Cellulonodin-2 and Lihuanodin: Lasso Peptides with an Aspartimide Post-Translational Modification. Author Li Cao, Moshe Beiser, Joseph Koos, Margarita Orlova, Hader Elashal, Hendrik Schröder, A James Link Publication Year 2021 Type Journal Article Abstract Lasso peptides are a family of ribosomally synthesized and post-translationally modified peptides (RiPPs) defined by their threaded structure. Besides the class-defining isopeptide bond, other post-translational modifications (PTMs) that further tailor lasso peptides have been previously reported. Using genome mining tools, we identified a subset of lasso peptide biosynthetic gene clusters (BGCs) that are colocalized with genes encoding protein l-isoaspartyl methyltransferase (PIMT) homologues. PIMTs have an important role in protein repair, restoring isoaspartate residues formed from asparagine deamidation to aspartate. Here we report a new function for PIMT enzymes in the post-translational modification of lasso peptides. The PIMTs associated with lasso peptide BGCs first methylate an l-aspartate side chain found within the ring of the lasso peptide. The methyl ester is then converted into a stable aspartimide moiety, endowing the lasso peptide ring with rigidity relative to its unmodified counterpart. We describe the heterologous expression and structural characterization of two examples of aspartimide-modified lasso peptides from thermophilic Gram-positive bacteria. The lasso peptide cellulonodin-2 is encoded in the genome of actinobacterium , while lihuanodin is encoded in the genome of firmicute . Additional genome mining revealed PIMT-containing lasso peptide BGCs in 48 organisms. In addition to heterologous expression, we have reconstituted PIMT-mediated aspartimide formation in vitro, showing that lasso peptide-associated PIMTs transfer methyl groups very rapidly as compared to canonical PIMTs. Furthermore, in stark contrast to other characterized lasso peptide PTMs, the methyltransferase functions only on lassoed substrates. Keywords Protein Processing, Post-Translational, Peptides, Aspartic Acid, Bacillales, Protein D-Aspartate-L-Isoaspartate Methyltransferase, Thermobifida Journal J Am Chem Soc Volume 143 Issue 30 Pages 11690-11702 Date Published 2021 Aug 04 ISSN Number 1520-5126 DOI 10.1021/jacs.1c05017 Alternate Journal J Am Chem Soc PMCID PMC9206484 PMID 34283601 PubMedPubMed CentralGoogle ScholarBibTeXEndNote X3 XML