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Biosynthesis of peptide–nucleobase hybrids in ribosomal peptides

Nature Chemical Biology volume 21, pages 143–154 (2025)Cite this article

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Abstract

The main biopolymers in nature are oligonucleotides and polypeptides. However, naturally occurring peptide–nucleobase hybrids are rare. Here we report the characterization of the founding member of a class of peptide–nucleobase hybrid natural products with a pyrimidone motif from a widely distributed ribosomally synthesized and post-translationally modified (RiPP) biosynthetic pathway. This pathway features two steps where a heteromeric RRE–YcaO–dehydrogenase complex catalyzes the formation of a six-membered pyrimidone ring from an asparagine residue on the precursor peptide, and an acyl esterase selectively recognizes this moiety to cleave the C-terminal follower peptide. Mechanistic studies reveal that the pyrimidone formation occurs in a substrate-assisted catalysis manner, requiring a His residue in the precursor to activate asparagine for heterocyclization. Our study expands the chemotypes of RiPP natural products and the catalytic scope of YcaO enzymes. This discovery opens avenues to create artificial biohybrid molecules that resemble both peptide and nucleobase, a modality of growing interest.

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Fig. 1: YcaO-catalyzed enzymatic reactions and spy BGC.
Fig. 2: MALDI-TOF mass spectral analysis of in vivo reaction catalyzed by SpyB, SpyC and SpyD.
Fig. 3: Structural elucidation of the peptide–nucleobase hybrid product Δ12SpyABCDE.
Fig. 4: In vitro reconstitution and mechanistic studies of pyrimidone modification.
Fig. 5: In vitro assay of protease NmSpyE.
Fig. 6: LC–MS analysis of pyrimidone-containing molecules.

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Data availability

All data supporting the findings of this study are presented in the main text and Supplementary Information file. NCBI (https://www.ncbi.nlm.nih.gov/) accessions and PDB (https://www.rcsb.org/) codes are referenced in the Supplementary Information file, and publicly accessible on the respective websites. Source data are provided with this paper. mzML versions of the LC–MS/MS files used for metabolomics are available from the MassIVE repository with accession MSV000094563 (ftp://massive.ucsd.edu/v07/MSV000094563/). Data is available from the corresponding author upon request.

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Acknowledgements

This work was supported by the National Institutes of Health (GM079038 to S.K.N.) and the Biotechnology and Biological Sciences Research Council via an institute strategic program grant (BB/X01097X/1) and research grant (BB/V016024/1) to A.W.T. and N.M.V. The Bruker UltrafleXtreme MALDI-TOF/TOF mass spectrometer was purchased in part with a grant from the National Institutes of Health (S10RR027109A). We thank J.S. Griffitts of Department of Microbiology and Molecular Biology at Brigham Young University for providing the DNA plasmids for the production of tclE/tclI/tclJ/tclN.

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Authors and Affiliations

  1. Department of Biochemistry, University of Illinois at Urbana–Champaign, Urbana, IL, USA

    Zeng-Fei Pei & Satish K. Nair

  2. Department of Molecular Microbiology, John Innes Centre, Norwich, UK

    Natalia M. Vior & Andrew W. Truman

  3. School of Chemical Sciences NMR Laboratory, University of Illinois at Urbana–Champaign, Urbana, IL, USA

    Lingyang Zhu

  4. Institute for Genomic Biology, University of Illinois at Urbana–Champaign, Urbana, IL, USA

    Satish K. Nair

  5. Center for Biophysics and Quantitative Biology, University of Illinois at Urbana-Champaign, Urbana, IL, USA

    Satish K. Nair

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Contributions

Z.P. and S.K.N. designed the studies. Z.P. performed the experiments, and L.Z. acquired and interpreted the NMR data. N.M.V. and A.W.T. performed the metabolic analysis. All authors analyzed data and assisted in the writing and editorial process. S.K.N. conceived and supervised the project.

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Correspondence to Satish K. Nair.

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Supplementary Methods, Supplementary Figs. 1–40, Supplementary Tables 1–6.

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Supplementary Data 1

Supporting data for Supplementary Fig. 28.

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Pei, ZF., Vior, N.M., Zhu, L. et al. Biosynthesis of peptide–nucleobase hybrids in ribosomal peptides. Nat Chem Biol 21, 143–154 (2025). https://doi.org/10.1038/s41589-024-01736-9

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