Investigations into PoyH, a promiscuous protease from polytheonamide biosynthesis

  • Maximilian J. Helf
  • Michael F. Freeman
  • Jörn PielEmail author
Natural Products - Original Paper


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.


Polytheonamide Ribosomal peptide RiPP Cysteine protease Post-translational modification 



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)


  1. 1.
    Arnison PG, Bibb MJ, Bierbaum G, Bowers AA, Bugni TS, Bulaj G, Camarero JA, Campopiano DJ, Challis GL, Clardy J, Cotter PD, Craik DJ, Dawson M, Dittmann E, Donadio S, Dorrestein PC, Entian K-D, Fischbach MA, Garavelli JS, Göransson U, Gruber CW, Haft DH, Hemscheidt TK, Hertweck C, Hill C, Horswill AR, Jaspars M, Kelly WL, Klinman JP, Kuipers OP, Link AJ, Liu W, Marahiel MA, Mitchell DA, Moll GN, Moore BS, Müller R, Nair SK, Nes IF, Norris GE, Olivera BM, Onaka H, Patchett ML, Piel J, Reaney MJT, Rebuffat S, Ross RP, Sahl H-G, Schmidt EW, Selsted ME, Severinov K, Shen B, Sivonen K, Smith L, Stein T, Süssmuth RD, Tagg JR, Tang G-L, Truman AW, Vederas JC, Walsh CT, Walton JD, Wenzel SC, Willey JM, van der Donk WA (2013) Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature. Nat Prod Rep 30:108–160. CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Begley M, Cotter PD, Hill C, Ross RP (2009) Identification of a novel two-peptide lantibiotic, lichenicidin, following rational genome mining for LanM proteins. Appl Environ Microbiol 75:5451–5460. CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Berger A, Schechter I (1970) Mapping the active site of papain with the aid of peptide substrates and inhibitors. Philos Trans R Soc B Biol Sci 257:249–264. CrossRefGoogle Scholar
  4. 4.
    Bowers AA, Acker MG, Koglin A, Walsh CT (2010) Manipulation of thiocillin variants by prepeptide gene replacement: structure, conformation, and activity of heterocycle substitution mutants. J Am Chem Soc 132:7519–7527. CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Bowers AA, Acker MG, Young TS, Walsh CT (2012) Generation of thiocillin ring size variants by prepeptide gene replacement and in vivo processing by Bacillus cereus. J Am Chem Soc 134:10313–10316. CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Burrage S, Raynham T, Williams G, Essex JW, Allen C, Cardno M, Swali V, Bradley M (2000) Biomimetic synthesis of lantibiotics. Chemistry (Easton) 6:1455–1466. Google Scholar
  7. 7.
    Buttle DJ (2013) Glycyl endopeptidase. In: Salvesen NDR (ed) Handbook of proteolytic enzymes. Academic Press, London, pp 1867–1870CrossRefGoogle Scholar
  8. 8.
    Chambers MC, MacLean B, Burke R, Amodei D, Ruderman DL, Neumann S, Gatto L, Fischer B, Pratt B, Egertson J, Hoff K, Kessner D, Tasman N, Shulman N, Frewen B, Baker TA, Brusniak MY, Paulse C, Creasy D, Flashner L, Kani K, Moulding C, Seymour SL, Nuwaysir LM, Lefebvre B, Kuhlmann F, Roark J, Rainer P, Detlev S, Hemenway T, Huhmer A, Langridge J, Connolly B, Chadick T, Holly K, Eckels J, Deutsch EW, Moritz RL, Katz JE, Agus DB, MacCoss M, Tabb DL, Mallick P (2012) A cross-platform toolkit for mass spectrometry and proteomics. Nat Biotechnol 30:918–920. CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Chatterjee C, Paul M, Xie L, van der Donk WA (2005) Biosynthesis and mode of action of lantibiotics. Chem Rev 105:633–684. CrossRefPubMedGoogle Scholar
  10. 10.
    Choe Y, Leonetti F, Greenbaum DC, Lecaille F, Bogyo M, Brömme D, Ellman JA, Craik CS (2006) Substrate profiling of cysteine proteases using a combinatorial peptide library identifies functionally unique specificities. J Biol Chem 281:12824–12832. CrossRefPubMedGoogle Scholar
  11. 11.
