Graspetides represent a class of ribosomally synthesized and post-translationally modified peptide (RiPP) that can contain macrolactone/macrolactam linkages from amino acid side chains. Many predicted graspetide biosynthetic gene clusters (BGCs) contain untapped tailoring enzymes, including some with the potential to modify macrocyclic peptide scaffolds. In this work, we investigated several of these BGCs and discovered the first examples of partner protein-dependent graspetide biosynthesis and the installation of an unprecedented cyclized 5-hydroxyisopeptide moiety. We first updated the bioinformatic tool RODEO to robustly identify diverse graspetides with additional tailoring enzymes. Using this algorithm to survey available genomic data, a data set of >20,000 predicted graspetides was generated and a large-scale bioinformatic analysis was performed on proteins in or near graspetide BGCs. From this analysis, two groups of graspetides with strictly conserved co-occurring proteins were prioritized for characterization. These graspetides contained novel ring connectivities and their biosynthesis was dependent on co-occurring partner proteins, a feature unprecedented among characterized graspetides. The first graspetide, rosaritide, features three interlocking macrolactone linkages. The activity and stability of the rosaritide graspetide synthetase was dependent on a partner protein which copurifies to form a catalytically active complex. The second graspetide, corallotide, is unusually large and contains five repeated motifs in which a Lys is first macrocyclized into an isopeptide bond and then hydroxylated at the δ-carbon by a divergent 2OG-Fe(II)-dependent oxygenase. The biosynthesis and biosynthetic enzymes from these BGCs were then characterized in vitro. Overall, this study expands our understanding of graspetide biosynthesis and the ability to predict graspetide BGCs.
Carter et al. (Sat,) studied this question.