Several classes of ribosomally synthesized and post-translationally modified peptides (RiPPs) are composed of multiple macrocycles. The enzymes that assemble these macrocycles must surmount the challenge of installing a single specific set of linkages out of dozens of distinct possibilities. One class of RiPPs that includes multiple macrocycles are the graspetides, named after the ATP-grasp enzymes that install ester or amide linkages between pairs of nucleophilic and electrophilic side chains. Here, using heterologous expression and NMR spectroscopy, we characterize the connectivity and structure of amycolimiditide, a 29 aa graspetide with a stem-loop structure. The stem includes four esters and extends over 20 Å. The loop of amycolimiditide is distinguished by the presence of an aspartimide moiety, installed by a dedicated O-methyltransferase enzyme. We further characterize the biosynthesis of amycolimiditide in vitro, showing that the amycolimiditide ATP-grasp enzyme AmdB operates in a strict vectorial manner, installing esters starting at the loop and proceeding down the stem. Surprisingly, the O-methyltransferase AmdM that aspartimidylates amycolimiditide prefers a substrate with all four esters installed, despite the fact that the most distal ester is ∼30 Å away from the site of aspartimidylation. This study provides insights into the structure and diversity of aspartimidylated graspetides and also provides fresh insights into how RiPP biosynthetic enzymes engage their peptide substrates.