A Versatile Enzymatic Pathway for Modification of Peptide C-Termini

Advances in bioinformatics have enabled the discovery of unique enzymatic reactions, particularly for ribosomally synthesized and post-translationally modified peptides (RiPPs). The recently discovered daptides, peptides with their C-terminus replaced by an amine, represent one such case, but the diversity, requirements, and engineering potential of daptide biosynthesis remain to be established. Using the daptide biosynthetic gene clusters from <i>Thermobifida fusca</i> and <i>Streptomyces azureus</i>, we reconstituted daptide biosynthesis <i>in vitro</i>, revealing the enzymatic requirements for successive oxidative decarboxylation, transamination, and <i>N</i>,<i>N</i>-dimethylation. <i>In vitro</i> and <i>in vivo</i> studies showed a tailoring family of YcaO enzymes convert a secondary amine intermediate to a C-terminal imidazoline. We further demonstrated enzymatic activity toward shortened, leader peptide-free, and non-native core peptides, highlighting a broad substrate tolerance. Using these insights, we directed the daptide pathway to install new C-termini, including a bioconjugation-compatible aminoacetone, on various peptide and protein substrates.