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.

Microviridins 和其他 ω-酯连接的肽，统称为gracesetides，其特征在于由 ATP-grasp 酶安装的侧链-侧链连接。在这里，我们报告了一个抓握肽家族的发现，该家族的基因簇还编码与蛋白质修复催化剂蛋白l-异天冬氨酰甲基转移酶同源的O-甲基转移酶。使用异源表达，我们生产了夫西米肽，一种核糖体合成的翻译后修饰肽（RiPP）。夫西米肽的 NMR 分析表明，该肽含有两个酯交联，形成茎环大环。此外，在环大环内发现了异常稳定的天冬酰亚胺部分。我们在体外完全重建了夫西米肽生物合成，包括酯和天冬酰亚胺部分的形成。嵌入夫西米肽中的天冬酰亚胺部分区域选择性地水解为异天冬氨酸。令人惊讶的是，这种含有异天冬氨酸的肽也是L-异天冬氨酰甲基转移酶同系物的底物，从而驱动任何水解产物回到天冬酰亚胺形式。虽然天冬酰亚胺通常被认为是蛋白质配方中的有害产品，但我们的数据表明，一些 RiPP 具有通过酶活性有意安装的天冬酰亚胺残基。