Intentional Aspartimidylation in Ribosomally Synthesized and Post-translationally Modified Peptides

Abstract

Aspartimides are well-known as undesired side products in solid-phase peptide synthesis and pharmaceutical formulations. In this dissertation, we discovered natural products, specifically ribosomally synthesized and post-translationally modified peptides (RiPPs), in which aspartimide is intentionally installed. Aspartimidylation, a novel posttranslational modification (PTM) in RiPP biosynthesis, was first demonstrated in the discovery of two lasso peptides, cellulonodin-2 and lihuanodin. The aspartimide in these peptides was installed by a dedicated O-methyltransferase, a protein L-isoaspartyl methyltransferase (PIMT) homolog. We confirmed the existence of the aspartimide moiety by solving the solution nuclear magnetic resonance (NMR) structure of lihuanodin and by analyzing the hydrolysis and hydrazinolysis products of both peptides. The two methyltransferases that install the aspartimide in cellulonodin-2 and lihuanodin, TceM and LihM respectively, are exceptionally fast methyl transfer catalysts compared to canonical PIMTs. Moreover, both TceM and LihM function on only lassoed substrates in stark contrast to other lasso peptide PTMs. The aspartimides in cellulonodin-2 and lihuanodin have two peculiarities: the recalcitrance of the aspartimide to hydrolysis and the regioselectivity of hydrolysis to only Asp when they do hydrolyze. We measured rates of methyl transfer, aspartimide formation, and aspartimide hydrolysis in the lihuanodin biosynthesis, and the relative magnitudes of these rates explained the accumulation and stability of the aspartimidylated lihuanodin. Additionally, we demonstrated that the residue C-terminal to the aspartimide controlled the regioselectivity of hydrolysis and thus the threadedness of the peptide. Leveraging the fact that PIMT homologs are also associated with other RiPPs families such as lanthipeptide and graspetide, we discovered a novel family of RiPPs using these PIMT homologs as bioinformatic seeds. Moreover, we provided experimental evidence by demonstrating the heterologous production of the first example of this novel RiPP family. The findings in this dissertation illustrate that the PIMT-mediated aspartimide formation is a widely adapted backbone modification strategy in RiPP biosynthesis. Future studies will be directed towards solving crystal structures of RiPP-associated PIMT homologs, discovering other aspartimide-containing RiPPs, and deciphering the physiological roles or bioactivities of these peptides.