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Discovery and biosynthesis of cyclic plant peptides via autocatalytic cyclases

Nature Chemical Biology volume 18, pages 18–28 (2022)Cite this article

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Abstract

Many bioactive plant cyclic peptides form side-chain-derived macrocycles. Lyciumins, cyclic plant peptides with tryptophan macrocyclizations, are ribosomal peptides (RiPPs) originating from repetitive core peptide motifs in precursor peptides with plant-specific BURP (BNM2, USP, RD22 and PG1beta) domains, but the biosynthetic mechanism for their formation has remained unknown. Here, we characterize precursor-peptide BURP domains as copper-dependent autocatalytic peptide cyclases and use a combination of tandem mass spectrometry-based metabolomics and plant genomics to systematically discover five BURP-domain-derived plant RiPP classes, with mono- and bicyclic structures formed via tryptophans and tyrosines, from botanical collections. As BURP-domain cyclases are scaffold-generating enzymes in plant specialized metabolism that are physically connected to their substrates in the same polypeptide, we introduce a bioinformatic method to mine plant genomes for precursor-peptide-encoding genes by detection of repetitive substrate domains and known core peptide features. Our study sets the stage for chemical, biosynthetic and biological exploration of plant RiPP natural products from BURP-domain cyclases.

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Fig. 1: Systematic discovery approach of side-chain-macrocyclic plant peptides.
Fig. 2: Discovery of side-chain-macrocyclic peptides in botanical collections.
Fig. 3: Transient expression of BURP-domain precursor peptides in N. benthamiana.
Fig. 4: In vitro reconstitution of AhyBURP as an autocatalytic peptide cyclase.
Fig. 5: In vitro reconstitution of SkrBURP domain.
Fig. 6: Genome mining of BURP-domain-derived peptides by plantiSMASH and RepeatFinder.

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Data availability

Plant genome sequences were derived from JGI Phytozome (v.13)45 and NCBI GenBank. Known peptide structures were derived from Pubchem62. Gene sequences of peptide precursors have been deposited in GenBank (CcaBURP1, MW570736; CcaBURP2, MW570737; AhyBURP, MW570734 and SkrBURP, MW570735). Transcriptomic data of S. kraussiana has been deposited in the NCBI-SRA (SRR11647978). Metabolomic datasets and reference peptide MS/MS spectra have been submitted to GNPS-MassIVE29 under the accession number MSV000087872. MS/MS spectra of characterized peptides have been submitted to GNPS-MS/MS Spectral Libraries. The molecular network can be found under https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=c530b123d3984aa58247d6bd2789c69f#. Plant genome analysis outputs by plantiSMASH-RepeatFinder are available at: https://doi.org/10.5281/zenodo.4687169. All other data supporting the findings of this study are presented in the published article including its supporting information or are available from the corresponding authors upon reasonable request.

Code availability

Updated plantiSMASH and RepeatFinder code are available on Github (https://github.com/plantismash/plantismash).

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Acknowledgements

We thank D. Michener and M. Palmer for access to the Matthaei Botanical Garden and C. Dick and B. Ruhfel for access to the University of Michigan Herbarium. We also thank G. Lomonosoff (John Innes Centre, UK) for sharing the pEAQ-HT vector. This work was supported by the Biological Sciences Scholar Program at the University of Michigan (R.D.K) and the Graduate School for Experimental Plant Sciences in the Netherlands (M.H.M.).

