Biosynthesis and characterization of fuscimiditide, an aspartimidylated graspetide. Author Hader Elashal, Joseph Koos, Wai Cheung-Lee, Brian Choi, Li Cao, Michelle Richardson, Heather White, A James Link Publication Year 2022 Type Journal Article Abstract Microviridins and other ω-ester-linked peptides, collectively known as graspetides, are characterized by side-chain-side-chain linkages installed by ATP-grasp enzymes. Here we report the discovery of a family of graspetides, the gene clusters of which also encode an O-methyltransferase with homology to the protein repair catalyst protein L-isoaspartyl methyltransferase. Using heterologous expression, we produced fuscimiditide, a ribosomally synthesized and post-translationally modified peptide (RiPP). NMR analysis of fuscimiditide revealed that the peptide contains two ester cross-links forming a stem-loop macrocycle. Furthermore, an unusually stable aspartimide moiety is found within the loop macrocycle. We fully reconstituted fuscimiditide biosynthesis in vitro including formation of the ester and aspartimide moieties. The aspartimide moiety embedded in fuscimiditide hydrolyses regioselectively to isoaspartate. Surprisingly, this isoaspartate-containing peptide is also a substrate for the L-isoaspartyl methyltransferase homologue, thus driving any hydrolysis products back to the aspartimide form. Whereas an aspartimide is often considered a nuisance product in protein formulations, our data suggest that some RiPPs have aspartimide residues intentionally installed via enzymatic activity. Keywords Amino Acid Sequence, Peptides, Methyltransferases, Protein D-Aspartate-L-Isoaspartate Methyltransferase, Isoaspartic Acid, Esters Journal Nat Chem Volume 14 Issue 11 Pages 1325-1334 Date Published 2022 Nov ISSN Number 1755-4349 DOI 10.1038/s41557-022-01022-y Alternate Journal Nat Chem PMCID PMC10078976 PMID 35982233 PubMedPubMed CentralGoogle ScholarBibTeXEndNote X3 XML