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Biosynthesis and characterization of fuscimiditide, an aspartimidylated graspetide

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

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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.

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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.

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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.

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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.

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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

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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.

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Correspondence to A. James Link.

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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

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