Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Biosynthesis and characterization of fuscimiditide, an aspartimidylated graspetide

Nature Chemistry volume 14, pages 1325–1334 (2022)Cite this article

Subjects

Abstract

Microviridins and other ω-ester-linked peptides, collectively known as graspetides, are characterized by side-chain–side-chain linkages installed by ATP-grasp enzymes. Here we report the discovery of a family of graspetides, the gene clusters of which also encode an O-methyltransferase with homology to the protein repair catalyst protein l-isoaspartyl methyltransferase. Using heterologous expression, we produced fuscimiditide, a ribosomally synthesized and post-translationally modified peptide (RiPP). NMR analysis of fuscimiditide revealed that the peptide contains two ester cross-links forming a stem–loop macrocycle. Furthermore, an unusually stable aspartimide moiety is found within the loop macrocycle. We fully reconstituted fuscimiditide biosynthesis in vitro including formation of the ester and aspartimide moieties. The aspartimide moiety embedded in fuscimiditide hydrolyses regioselectively to isoaspartate. Surprisingly, this isoaspartate-containing peptide is also a substrate for the l-isoaspartyl methyltransferase homologue, thus driving any hydrolysis products back to the aspartimide form. Whereas an aspartimide is often considered a nuisance product in protein formulations, our data suggest that some RiPPs have aspartimide residues intentionally installed via enzymatic activity.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Get just this article for as long as you need it

$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Gene clusters and precursors for graspetides.
Fig. 2: PTMs on ThfA.
Fig. 3: NMR structures of pre-fuscimiditide and fuscimiditide.
Fig. 4: Characterization of the major fuscimiditide hydrolysis product, iso pre-fuscimiditide.
Fig. 5: Analysis of ThfB and ThfM activity in vitro.

Data availability

Structure data for fuscimiditide and pre-fuscimiditide has been deposited in the Protein Data Bank (PDB) with accession codes 7LIF and 7LI2, respectively. NMR data has been deposited to the Biological Magnetic Resonance Bank (BMRB); the accession numbers are 30851 for fuscimiditide and 30849 for pre-fuscimiditide. Mass spectra are shown within the article and its Supporting Information; raw mass spectrometry data underlying figures will be provided upon request due to the large file sizes for this data. All other data are present in the main text or the Supplementary Information. Source data are provided with this paper.

References

  1. Arnison, P. G. et al. Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature. Nat. Prod. Rep. 30, 108–160 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Tan, N. H. & Zhou, J. Plant cyclopeptides. Chem. Rev. 106, 840–895 (2006).

    Article  CAS  PubMed  Google Scholar 

  3. Gunasekera, S., Daly, N. L., Anderson, M. A. & Craik, D. J. Chemical synthesis and biosynthesis of the cyclotide family of circular proteins. IUBMB Life 58, 515–524 (2006).

    Article  CAS  PubMed  Google Scholar 

  4. Cascales, L. & Craik, D. J. Naturally occurring circular proteins: distribution, biosynthesis and evolution. Org. Biomol. Chem. 8, 5035–5047 (2010).

    Article  CAS  PubMed  Google Scholar 

  5. Lee, J., Mcintosh, J., Hathaway, B. J. & Schmidt, E. W. Using marine natural products to discover a protease that catalyzes peptide macrocyclization of diverse substrates. J. Am. Chem. Soc. 131, 2122–2124 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Hegemann, J. D., Zimmermann, M., Xie, X. & Marahiel, M. A. Lasso peptides: an intriguing class of bacterial natural products. Acc. Chem. Res. 48, 1909–1919 (2015).

    Article  CAS  PubMed  Google Scholar 

  7. Maksimov, M. O. & Link, A. J. Prospecting genomes for lasso peptides. J. Ind. Microbiol. Biotechnol. 41, 333–344 (2014).

    Article  CAS  PubMed  Google Scholar 

  8. Cheung-Lee, W. L. & Link, A. J. Genome mining for lasso peptides: past, present, and future. J. Ind. Microbiol. Biotechnol. 46, 1371–1379 (2019).

    Article  CAS  PubMed  Google Scholar 

  9. Kawulka, K. et al. Structure of subtilosin A, an antimicrobial peptide from Bacillus subtilis with unusual posttranslational modifications linking cysteine sulfurs to α-carbons of phenylalanine and threonine. J. Am. Chem. Soc. 125, 4726–4727 (2003).