Author information

Author notes

  1. These authors contributed equally: Desnor N. Chigumba, Lisa S. Mydy, Floris de Waal.

Authors and Affiliations

  1. Department of Medicinal Chemistry, University of Michigan, Ann Arbor, MI, USA

    Desnor N. Chigumba, Lisa S. Mydy, Wenjie Li, Khadija Shafiq, Jesse W. Wotring, Tim Mladenovic, Ashootosh Tripathi, Jonathan Z. Sexton & Roland D. Kersten

  2. Bioinformatics Group, Wageningen University, Wageningen, Netherlands

    Floris de Waal, Satria Kautsar & Marnix H. Medema

  3. Natural Products Discovery Core, Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA

    Osama G. Mohamed & Ashootosh Tripathi

  4. Pharmacognosy Department, Faculty of Pharmacy, Cairo University, Cairo, Egypt

    Osama G. Mohamed

  5. Department of Internal Medicine, University of Michigan, Ann Arbor, MI, USA

    Jonathan Z. Sexton

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Contributions

D.N.C., L.S.M. and F.d.W. contributed equally to this work. R.D.K. and M.H.M. conceived the idea. D.N.C., W.L., K.S. and R.D.K. collected plant samples, prepared and analyzed plant extracts by metabolomics, isolated and structurally elucidated plant peptides. R.D.K. expressed plant proteins in tobacco, prepared and analyzed transgenic tobacco leaf extracts by metabolomics. L.S.M. and R.D.K. expressed proteins in E. coli, purified proteins, performed and analyzed in vitro enzyme assays by bottom-up proteomics. T.M. analyzed known plant cyclic peptide structures. K.S., J.W.W., O.G.M., A.T. and J.Z.S. performed peptide bioactivity assays. M.H.M., S.K. and F.d.W. designed and wrote RepeatFinder code and integrated RepeatFinder into plantiSMASH. M.H.M., S.K., F.d.W. and R.D.K. analyzed plant genomes via plantiSMASH-RepeatFinder. R.D.K., M.H.M., D.N.C., L.S.M. and F.d.W. wrote the paper.

Corresponding authors

Correspondence to Marnix H. Medema or Roland D. Kersten.

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The authors declare no competing interests.

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Peer review information Nature Chemical Biology thanks Yit-Heng Chooi and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Peptides with side-chain-macrocyclizations from plants, bacteria and fungi.

Representative chemical structures are shown. Macrocyclic bonds are highlighted in red.

Extended Data Fig. 2 Extraction analysis of reference plant peptides with side-chain-macrocyclic bonds from source plant material.

Reference peptides were monitored in extract fractions based on corresponding extracted ion chromatogram (EIC) peak areas. EIC peak area data were plotted as dot plots overlayed with bar graphs representing the mean of three extractions of tissue from three biologically different plants (N = 3). Error bars of the bar graph represent one standard deviation (±SD). Abbreviation: EA - ethyl acetate, nBuOH - n-butanol, A. hypochondriacus – Amaranthus hypochondriacus, S. floribunda – Stephanotis floribunda, C. argentea – Celosia argentea, R. cordifolia – Rubia cordifolia, C. americanus.

Extended Data Fig. 3 Molecular network of peptide-filtered LC-MS/MS datasets of UM botanical collections.

Full molecular network of peptide-filtered datasets with characterized spectral clusters highlighted including new cyclic plant peptides.

Extended Data Fig. 4 Cyclopeptide alkaloid-containing spectral cluster of peptide-filtered LC-MS/MS datasets of UM botanical collections.

Nodes are colored according to source plant as shown in the top box. Node sizes represent relative precursor ion intensity. Node values are precursor ion mass values. Pie charts represent relative spectral counts of defined groups. Nodes of reference peptides and characterized peptides are indicated with arrows.

Extended Data Fig. 5 Candidate BURP domain precursor peptides of target cyclic plant peptides from botanical collections.

BURP domains are underlined, putative core peptides are highlighted in red.

Extended Data Fig. 6 Bioactivity evaluation of side-chain-macrocyclic plant peptides.

