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bioRxiv · 10.1101/2023.06.21.545981

A large-scale bioinformatic study of graspimiditides and structural characterization of albusimiditide

Choi, B.·Acuna, A.·Koos, J. D.·Link, A. J.
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Abstract

Graspetides are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) that exhibits an impressive diversity in patterns of side chain-to-side chain {omega}-ester or {omega}-amide linkages. Recent studies have uncovered a significant portion of graspetides to contain an additional post-translational modification involving aspartimidylation catalyzed by an O-methyltransferase, predominantly found in the genomes of Actinomycetota. Here, we present a comprehensive bioinformatic analysis focused on graspetides harboring aspartimide for which we propose the name graspimiditides. From Protein BLAST results of 5,000 methyltransferase sequences, we identified 962 unique putative graspimiditides, which we further classified into eight main clusters based on sequence similarity along with several smaller clusters and singletons. The previously studied graspimiditides, fuscimiditide and amycolimiditide, are identified in this analysis; fuscimiditide is a singleton while amycolimiditide is in the fifth largest cluster. Cluster 1, by far the largest cluster, contains 641 members, encoded almost exclusively in the Streptomyces genus. To characterize an example of a graspimiditide in Cluster 1, we conducted experimental studies on the peptide from Streptomyces albus J1074, which we named albusimiditide. By tandem mass spectrometry, hydrazinolysis, and amino acid substitution experiments, we elucidated the structure of albusimiditide to be a large tetracyclic peptide with four {omega}-ester linkages generating a stem-loop structure with one aspartimide. The ester crosslinks form 22-, 46-, 22-, and 44-atom macrocycles, last of which, the loop, contains the enzymatically installed aspartimide. Further in vitro experiments revealed that the aspartimide hydrolyzes in a 3:1 ratio of isoaspartate to aspartate residues. Overall, this study offers a comprehensive insight into the diversity and structural features of graspimiditides, paving the way for future investigations of this unique class of natural product. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/545981v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@bd68f2org.highwire.dtl.DTLVardef@1350705org.highwire.dtl.DTLVardef@5ff593org.highwire.dtl.DTLVardef@1f3c4a9_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Choi, B., Acuna, A., Koos, J. D., Link, A. J.. 2023-06-22. A large-scale bioinformatic study of graspimiditides and structural characterization of albusimiditide. https://doi.org/10.1101/2023.06.21.545981

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