    Cimermancic P, Medema MH, Claesen J, Kurita K, Wieland Brown LC, Mavrommatis K, Pati A, Godfrey PA, Koehrsen M, Clardy J, Birren BW, Takano E, Sali A, Linington RG, Fischbach MA (2014) Insights into secondary metabolism from a global analysis of prokaryotic biosynthetic gene clusters. Cell 158:412–421. CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Cogan DP, Hudson GA, Zhang Z, Pogorelov TV, van der Donk WA, Mitchell DA, Nair SK (2017) Structural insights into enzymatic [4+2] aza -cycloaddition in thiopeptide antibiotic biosynthesis. Proc Natl Acad Sci 114:201716035. CrossRefGoogle Scholar
  13. 13.
    Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32:1792–1797. CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Edgar RC (2004) MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinform 5:113. CrossRefGoogle Scholar
  15. 15.
    Freeman MF, Gurgui C, Helf MJ, Morinaka BI, Uria AR, Oldham NJ, Sahl H-G, Matsunaga S, Piel J (2012) Metagenome mining reveals polytheonamides as posttranslationally modified ribosomal peptides. Science 338:387–390. CrossRefPubMedGoogle Scholar
  16. 16.
    Freeman MF, Helf MJ, Bhushan A, Morinaka BI, Piel J (2017) Seven enzymes create extraordinary molecular complexity in an uncultivated bacterium. Nat Chem 9:387–395. CrossRefPubMedGoogle Scholar
  17. 17.
    Fuchs SW, Lackner G, Morinaka BI, Morishita Y, Asai T, Riniker S, Piel J (2016) A Lanthipeptide-like N-Terminal leader region guides peptide epimerization by radical SAM epimerases: implications for RiPP evolution. Angew Chem Int Ed Engl 55:12330–12333. CrossRefPubMedGoogle Scholar
  18. 18.
    Furgerson Ihnken LA, Chatterjee C, van der Donk WA (2008) In vitro reconstitution and substrate specificity of a lantibiotic protease. Biochemistry 47:7352–7363. CrossRefPubMedGoogle Scholar
  19. 19.
    Gouet P, Courcelle E, Stuart D, Metoz F (1999) ESPript: analysis of multiple sequence alignments in PostScript. Bioinformatics 15:305–308. CrossRefPubMedGoogle Scholar
  20. 20.
    Haft DH, Basu MK, Mitchell DA (2010) Expansion of ribosomally produced natural products: a nitrile hydratase- and Nif11-related precursor family. BMC Biol 8:70. CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Hamada T, Matsunaga S, Yano G, Fusetani N (2005) Polytheonamides A and B, highly cytotoxic, linear polypeptides with unprecedented structural features, from the marine sponge, Theonella swinhoei. J Am Chem Soc 127:110–118. CrossRefPubMedGoogle Scholar
  22. 22.
    Ishii S, Yano T, Ebihara A, Okamoto A, Manzoku M, Hayashi H (2010) Crystal structure of the peptidase domain of Streptococcus ComA, a bifunctional ATP-binding cassette transporter involved in the quorum-sensing pathway. J Biol Chem 285:10777–10785. CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Ishii S, Yano T, Hayashi H (2006) Expression and characterization of the peptidase domain of Streptococcus pneumoniae ComA, a bifunctional ATP-binding cassette transporter involved in quorum sensing pathway. J Biol Chem 281:4726–4731. CrossRefPubMedGoogle Scholar
  24. 24.
    Jones DT (1999) Protein secondary structure prediction based on position-specific scoring matrices. J Mol Biol 292:195–202. CrossRefPubMedGoogle Scholar
  25. 25.
    Kelley LA, Sternberg MJE (2009) Protein structure prediction on the Web: a case study using the Phyre server. Nat Protoc 4:363–371. CrossRefPubMedGoogle Scholar
  26. 26.
    Kotake Y, Ishii S, Yano T, Katsuoka Y, Hayashi H (2008) Substrate recognition mechanism of the peptidase domain of the quorum-sensing-signal–producing ABC transporter ComA from Streptococcus. Biochemistry 47:2531–2538. CrossRefPubMedGoogle Scholar
  27. 27.
    Kuipers A, de Boef E, Rink R, Fekken S, Kluskens LD, Driessen AJM, Leenhouts K, Kuipers OP, Moll GN (2004) NisT, the transporter of the lantibiotic nisin, can transport fully modified, dehydrated, and unmodified prenisin and fusions of the leader peptide with non-lantibiotic peptides. J Biol Chem 279:22176–22182. CrossRefPubMedGoogle Scholar
  28. 28.
    Lackner G, Peters EE, Helfrich EJN, Piel J (2017) Insights into the lifestyle of uncultured bacterial natural product factories associated with marine sponges. Proc Natl Acad Sci 114:E347–E356. CrossRefPubMedGoogle Scholar
  29. 29.
