Large-scale Bioinformatic Study of Graspimiditides and Structural Characterization of Albusimiditide

Brian Choi, Arthur Acuña, Joseph D. Koos, A. James Link

Research output: Contribution to journalArticlepeer-review

Abstract

Graspetides are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) that exhibit an impressive diversity in patterns of side chain-to-side chain ω-ester or ω-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 5000 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 ω-ester linkages generating a stem-loop structure with one aspartimide. The ester cross-links form 22-, 46-, 22-, and 44-atom macrocycles, the 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 comprehensive insight into the diversity and structural features of graspimiditides, paving the way for future investigations of this unique class of natural products.

Original languageEnglish (US)
Pages (from-to)2394-2404
Number of pages11
JournalACS chemical biology
Volume18
Issue number11
DOIs
StatePublished - Nov 17 2023

All Science Journal Classification (ASJC) codes

  • Molecular Medicine
  • Biochemistry

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