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Biosynthesis-guided discovery reveals enteropeptins as alternative sactipeptides containing N-methylornithine

Nature Chemistry volume 14, pages 1390–1398 (2022)Cite this article

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Abstract

The combination of next-generation DNA sequencing technologies and bioinformatics has revitalized natural product discovery. Using a bioinformatic search strategy, we recently identified ∼600 gene clusters in otherwise overlooked streptococci that code for ribosomal peptide natural products synthesized by radical S-adenosylmethionine enzymes. These grouped into 16 subfamilies and pointed to an unexplored microbiome biosynthetic landscape. Here we report the structure, biosynthesis and function of one of these natural product groups, which we term enteropeptins, from the gut microbe Enterococcus cecorum. We show three reactions in the biosynthesis of enteropeptins that are each catalysed by a different family of metalloenzymes. Among these, we characterize the founding member of a widespread superfamily of Fe–S-containing methyltransferases, which, together with an Mn2+-dependent arginase, installs N-methylornithine in the peptide sequence. Biological assays with the mature product revealed bacteriostatic activity only against the producing strain, extending an emerging theme of fratricidal or self-inhibitory metabolites in microbiome firmicutes.

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Fig. 1: Streptococcal RaS-RiPPs and the kgr gene cluster.
Fig. 2: Reactions of KgrBCD characterized by HR-MS and HR-MS/MS.
Fig. 3: NMR spectral analysis of the product of the reaction of KgrA with KgrBCD.
Fig. 4: Orn N-methylation catalysed by KgrB.
Fig. 5: Discovery of enteropeptins, the mature products of the kgr cluster, from E. cecorum.
Fig. 6: Enteropeptin activity and compound class.

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Data availability

The Uniprot ID for KgrC is S1RLF6. Plasmids and strains used in this study are described in Supplementary Table 2. All oligonucleotides are shown in Supplementary Table 3. The sequences of codon-optimized gene fragments are provided in Supplementary Note 1. Other relevant data supporting the findings of this study are available within the paper and the supplementary material. NMR spectra are included in Supplementary Information. Raw NMR data used to elucidate natural product structures are available from the corresponding author upon reasonable request.

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Acknowledgements

We thank the Edward C. Taylor-Eli Lilly Fellowship in Chemistry (to K.A.C.) and the National Science Foundation (NSF CAREER Award 1847932 to M.R.S.) for support of this work.

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Authors and Affiliations

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

    Kenzie A. Clark, Brett C. Covington & Mohammad R. Seyedsayamdost

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

    Mohammad R. Seyedsayamdost

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Contributions

K.A.C. and M.R.S. conceived of the study. K.A.C. carried out in vitro characterization and structural elucidation of the reactions of all enzymes. B.C.C. identified the mature natural products from the native organism. K.A.C. and B.C.C. carried out bioactivity assays. K.A.C. and M.R.S. wrote the manuscript, with contributions from B.C.C.

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Correspondence to Mohammad R. Seyedsayamdost.

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Nature Chemistry thanks Qi Zhang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary Note 1, Tables 1–18 and Figs. 1–15.

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Clark, K.A., Covington, B.C. & Seyedsayamdost, M.R. Biosynthesis-guided discovery reveals enteropeptins as alternative sactipeptides containing N-methylornithine. Nat. Chem. 14, 1390–1398 (2022). https://doi.org/10.1038/s41557-022-01063-3

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