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Investigations into PoyH, a promiscuous protease from polytheonamide biosynthesis

  • Maximilian J. Helf
  • Michael F. Freeman
  • Jörn Piel
Natural Products - Original Paper
  • 98 Downloads

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.

Keywords

Polytheonamide Ribosomal peptide RiPP Cysteine protease Post-translational modification 

Notes

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.

Supplementary material

10295_2018_2129_MOESM1_ESM.docx (17.7 mb)
Supplementary material 1 (DOCX 18156 kb)

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Copyright information

© Society for Industrial Microbiology and Biotechnology 2019

Authors and Affiliations

  1. 1.Institute of MicrobiologyEidgenössische Technische Hochschule (ETH) ZurichZurichSwitzerland
  2. 2.Boyce Thompson InstituteCornell UniversityIthacaUSA
  3. 3.Department of Biochemistry, Molecular Biology, and Biophysics and BioTechnology InstituteUniversity of Minnesota-Twin CitiesSt. PaulUSA

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