precursor in Escherichia coli and analyzed the modifications in the precursor peptide using LC-MS. The activity of the cyclodehydratase and methyltransferase present in the gene cluster could be successfully reconstituted in E. coli . The resulting modifications were localized at the C -terminus of the precursor and a dehydrated cysteine and a methylated threonine were observed. Size-exclusion chromatography unexpectedly revealed that the precursor assembles into a stable tetramer of 120 kDa. Such multimeric assemblies are surprising for a RiPP precursor and we are currently investigating the role of these structures. We further aim to characterize the last enzyme in the gene cluster—a large kinase/cyclase protein—and to isolate the RiPP from the native producer. References 1. Haft et al., BMC Biology 8, 70 (2010).
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