    Article  CAS  PubMed  Google Scholar 

  10. Kawulka, K. E. et al. Structure of subtilosin A, a cyclic antimicrobial peptide from Bacillus subtilis with unusual sulfur to α-carbon cross-links: formation and reduction of α-thio-α-amino acid derivatives. Biochemistry 43, 3385–3395 (2004).

    Article  CAS  PubMed  Google Scholar 

  11. Brown, L. C. W., Acker, M. G., Clardy, J., Walsh, C. T. & Fischbach, M. A. Thirteen posttranslational modifications convert a 14-residue peptide into the antibiotic thiocillin. Proc. Natl Acad. Sci. USA 106, 2549–2553 (2009).

    Article  Google Scholar 

  12. Kelly, W. L., Pan, L. & Li, C. Thiostrepton biosynthesis: prototype for a new family of bacteriocins. J. Am. Chem. Soc. 131, 4327–4334 (2009).

    Article  CAS  PubMed  Google Scholar 

  13. Bagley, M. C., Dale, J. W., Merritt, E. A. & Xiong, X. Thiopeptide antibiotics. Chem. Rev. 105, 685–714 (2005).

    Article  CAS  PubMed  Google Scholar 

  14. Willey, J. M. & van der Donk, W. A. Lantibiotics: peptides of diverse structure and function. Annu. Rev. Microbiol. 61, 477–501 (2007).

    Article  CAS  PubMed  Google Scholar 

  15. Knerr, P. J. & van der Donk, W. A. Discovery, biosynthesis, and engineering of lantipeptides. Annu. Rev. Microbiol. 81, 479–505 (2012).

    CAS  Google Scholar 

  16. Bierbaum, G. & Sahl, H. Lantibiotics: mode of action, biosynthesis and bioengineering. Curr. Pharm. Biotechnol. 10, 2–18 (2009).

    Article  CAS  PubMed  Google Scholar 

  17. Schramma, K. R., Bushin, L. B. & Seyedsayamdost, M. R. Structure and biosynthesis of macrocyclic peptide containing an unprecedented lysine-to-tryptophan crosslink. Nat. Chem. 7, 431–437 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Okino, T., Matsuda, H., Murakami, M. & Yamaguchi, K. New microviridins, elastase inhibitors from the blue-green alga Microcystis aeruginosa. Tetrahedron 51, 10679–10686 (1995).

    Article  CAS  Google Scholar 

  19. Li, K., Condurso, H. L., Li, G., Ding, Y. & Bruner, S. D. Structural basis for precursor protein-directed ribosomal peptide macrocyclization. Nat. Chem. Biol. 12, 973–979 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  20. Liao, R. et al. Thiopeptide biosynthesis featuring ribosomally synthesized precursor peptides and conserved posttranslational modifications. Chem. Biol. 16, 141–147 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Walsh, C. T., Malcolmson, S. J. & Young, T. S. Three ring posttranslational circuses: insertion of oxazoles, thiazoles, and pyridines into protein-derived frameworks. ACS Chem. Biol. 7, 429–442 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Ireland, C. & Scheuer, P. J. Ulicyclamide and ulithiacyclamide, two new small peptides from a marine tunicate. J. Am. Chem. Soc. 102, 5688 (1980).

    Article  CAS  Google Scholar 

  23. Morris, R. P. et al. Ribosomally synthesized thiopeptide antibiotics targeting elongation factor Tu. J. Am. Chem. Soc. 131, 5946–5955 (2009).

    Article  CAS  PubMed  Google Scholar 

  24. Freeman, M. F. et al. Metagenome mining reveals polytheonamides as posttranslationally modified ribosomal peptides. Science 338, 387–391 (2012).

    Article  CAS  PubMed  Google Scholar 

  25. Maksimov, M. O., Pan, S. J. & Link, A. J. Lasso peptides: structure, function, biosynthesis, and engineering. Nat. Prod. Rep. 29, 996–1006 (2012).

    Article  CAS  PubMed  Google Scholar 

  26. Braffman, N. R. et al. Structural mechanism of transcription inhibition by lasso peptides microcin J25 and capistruin. Proc. Natl Acad. Sci. USA 116, 1273–1278 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Wilson, K. A. et al. Structure of microcin J25, a peptide inhibitor of bacterial RNA polymerase, is a lassoed tail. J. Am. Chem. Soc. 125, 12475–12483 (2003).

