Journal Article

Peptide and Protein Cyclization by a Promiscuous Graspetide Synthetase

Johnson, Toby G., Link, A. James, Elashal, Hader E., Choi, Brian, Acuña, Arthur

2025

Overview

Macrocyclic peptides have drawn considerable interest as modalities for drug discovery. Graspetides are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) that harbor one or more macrocycles. These macrocycles are built via side chain-side chain linkages that are installed by ATP-grasp enzymes, giving the peptide family their name. We recently reported on the discovery, structure, and biosynthesis of the graspetide pre-fuscimiditide which is comprised of a stem covalently linked by two ester moieties and a 10 aa loop. Here we probe the substrate tolerance of the fuscimiditide ATP-grasp enzyme, ThfB, and show that it is highly promiscuous. ThfB can cyclize substrates with substitutions to or extensions of the stem region as well as generate multivalent cyclic structures. ThfB also shows remarkable tolerance to substitutions in the loop of pre-fuscimiditide. Loops comprised of flexible glycine-serine sequences ranging from 4 aa to 72 aa were efficiently cyclized by ThfB. Even substrates in which full-length proteins were swapped for the 10 aa loop of pre-fuscimiditide could be cyclized by ThfB. We also show that ThfB can covalently cross-link supramolecularly assembled protein chains. These data show that ThfB is a highly generalizable biocatalyst for both peptide and protein macrocyclization as well for intermolecular protein cross-linking.

• Publisher: American Chemical Society