Biomolecular NMR studies of the structure and biosynthesis of RiPP natural products

Natural products have been a rich source of new chemical matter with clinical applications. These compounds are broadly defined as small molecules generated by organisms from multiple kingdoms including plants, fungi, and prokaryotes. The outsized impact natural products have had on modern medicine has inspired ongoing efforts to find and characterize new natural products. The vast increase in sequenced genomes has allowed natural product discovery to move from a blind screening approach to a bioinformatics guided approach. Screening of extracts for bioactivity is not only laborious, but also leads to the rediscovery of known compounds. With a genome-mining approach, bioinformatic analysis of natural product gene clusters empowers researchers to identify producer strains and novel products. This eliminates the rediscovery problem and reduces the throughput necessary for novel natural product isolation. Within the large family of natural products, the Ribosomally-produced and Posttranslationally modified Peptides (RiPPs) are a particularly intriguing class. These compounds begin as a genetically encoded precursor, which is translated into a short (~50) residue peptide. This precursor peptide is then modified by genetically co-located enzymes to generate a mature natural product. The diverse, enzymatically-derived modifications to RiPP precursors include both sidechain and backbone modifications and generate a large and chemically diverse class of natural products. RiPPs are a potentially fruitful source of new chemical matter for clinical application for several reasons. First, the fact that RiPP precursors are genetically encoded means that there is a direct link between genetic sequence and chemical structure. Because of this, bioinformatic analysis allows relatively easy prediction of final chemical structure, and opens the possibility for analog generation through relatively simple genetic modifications. Secondly, recent advances in genome sequencing have greatly increased the known space of organismal genomes. As far as RiPPs are concerned, this offers plentiful opportunity for genome mining to identify novel RiPP biosynthetic gene clusters with potentially novel bioactivities. In addition to leveraging bioinformatics to discover novel RiPPs, this class of natural product has an increasingly diverse range of bioactivities. The wealth of information regarding known mechanisms of action of RiPPs serves as further justification for the discovery and characterization of novel RiPPs. In Chapter 1, I discuss the current state of understanding of the mechanisms of action of several classes of RiPP. This introductory chapter is an excerpt from a comprehensive review of the mechanisms of action of all known RiPPs. In Chapter 2, we report the bioinformatics-guided discovery and biosynthetic reconstitution of the lasso peptide fusilassin. This biosynthetic gene cluster was shown to possess exceptional plasticity, and was used to generate a number of non-native lasso peptides, including lasso peptides from separate pathways. The enzymes in this pathway also exhibited the greatest in vitro stability of known lasso peptides and served as a platform for a number of additional efforts aimed at characterizing interactions of biosynthetic enzymes and generating lasso peptide analog libraries. In Chapter3, we report the NMR-based characterization of protein-protein interactions governing the biosynthesis of the lasso peptide fusilassin. We accomplished the full backbone assignment of the FusE RiPP recognition element and utilized chemical shift perturbations to characterize the precursor peptide-FusE interactions. Solvent paramagnetic relaxation enhancements were used to characterize the interactions between FusE and the leader peptidase FusB. In Chapter 4, we described the discovery of two novel thioether-containing RiPPs discovered through genome mining. The sactipeptide huazacin was characterized by HRMS/MS and NMR spectroscopy and was shown to have bioactivity against Clostridia sp. Utilizing sequence homology and HRMS/MS, the natural product freyrasin was hypothesized to contain unprecedented alpha-to-beta carbon thioether linkages. This novel structure was confirmed by the complete assignment of freyrasin and TOCSY and NOESY-based experimental characterization of this novel post-translational modification. Based on the NMR structure elucidation, this natural product served as the first fully characterized member of a novel class of RiPPs termed the ranthipeptides. In Chapter 5 we utilize reactivity-based screening to characterize the post-translational modification of the citrulassin lasso peptides which contain a non-proteinogenic citrulline residue. Comparative genomic analyses and complementation experiments identified the arginine deiminase responsible for the installation of this functional groups. Solution NMR analysis generated a 3D solution NMR ensemble structure for this peptide. In Chapter 6 we report the discovery of two novel thiopeptide RiPPs. Genome-mining identified a minimal thiopeptide biosynthetic gene cluster with two predicted precursor peptides. These compounds were produced synthetically and chemoenzymatically and fully characterized by HRMS/MS and NMR. The structure elucidation of these compounds led to the reclassification of the thiopeptides as pyritides. In Chapter 7 genome mining approaches were used to identify and isolate a novel graspetide termed thatisin A. Results from HPLC purifications demonstrated a thermallydependent atropisomerization. Temperature-controlled NMR structure elucidation accomplished the structure elucidation of thatisin A and demonstrated the presence of a cis-proline residue located within a constrained macrocycle. Computational and MS-based methanolysis experiments further confirmed our proline isomerization conformational exchange hypothesis