    Article  CAS  PubMed  Google Scholar 

  28. Travin, D. Y. et al. Structure of ribosome-bound azole-modified peptide phazolicin rationalizes its species-specific mode of bacterial translation inhibition. Nat. Commun. 10, 4563 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  29. Metelev, M. et al. Klebsazolicin inhibits 70S ribosome by obstructing the peptide exit tunnel. Nat. Chem. Biol. 13, 1129–1136 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Ishitsuka, M. O., Kusumi, T., Kakisawa, H., Kaya, K. & Watanabe, M. M. Microviridin: a novel tricyclic depsipeptide from the toxic cyanobacterium Microcystis viridis. J. Am. Chem. Soc. 112, 8180–8182 (1990).

    Article  CAS  Google Scholar 

  31. Ahmed, M. N. et al. Phylogenomic analysis of the microviridin biosynthetic pathway coupled with targeted chemo-enzymatic synthesis yields potent protease inhibitors. ACS Chem. Biol. 12, 1538–1546 (2017).

    Article  CAS  PubMed  Google Scholar 

  32. Ziemert, N., Ishida, K., Weiz, A., Hertweck, C. & Dittmann, E. Exploiting the natural diversity of microviridin gene clusters for discovery of novel tricyclic depsipeptides. Appl. Environ. Microbiol. 76, 3568–3574 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Ziemert, N., Ishida, K., Liaimer, A., Hertweck, C. & Dittmann, E. Ribosomal synthesis of tricyclic depsipeptides in bloom-forming cyanobacteria. Angew. Chem. Int. Ed. 47, 7756–7759 (2008).

    Article  CAS  Google Scholar 

  34. Philmus, B., Christiansen, G., Yoshida, W. Y. & Hemscheidt, T. K. Post-translational modification in microviridin biosynthesis. ChemBioChem 9, 3066–3073 (2008).

    Article  CAS  PubMed  Google Scholar 

  35. Philmus, B., Guerrette, J. P. & Hemscheidt, T. K. Substrate specificity and scope of MvdD, a GRASP-like ligase from the microviridin biosynthetic gene cluster. ACS Chem. Biol. 4, 429–434 (2009).

    Article  CAS  PubMed  Google Scholar 

  36. Lee, H., Park, Y. & Kim, S. Enzymatic cross-linking of side chains generates a modified peptide with four hairpin-like bicyclic repeats. Biochemistry 56, 4927–4930 (2017).

    Article  CAS  PubMed  Google Scholar 

  37. Lee, C., Lee, H., Park, J. U. & Kim, S. Introduction of bifunctionality into the multidomain architecture of the ω-ester-containing peptide plesiocin. Biochemistry 59, 285–289 (2020).

    Article  CAS  PubMed  Google Scholar 

  38. Roh, H., Han, Y., Lee, H. & Kim, S. A topologically distinct modified peptide with multiple bicyclic core motifs expands the diversity of microviridin-like peptides. ChemBioChem 20, 1051–1059 (2019).

    Article  CAS  PubMed  Google Scholar 

  39. Lee, H., Choi, M., Park, J. U., Roh, H. & Kim, S. Genome mining reveals high topological diversity of ω-ester-containing peptides and divergent evolution of ATP-grasp macrocyclases. J. Am. Chem. Soc. 142, 3013–3023 (2020).

    Article  CAS  PubMed  Google Scholar 

  40. Ramesh, S. et al. Bioinformatics-guided expansion and discovery of graspetides. ACS Chem. Biol. 16, 2787–2797 (2021).

    Article  CAS  PubMed  Google Scholar 

  41. Kaweewan, I., Nakagawa, H. & Kodani, S. Heterologous expression of a cryptic gene cluster from Marinomonas fungiae affords a novel tricyclic peptide marinomonasin. Appl. Microbiol. Biotechnol. 105, 7241–7250 (2021).

    Article  CAS  PubMed  Google Scholar 

  42. Unno, K. & Kodani, S. Heterologous expression of cryptic biosynthetic gene cluster from Streptomyces prunicolor yields novel bicyclic peptide prunipeptin. Microbiol. Res. 244, 126669 (2021).