    Lagedroste M, Smits SHJ, Schmitt L (2017) Substrate specificity of the secreted nisin leader peptidase NisP. Biochemistry 56:4005–4014. CrossRefPubMedGoogle Scholar
  30. 30.
    Li B, Sher D, Kelly L, Shi Y, Huang K, Knerr PJ, Joewono I, Rusch D, Chisholm SW, van der Donk WA (2010) Catalytic promiscuity in the biosynthesis of cyclic peptide secondary metabolites in planktonic marine cyanobacteria. Proc Natl Acad Sci 107:10430. CrossRefPubMedGoogle Scholar
  31. 31.
    Ljunggren A, Redzynia I, Alvarez-Fernandez M, Abrahamson M, Mort JS, Krupa JC, Jaskolski M, Bujacz G (2007) Crystal structure of the parasite protease inhibitor chagasin in complex with a host target cysteine protease. J Mol Biol 371:137–153. CrossRefPubMedGoogle Scholar
  32. 32.
    Loos M, Gerber C, Corona F, Hollender J, Singer H (2015) Accelerated isotope fine structure calculation using pruned transition trees. Anal Chem 87:5738–5744. CrossRefPubMedGoogle Scholar
  33. 33.
    Marchand JA, Peccoud J (2012) Gene synthesis. In: Peccoud J (ed) Methods in molecular biology. Humana, Totowa, pp 3–10Google Scholar
  34. 34.
    Marchler-Bauer A, Anderson JB, Chitsaz F, Derbyshire MK, DeWeese-Scott C, Fong JH, Geer LY, Geer RC, Gonzales NR, Gwadz M, He S, Hurwitz DI, Jackson JD, Ke Z, Lanczycki CJ, Liebert CA, Liu C, Lu F, Lu S, Marchler GH, Mullokandov M, Song JS, Tasneem A, Thanki N, Yamashita RA, Zhang D, Zhang N, Bryant SH (2009) CDD: specific functional annotation with the Conserved Domain Database. Nucleic Acids Res 37:D205–D210. CrossRefPubMedGoogle Scholar
  35. 35.
    Marchler-Bauer A, Bryant SH (2004) CD-search: protein domain annotations on the fly. Nucleic Acids Res 32:W327–W331. CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Marchler-Bauer A, Lu S, Anderson JB, Chitsaz F, Derbyshire MK, DeWeese-Scott C, Fong JH, Geer LY, Geer RC, Gonzales NR, Gwadz M, Hurwitz DI, Jackson JD, Ke Z, Lanczycki CJ, Lu F, Marchler GH, Mullokandov M, Omelchenko MV, Robertson CL, Song JS, Thanki N, Yamashita RA, Zhang D, Zhang N, Zheng C, Bryant SH (2011) CDD: a Conserved Domain Database for the functional annotation of proteins. Nucleic Acids Res 39:D225–D229. CrossRefPubMedGoogle Scholar
  37. 37.
    Medema MH, Kottmann R, Yilmaz P, Cummings M, Biggins JB, Blin K, de Bruijn I, Chooi YH, Claesen J, Coates RC, Cruz-Morales P, Duddela S, Düsterhus S, Edwards DJ, Fewer DP, Garg N, Geiger C, Gomez-Escribano JP, Greule A, Hadjithomas M, Haines AS, Helfrich EJN, Hillwig ML, Ishida K, Jones AC, Jones CS, Jungmann K, Kegler C, Kim HU, Kötter P, Krug D, Masschelein J, Melnik AV, Mantovani SM, Monroe EA, Moore M, Moss N, Nützmann H-W, Pan G, Pati A, Petras D, Reen FJ, Rosconi F, Rui Z, Tian Z, Tobias NJ, Tsunematsu Y, Wiemann P, Wyckoff E, Yan X, Yim G, Yu F, Xie Y, Aigle B, Apel AK, Balibar CJ, Balskus EP, Barona-Gómez F, Bechthold A, Bode HB, Borriss R, Brady SF, Brakhage AA, Caffrey P, Cheng Y-Q, Clardy J, Cox RJ, De Mot R, Donadio S, Donia MS, van der Donk WA, Dorrestein PC, Doyle S, Driessen AJM, Ehling-Schulz M, Entian K-D, Fischbach MA, Gerwick L, Gerwick WH, Gross H, Gust B, Hertweck C, Höfte M, Jensen SE, Ju J, Katz L, Kaysser L, Klassen JL, Keller NP, Kormanec J, Kuipers OP, Kuzuyama T, Kyrpides NC, Kwon H-J, Lautru S, Lavigne R, Lee CY, Linquan B, Liu X, Liu W, Luzhetskyy A, Mahmud T, Mast Y, Méndez C, Metsä-Ketelä M, Micklefield J, Mitchell DA, Moore BS, Moreira