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An intramolecular macrocyclase in plant ribosomal peptide biosynthesis

Nature Chemical Biology volume 20, pages 530–540 (2024)Cite this article

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Abstract

The biosynthetic dogma of ribosomally synthesized and posttranslationally modified peptides (RiPP) involves enzymatic intermolecular modification of core peptide motifs in precursor peptides. The plant-specific BURP-domain protein family, named after their four founding members, includes autocatalytic peptide cyclases involved in the biosynthesis of side-chain-macrocyclic plant RiPPs. Here we show that AhyBURP, a representative of the founding Unknown Seed Protein-type BURP-domain subfamily, catalyzes intramolecular macrocyclizations of its core peptide during the sequential biosynthesis of monocyclic lyciumin I via glycine-tryptophan crosslinking and bicyclic legumenin via glutamine-tyrosine crosslinking. X-ray crystallography of AhyBURP reveals the BURP-domain fold with two type II copper centers derived from a conserved stapled-disulfide and His motif. We show the macrocyclization of lyciumin-C(sp3)-N-bond formation followed by legumenin-C(sp3)-O-bond formation requires dioxygen and radical involvement based on enzyme assays in anoxic conditions and isotopic labeling. Our study expands enzymatic intramolecular modifications beyond catalytic moiety and chromophore biogenesis to RiPP biosynthesis.

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Fig. 1: The BURP-domain protein family and USP-type BURP-domain legumenin cyclase AhyBURP.
Fig. 2: Crystal structure of AhyBURP and copper sites.
Fig. 3: AhyBURP reaction.
Fig. 4: AhyBURP mechanistic investigation.
Fig. 5: Intramolecular macrocyclization in AhyBURP.

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Data availability

Atomic coordinates and processed diffraction data have been deposited in the PDB under accession codes 8SY2 and 8SY3. Proteomic datasets for Figs. 4 and 5 were uploaded to MassIVE, accession code MSV000093708. The data that support the findings of this study are available within the main text and its Supplementary Information. Source data are provided with this paper. Data are also available from the corresponding authors upon request.

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Acknowledgements

This study was supported by NIGMS (grant nos. R35GM146934 to R.D.K., F32GM146395 to L.S.M.), NIDDK (grant no. R01DK042303 to J.L.S.), the Hermann Frasch Foundation (R.D.K.) and the PhRMA foundation (predoctoral fellowship, D.N.C.). We thank the University of Michigan Center for Structural Biology and E. Scott for crystallography resources and for fast protein liquid chromatography (FPLC) use. We appreciate T. Cernak for access to synthetic resources, B. Palfey for spectrophotometer and fluorometer access, J. Bridwell-Rabb for glove box access and M. Knapp for training L.S.M. We are grateful to T. Cernak, B. Palfey and J. Bridwell-Rabb for helpful discussions as well. We also thank G. Lomonosoff (John Innes Centre, UK) for sharing the pEAQ-HT vector. GM/CA at the Advanced Photon Source has been funded by the National Cancer Institute (grant no. ACB-12002) and the National Institute of General Medical Sciences (grant nos. AGM-12006, P30GM138396). This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract no. DE-AC02-06CH11357. The Eiger 16M detector at GM/CA-XSD was funded by a National Institutes of Health grant no. S10 OD012289. L.S.M. thanks Q. Xu for aid in determining anomalous difference density.

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Authors and Affiliations

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

    Lisa S. Mydy, Jordan Hungerford, Desnor N. Chigumba, Di Wang & Roland D. Kersten

  2. Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA

    Jamie R. Konwerski & Janet L. Smith

  3. Plasma Chemistry Laboratory, Center for Applied Isotope Studies, University of Georgia, Athens, GA, USA

    Sarah C. Jantzi

  4. Department of Biological Chemistry, University of Michigan, Ann Arbor, MI, USA

    Janet L. Smith

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Contributions

L.S.M., J.L.S. and R.D.K. designed experiments, interpreted data and wrote initial and final drafts of the manuscript. L.S.M., J.H., J.R.K., S.C.J., D.W. and R.D.K. performed experiments and analyzed data. D.N.C. generated the sequence similarity network and its analysis. All authors reviewed and edited the manuscript.

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Correspondence to Lisa S. Mydy or Roland D. Kersten.

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Nature Chemical Biology thanks Ninian Blackburn and the other, anonymous, reviewers for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Methods, Tables 1–4, Figs. 1–35 and references.

Reporting Summary

Supplementary Data 1

PDB Validation report for PDB ID 8SY2.

Supplementary Data 2

PDB Validation report for PDB ID 8SY3.

Source data

Source Data Fig. 3

AhyBURP genes and EIC peak areas in Fig. 3.

Source Data Fig. 4

MassIVE datasets for Fig. 4.

Source Data Fig. 5

MassIVE datasets for Fig. 5.

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Mydy, L.S., Hungerford, J., Chigumba, D.N. et al. An intramolecular macrocyclase in plant ribosomal peptide biosynthesis. Nat Chem Biol 20, 530–540 (2024). https://doi.org/10.1038/s41589-024-01552-1

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