    Article  CAS  PubMed  Google Scholar 

  43. Montalbán-López, M. et al. New developments in RiPP discovery, enzymology and engineering. Nat. Prod. Rep. 38, 130–239 (2021).

    Article  PubMed  Google Scholar 

  44. Murray, E. D. & Clarke, S. Metabolism of a synthetic l-isoaspartyl-containing hexapeptide in erythrocyte extracts: enzymatic methyl esterification is followed by nonenzymatic succinimide formation. J. Biol. Chem. 261, 306–312 (1986).

    Article  CAS  PubMed  Google Scholar 

  45. McFadden, P. N. & Clarke, S. Conversion of isoaspartyl peptides to normal peptides: implications for the cellular repair of damaged proteins. Proc. Natl Acad. Sci. USA 84, 2595–2599 (1987).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Ingrosso, D. et al. Increased methyl esterification of altered aspartyl residues erythrocyte membrane proteins in response to oxidative stress. Eur. J. Biochem. 267, 4397–4405 (2000).

    Article  CAS  PubMed  Google Scholar 

  47. Young, A. L., Carter, W. G., Doyle, H. A., Mamula, M. J. & Aswad, D. W. Structural integrity of histone H2B in vivo requires the activity of protein l-isoaspartate O-methyltransferase, a putative protein repair enzyme. J. Biol. Chem. 276, 37161–37165 (2001).

    Article  CAS  PubMed  Google Scholar 

  48. Koos, J. D. & Link, A. J. Heterologous and in vitro reconstitution of fuscanodin, a lasso peptide from Thermobifida fusca. J. Am. Chem. Soc. 141, 928–935 (2019).

    Article  CAS  PubMed  Google Scholar 

  49. Cao, L. et al. Cellulonodin-2 and lihuanodin: lasso peptides with an aspartimide post-translational modification. J. Am. Chem. Soc. 143, 11690–11702 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Iyer, L. M., Abhiman, S., Burroughs, A. M. & Aravind, L. Amidoligases with ATP-grasp, glutamine synthetase-like and acetyltransferase-like domains: synthesis of novel metabolites and peptide modifications of proteins. Mol. Biosyst. 5, 1636–1660 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Vaisar, T. & Urban, J. Probing proline effect in CID of protonated peptides. J. Mass Spectrom. 31, 1185–1187 (1996).

    Article  CAS  PubMed  Google Scholar 

  52. Someya, C. I., Inoue, S., Irran, E., Krackl, S. & Enthaler, S. New binding modes of 1-acetyl- and 1-benzoyl-5-hydroxypyrazolines—synthesis and characterization of O,O′-pyrazoline– and N,O-pyrazoline–zinc complexes. Eur. J. Inorg. Chem. 2011, 2691–2697 (2011).

    Article  Google Scholar 

  53. Klaene, J. J., Ni, W., Alfaro, J. F. & Zhou, Z. S. Detection and quantitation of succinimide in intact protein via hydrazine trapping and chemical derivatization. J. Pharm. Sci. 103, 3033–3042 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Geiger, T. & Clarke, S. Deamidation, isomerization, and racemization at asparaginyl and aspartyl residues in peptides. J. Biol. Chem. 262, 785–794 (1987).

    Article  CAS  PubMed  Google Scholar 

  55. Ulrich, E. L. et al. BioMagResBank. Nucleic Acids Res. 36, 402–408 (2008).

  56. Grassi, L. et al. Complete NMR assignment of succinimide and its detection and quantification in peptides and intact proteins. Anal. Chem. 89, 11962–11970 (2017).

    Article  CAS  PubMed  Google Scholar 

  57. Cheung, W. L., Pan, S. J. & Link, A. J. Much of the Microcin J25 leader peptide is dispensable. J. Am. Chem. Soc. 25, 2514–2515 (2010).

    Article  Google Scholar 

  58. Xie, L. et al. Lacticin 481: in vitro reconstitution of lantibiotic synthetase activity. Science 303, 679–681 (2004).