(a) Antibacterial assays show no significant growth inhibition of Bacillus subtilis 168, Staphylococcus aureus ATCC43300 or Shigella flexneri BS103 at 100 μM compound concentration compared to 1% DMSO control after 24 h incubation at 37 °C compared to antibiotic positive controls of ciprofloxacin and rifampicin. Data represents mean of OD600 of cultures (N = 2) ± SEM. (b) Compounds were assessed for anti-cancer activity at a concentration of 100 µg/mL in four different human cell lines including H1437 (lung adenocarcinoma), Caco-2 (colorectal adenocarcinoma), LNCaP (prostate adenocarcinoma), and Huh7 (hepatocellular carcinoma). Data represents the mean ± SEM of N = 68 vehicle controls and N = 3 treatments. Statistically significant reductions in cell viability were determined using unpaired two-tailed t-tests in Graphpad Prism 9 (p < 0.0001 = ****, p < 0.001 = ***, p < 0.01 = **). (c) Sequences of BURP domain protein homologs of selanine precursor peptide SkrBURP. SkrBURP homologs from bruchid-resistant Vigna radiata contain core peptides which match the insecticidal cyclopeptide alkaloid vignatic acid A from bruchid-resistant Vigna radiata strain TC1966.

Extended Data Fig. 7 In vitro reconstitution of SkrBURP and CcaBURP domains.

(a) Detection of monocyclic core peptide modifications in purified MBP-CcaBURP2-[1xQLLVW] after incubation with Cu(II) at pH 7. (b) Detection of monocyclic core peptide modification in purified CcaBURP1-[1xQILFW] after incubation with Cu(II) at pH 7. (c) Detection of desmethyl-selanine A and B after incubation of tryptic bicyclic core peptide iii of SkrBURP-[1xVLFYPSY] with aminopeptidase (AP) and carboxypeptidase (CP). Asterisks (*) notes a cyclic peptide with a different MS/MS spectrum than desmethyl-selanine B. (d) Detection of cercic acid after incubation of tryptic monocyclic core peptide ii of CcaBURP1-[1xQILFW] with aminopeptidase and carboxypeptidase. BURP-domain-sequences are underlined, core peptides are highlighted in red, bold sequences are characterized tryptic peptides including core peptides. Abbreviation: MBP - maltose-binding protein.

Extended Data Fig. 8 Biosynthetic proposal for BURP-domain-derived plant RiPPs legumenin, lyciumin and stephanotic acid-[LV].

Biosynthesis of BURP-domain-derived plant RiPPs is proposed to proceed through BURP domain cyclization of core peptide motifs, N-terminal proteolysis, N-terminal protection and C-terminal proteolysis. Legumenin biosynthesis involves two sequential cyclizations, in which the C-terminal macrocycle is being formed first. Formed macrocyclic bonds in core peptide motifs are highlighted in red.

Extended Data Fig. 9 Biosynthetic proposal for BURP-domain-derived plant RiPPs selanine A (bicyclopeptide alkaloid), selanine B (cyclopeptide alkaloid) and cercic acid.

Biosynthesis of BURP-domain-derived plant RiPPs is proposed to proceed through BURP domain cyclization of core peptide motifs, N-terminal proteolysis, N-terminal protection and C-terminal proteolysis. Selanine A biosynthesis involves two sequential cyclizations, in which the N-terminal macrocycle is formed first. Formed macrocyclic bonds in core peptide motifs are highlighted in red.

Supplementary information

Supplementary Information

Supplementary Tables 1–11, Figs. 1–13 and Notes 1–11.

Reporting Summary

Supplementary Data 1

Side-chain-macrocyclic peptide analysis.

Supplementary Data 2

Source plants of metabolomic datasets.

Supplementary Data 3

Molecular cluster analysis with reference peptide spectra from Extended Data Fig. 3.

Supplementary Data 4

plantiSMASH-RepeatFinder genome analysis.

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Chigumba, D.N., Mydy, L.S., de Waal, F. et al. Discovery and biosynthesis of cyclic plant peptides via autocatalytic cyclases. Nat Chem Biol 18, 18–28 (2022). https://doi.org/10.1038/s41589-021-00892-6

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