(663e) Genome Mining Unveils Class of Ribosomal Peptides with Two Amino Termini

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a family of natural products growing rapidly in the genomics era. However, genome mining of first-in-class RiPPs remains an unsolved challenge. The few successful examples of bioinformatically discovered, first-in-class RiPPs exclusively focused on profiling homologs of known RiPP biosynthetic enzymes. Here we report a new RiPP class defined by an unusual (2S)-2-(dimethylamino)propylamine (Dap)-modified C-terminus, which we term the daptides. Biosynthetic gene clusters (BGCs) associated with daptide production were bioinformatically identified through a class-independent approach using analysis of the locally encoded RiPP precursor peptide with a common substrate-binding domain called the RiPP Recognition Element (RRE). The validity of our predicted BGCs were confirmed using synthetic biology techniques for heterologous expression of a representative BGC from Microbacterium paraoxydans DSM 15019. We then employed mass spectrometry, NMR, and synthesis of authentic standards to determine the structural modifications to the peptide C-terminus. Derived from a C-terminal threonine residue, the class-defining Dap residue is installed over three steps by an oxidative decarboxylase, aminotransferase, and methyltransferase. The genes encoding these enzymes, along with two genes encoding RRE domains, are found in nearly 500 daptide BGCs. In-depth characterization of a domain of unknown function-RRE fusion protein that is essential in the oxidative decarboxylation step reveals its function as a scaffold protein that directs the reaction in a leader peptide-dependent manner. This discovery of a class-defining post-translational modification in RiPPs may serve as a prototype for unveiling additional first-in-class RiPPs through genome mining.