    Article  CAS  PubMed  Google Scholar 

  59. Oman, T. J., Knerr, P. J., Bindman, N. A., Vela, J. E. & Van Der Donk, W. A. An engineered lantibiotic synthetase that does not require a leader peptide on its substrate. J. Am. Chem. Soc. 134, 6952–6955 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Koehnke, J. et al. Structural analysis of leader peptide binding enables leader-free cyanobactin processing. Nat. Chem. Biol. 11, 558–563 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Reyna-González, E., Schmid, B., Petras, D., Süssmuth, R. D. & Dittmann, E. Leader peptide-free in vitro reconstitution of microviridin biosynthesis enables design of synthetic protease-targeted libraries. Angew. Chem. Int. Ed. 55, 9398–9401 (2016).

    Article  Google Scholar 

  62. Weiz, A. R. et al. Leader peptide and a membrane protein scaffold guide the biosynthesis of the tricyclic peptide microviridin. Chem. Biol. 18, 1413–1421 (2011).

    Article  CAS  PubMed  Google Scholar 

  63. Pederick, J. L., Thompson, A. P., Bell, S. G. & Bruning, J. B. d-Alanine–d-alanine ligase as a model for the activation of ATP-grasp enzymes by monovalent cations. J. Biol. Chem. 295, 7894–7904 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Ghilarov, D. et al. Architecture of microcin B17 synthetase: an octameric protein complex converting a ribosomally synthesized peptide into a DNA gyrase poison. Mol. Cell 73, 749–762 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Neumann, K., Farnung, J., Baldauf, S. & Bode, J. W. Prevention of aspartimide formation during peptide synthesis using cyanosulfurylides as carboxylic acid-protecting groups. Nat. Commun. 11, 1–10 (2020).

    Article  Google Scholar 

  66. Ren, H. et al. Discovery and characterization of a class IV lanthipeptide with a nonoverlapping ring pattern. ACS Chem. Biol. 15, 1642–1649 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Hegemann, J. D. & Sussmuth, R. D. Matters of class: coming of age of class III and IV lanthipeptides. RSC Chem. Biol. 1, 110–127 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Acedo, J. Z. et al. O-Methyltransferase-mediated incorporation of a β-amino acid in lanthipeptides. J. Am. Chem. Soc. 141, 16790–16801 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank I. Pelczer (Princeton University NMR Facility) for help with acquiring NMR spectra, H. Schroeder for assistance with tandem mass spectrometry and R. Cohen and X. Wang (Merck) for their advice on conducting Marfey’s analysis. This work was supported by National Institutes of Health grant GM107036 and a grant from Princeton University School of Engineering and Applied Sciences (Focused Research Team on Precision Antibiotics). L.C. was supported by an NSF Graduate Research Fellowship Program under grant DGE-1656466. J.D.K. was supported in part by training grant T32 GM7388.

Author information

Authors and Affiliations

  1. Department of Chemical and Biological Engineering, Princeton University, Princeton, NJ, USA

    Hader E. Elashal, Wai Ling Cheung-Lee, Brian Choi, Li Cao, Michelle A. Richardson, Heather L. White & A. James Link

  2. Department of Molecular Biology, Princeton University, Princeton, NJ, USA

    Joseph D. Koos & A. James Link

  3. Department of Chemistry, Princeton University, Princeton, NJ, USA

    A. James Link

Authors
  1. Hader E. Elashal
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Joseph D. Koos
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Wai Ling Cheung-Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Brian Choi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Li Cao
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Michelle A. Richardson
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Heather L. White
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. A. James Link
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

H.E.E., J.D.K. and A.J.L. conceived of the project. H.E.E, J.D.K., W.L.C-L., B.C., L.C., M.A.R. and H.L.W. carried out experiments. H.E.E. and A.J.L wrote the initial draft of the paper, and all authors participated in revision of the paper. A.J.L. acquired funding and supervised the project.

Corresponding author

Correspondence to A. James Link.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Chemistry thanks the anonymous reviewer(s) for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary methods and materials, Figs. 1–40 and Tables 1–9.

Reporting Summary

Source data

Source Data Fig. 3

Excel data underlying graph

Source Data Fig. 4

Excel data underlying graph

Rights and permissions

Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Elashal, H.E., Koos, J.D., Cheung-Lee, W.L. et al. Biosynthesis and characterization of fuscimiditide, an aspartimidylated graspetide. Nat. Chem. 14, 1325–1334 (2022). https://doi.org/10.1038/s41557-022-01022-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41557-022-01022-y

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing