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Investigations into PoyH, a promiscuous protease from polytheonamide biosynthesis

  • Natural Products - Original Paper
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Journal of Industrial Microbiology & Biotechnology

Abstract

Polytheonamides are the most extensively modified ribosomally synthesized and post-translationally modified peptide natural products (RiPPs) currently known. In RiPP biosynthesis, the processed peptide is usually released from a larger precursor by proteolytic cleavage to generate the bioactive terminal product of the pathway. For polytheonamides, which are members of a new RiPP family termed proteusins, we have recently shown that such cleavage is catalyzed by the cysteine protease PoyH acting on the precursor PoyA, both encoded in the polytheonamide biosynthetic gene cluster. We now report activity for PoyH under a variety of reaction conditions for different maturation states of PoyA and demonstrate a potential use of PoyH as a promiscuous protease to liberate and characterize RiPPs from other pathways. As a proof of concept, the identified recognition motif was introduced into precursors of the thiopeptide thiocillin and the lanthipeptide lichenicidin VK1, allowing for their site-specific cleavage with PoyH. Additionally, we show that PoyH cleavage is inhibited by PoyG, a previously uncharacterized chagasin-like protease inhibitor encoded in the polytheonamide gene cluster.

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Acknowledgements

JP acknowledges funding from the SNF (31003A_146992/1), the EU (SYNPEPTIDE), and the Helmut Horten Foundation. This work was supported by the Studienstiftung des Deutschen Volkes (PhD fellowship to MJH), the Human Frontier Science Program (Postdoctoral fellowship to MFF). We thank Alexander Brachmann and Brandon Morinaka for support with mass spectrometry.

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  1. Maximilian J. Helf

    Present address: Boyce Thompson Institute, Cornell University, 533 Tower Road, Ithaca, 14853, USA

  2. Michael F. Freeman

    Present address: Department of Biochemistry, Molecular Biology, and Biophysics and BioTechnology Institute, University of Minnesota-Twin Cities, St. Paul, MN, 55108, USA

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  1. Institute of Microbiology, Eidgenössische Technische Hochschule (ETH) Zurich, Vladimir-Prelog-Weg 4, 8093, Zurich, Switzerland

    Maximilian J. Helf, Michael F. Freeman & Jörn Piel

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Helf, M.J., Freeman, M.F. & Piel, J. Investigations into PoyH, a promiscuous protease from polytheonamide biosynthesis. J Ind Microbiol Biotechnol 46, 551–563 (2019). https://doi.org/10.1007/s10295-018-02129-3

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