biostudies-literatureChoi BPrinceton UniversityNational Institute of General Medical SciencesNIGMS NIH HHS11605-11609https://www.ebi.ac.uk/biostudies/studies/S-EPMC11064158biostudies-literatureUnknown146(17)The ribosomally synthesized and post-translationally modified peptide (RiPP) superfamily of natural products includes many examples of cyclic peptides with diverse macrocyclization chemistries. The graspetides, one family of macrocyclized RiPPs, harbor side chain-side chain ester or amide linkages. We recently reported the structure and biosynthesis of the graspetide pre-fuscimiditide, a 22-amino-acid (aa) peptide with two ester cross-links forming a stem-loop structure. These cross-links are introduced by a single graspetide synthetase, the ATP-grasp enzyme ThfB. Here we show that ThfB can also catalyze the formation of amide or thioester cross-links in prefuscimiditide, with thioester formation being especially efficient. We further show that upon proteolysis to reveal an N-terminal cystJournal of the American Chemical SocietyCyclic Peptides from Graspetide Biosynthesis and Native Chemical Ligation.PMC11064158R01 GM107036GM107036Choi BLink AJAcuna AfalseCyclic Peptides from Graspetide Biosynthesis and Native Chemical Ligation.The ribosomally synthesized and post-translationally modified peptide (RiPP) superfamily of natural products includes many examples of cyclic peptides with diverse macrocyclization chemistries. The graspetides, one family of macrocyclized RiPPs, harbor side chain-side chain ester or amide linkages. We recently reported the structure and biosynthesis of the graspetide pre-fuscimiditide, a 22-amino-acid (aa) peptide with two ester cross-links forming a stem-loop structure. These cross-links are introduced by a single graspetide synthetase, the ATP-grasp enzyme ThfB. Here we show that ThfB can also catalyze the formation of amide or thioester cross-links in prefuscimiditide, with thioester formation being especially efficient. We further show that upon proteolysis to reveal an N-terminal cyst2024-01-01T00:00:00Z2024 May2026-06-02T20:58:17.866Z2025-06-25T03:06:22.194ZS-EPMC110641583863464710.1021/jacs.4c02745