Fuscimiditide: a RiPP with Ω-Ester and Aspartimide Post-translational Modifications

Abstract

AbstractMicroviridins and other ω−ester linked peptides (OEPs) are characterized by sidechain-sidechain linkages installed by ATP-grasp enzymes. Here we describe the discovery of a new family of OEPs, the gene clusters of which also encode an O-methyltransferase with homology to the protein repair catalyst protein L-isoaspartyl methyltransferase (PIMT). We produced the first example of this new ribosomally synthesized and post-translationally modified peptide (RiPP), fuscimiditide, via heterologous expression. NMR analysis of fuscimiditide revealed that the peptide contains two ester crosslinks forming a stem-loop macrocycle. Furthermore, an unusually stable aspartimide moiety is found within the loop macrocycle. We have also fully reconstituted fuscimiditide biosynthesis in vitro establishing that ester formation catalyzed by the ATP-grasp enzyme is an obligate, rate-limiting first biosynthetic step. Aspartimide formation from aspartate is catalyzed by the PIMT homolog in the second step. The aspartimide moiety embedded in fuscimiditide hydrolyzes regioselectively to isoaspartate (isoAsp). Surprisingly, this isoAsp-containing protein is also a substrate for the PIMT homolog, thus driving any hydrolysis products back to the aspartimide form. Whereas aspartimide is often considered a nuisance product in protein formulations, our data here suggest that some RiPPs have aspartimide residues intentionally installed via enzymatic activity.Abstract Figure