Bioinformatics-Guided Expansion and Discovery of Graspetides

Abstract

Graspetides are a class of ribosomally synthesized and post-translationally modified peptide natural products featuring ATP-grasp ligase-dependent formation of macrolactones/macrolactams. These modifications arise from serine, threonine, or lysine donor residues linked to aspartate or glutamate acceptor residues. Characterized graspetides include serine protease inhibitors such as the microviridins and plesiocin. Here, we report an update to Rapid ORF Description and Evaluation Online (RODEO) for the automated detection of graspetides, which identified 3,923 high-confidence graspetide biosynthetic gene clusters. Sequence and co-occurrence analyses doubled the number of graspetide groups from 12 to 24, defined based on core consensus sequence and putative secondary modification. Bioinformatic analyses of the ATP-grasp ligase superfamily suggest that extant graspetide synthetases diverged once from an ancestral ATP-grasp ligase and later evolved to introduce a variety of ring connectivities. Furthermore, we characterized thatisin and iso-thatisin, two graspetides related by conformational stereoisomerism from Lysobacter antibioticus. Derived from a newly identified graspetide group, thatisin and iso-thatisin feature two interlocking macrolactones with identical ring connectivity, as determined by a combination of tandem mass spectrometry (MS/MS), methanolytic, and mutational analyses. NMR spectroscopy of thatisin revealed a cis conformation for a key proline residue, while molecular dynamics simulations, solvent-accessible surface area calculations, and partial methanolytic analysis coupled with MS/MS support a trans conformation for iso-thatisin at the same position. Overall, this work provides a comprehensive overview of the graspetide landscape, and the improved RODEO algorithm will accelerate future graspetide discoveries by enabling open-access analysis of existing and emerging genomes.

Mechanism of Action of Ribosomally Synthesized and Post-Translationally Modified Peptides

“…Initially, graspetides were largely identified in various genera of freshwater Cyanobacteria, including the bloom-forming Microcystis and Planktothrix. More recent genome mining efforts have identified graspetides in phyla outside of Cyanobacteria, including numerous terrestrial species. − In general, the graspetides are known serine protease inhibitors (Table ).…”

One‐Pot Chemoenzymatic Synthesis of Microviridin Analogs Containing Functional Tags

“…To fill these knowledge gaps, effective tools are urgently needed to monitor the intracellular and extracellular distribution of microviridins and to track the protein targets of microviridin ligands. In addition, 23 further groups of graspetides have recently been discovered or predicted, all of which still await functional characterization [6d] . As an initial step, we planned to develop microviridin chemical probes with functional groups to utilize in biological assays.…”

A scalable platform to discover antimicrobials of ribosomal origin

“…Graspetides are defined by the ATP-grasp ligase-dependent formation of macrolactone and macrolactam linkages between the side chains of donor Ser, Thr, or Lys residues and acceptor Asp or Glu residues 2 . Recent bioinformatic studies identified ~4000 graspetide BGCs, split into 24 groups based on sequence similarity of the ATP-grasp enzymes and the presence of conserved motifs within the predicted precursor peptides 14 , 48 , 49 . Eleven graspetide BGCs were refactored in this study and expressed under the four standard conditions.…”

“…In the graspetides class of RiPPs, only nine of the 24 bioinformatically predicted groups of graspetides have characterized members 14 . Using the FAST-RiPPs pipeline, graspetides were obtained from groups 8 (Gra-7A and Gra-7B), group 19 (Gra-8), and an unnumbered group (Gra-4), expanding the number of characterized groups to 11.…”