Our group seeks to identify new molecular structures formed by unusual enzymatic transformations. We focus on the ribosomally synthesized and post-translationally modified peptides (RiPPs), of which nearly 50 distinct structural classes exist. Decades of research show that while RiPPs harbor diverse structures and functions, the biosynthetic routes share a blueprint. Typical RiPP precursor peptides contain fewer than 60 amino acids. The modification enzymes engage the N-terminal portion, while the C-terminal region receives all post-trans- lational modifications (PTMs). Genome mining for RiPP biosynthetic gene clusters (BGCs) had been ex- tremely time-consuming and often unsuccessful owing to the difficulty of locating the requisite substrate pep- tide(s). The short length and hypervariability of RiPP precursor peptides frequently preclude their detection by automated gene finders. RODEO, an AI-based genome mining tool, has largely solved this problem for a subset of RiPP classes. The current proposal unites RODEO-enabled genomics analysis, enzyme chemistry, structural biology, and microbial physiology to characterize several “RiPP-like” BGCs that do not conform to the current definition of a RiPP natural product. With the breadth and depth of RiPP genomics coming into sharper focus, it has become increasingly clear that many “RiPP-like” BGCs lack a canonical precursor peptide. Rare PTMs such as backbone thioamidation and thioether crosslinks between cysteine and the side chains of other amino acids are known on larger protein substrates, such as methyl-coenzyme M reductase, ribosomal protein uL16, and quinohemoprotein amine dehydrogenase. These examples establish limited but critical precedent, and we herein predict that many more “RiPP-like PTMs” occur on protein substrates. Comparative analysis in prokaryotes supports this prediction, as a substantial level of sequence and genome neighborhood similarity exists between pathways encoding a canonical precursor peptide versus a larger protein substrate. This renewal project tackles several questions of outstanding interest regarding the interplay and utility of RiPP-like PTMs on non-canonical substrates. The specific aims are independently achievable and robustly combine in silico, in vitro, and in vivo methods. In one aspect, we will elucidate the biochemical function of ribosome thioamidation and the extent to which this PTM is propagated among other prokaryotes. Additional thioamidated proteins and their physiological roles will be discovered during this project. Further, we will pur- sue biosynthetic pathways predicted to produce poly-thioether-stabilized protein nanotubes and poly-isopep- tide-containing branched copolymers. The enzymes performing this usual PTM chemistry will be thoroughly evaluated, and the biological roles of the protein ultrastructures will be determined. Our preliminary data, rich environment, and strong investigative team place us in an ideal position to address these a...