LM, Müller R, Neilan BA, Nett M, Nielsen J, O’Gara F, Oikawa H, Osbourn A, Osburne MS, Ostash B, Payne SM, Pernodet J-L, Petricek M, Piel J, Ploux O, Raaijmakers JM, Salas JA, Schmitt EK, Scott B, Seipke RF, Shen B, Sherman DH, Sivonen K, Smanski MJ, Sosio M, Stegmann E, Süssmuth RD, Tahlan K, Thomas CM, Tang Y, Truman AW, Viaud M, Walton JD, Walsh CT, Weber T, van Wezel GP, Wilkinson B, Willey JM, Wohlleben W, Wright GD, Ziemert N, Zhang C, Zotchev SB, Breitling R, Takano E, Glöckner FO (2015) Minimum information about a biosynthetic gene cluster. Nat Chem Biol 11:625–631. CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Montalbán-López M, Deng J, van Heel AJ, Kuipers OP (2018) Specificity and application of the lantibiotic protease NisP. Front Microbiol 9:1–16. CrossRefGoogle Scholar
  39. 39.
    Morinaka BI, Verest M, Freeman MF, Gugger M, Piel J (2016) An orthogonal D2O-based induction system that provides insights into d-Amino acid pattern formation by radical S-adenosylmethionine peptide epimerases. Angew Chemie Int Ed. Google Scholar
  40. 40.
    Okeley NM, Zhu Y, van der Donk WA (2000) Facile chemoselective synthesis of dehydroalanine-containing peptides. Org Lett 2:3603–3606. CrossRefPubMedGoogle Scholar
  41. 41.
    Oman TJ, van der Donk WA (2010) Follow the leader: the use of leader peptides to guide natural product biosynthesis. Nat Chem Biol 6:9–18. CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Ortega MA, Velásquez JE, Garg N, Zhang Q, Joyce RE, Nair SK, Van Der Donk WA (2014) Substrate specificity of the lanthipeptide peptidase ElxP and the oxidoreductase ElxO. ACS Chem Biol 9:1718–1725. CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Rawlings ND, Barrett AJ (2013) The Clans and families of cysteine peptidases. In: Salvesen NDR (ed) Handbook of proteolytic enzymes. Academic Press, London, pp 1743–1773CrossRefGoogle Scholar
  44. 44.
    dos Reis FCG, Smith BO, Santos CC, Costa TFR, Scharfstein J, Coombs GH, Mottram JC, Lima APCA (2008) The role of conserved residues of chagasin in the inhibition of cysteine peptidases. FEBS Lett 582:485–490. CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Shenkarev ZO, Finkina EI, Nurmukhamedova EK, Balandin SV, Mineev KS, Nadezhdin KD, Yakimenko ZA, Tagaev AA, Temirov YV, Arseniev AS, Ovchinnikova TV (2010) Isolation, structure elucidation, and synergistic antibacterial activity of a novel two-component lantibiotic lichenicidin from Bacillus licheniformis VK21. Biochemistry 49:6462–6472. CrossRefPubMedGoogle Scholar
  46. 46.
    Smith CA, Want EJ, O’Maille G, Abagyan R, Siuzdak G (2006) XCMS: processing mass spectrometry data for metabolite profiling using nonlinear peak alignment, matching, and identification. Anal Chem 78:779–787. CrossRefPubMedGoogle Scholar
  47. 47.
    Turk D, Gunčar G, Podobnik M, Turk B (1998) Revised definition of substrate binding sites of papain-like cysteine proteases. Biol Chem 379:137–147. CrossRefPubMedGoogle Scholar
  48. 48.
    Völler GH, Krawczyk B, Ensle P, Süssmuth RD (2013) Involvement and unusual substrate specificity of a prolyl oligopeptidase in class III lanthipeptide maturation. J Am Chem Soc 135:7426–7429. CrossRefPubMedGoogle Scholar
  49. 49.
    Weber T, Blin K, Duddela S, Krug D, Kim HU, Bruccoleri R, Lee SY, Fischbach MA, Müller R, Wohlleben W, Breitling R, Takano E, Medema MH (2015) AntiSMASH 3.0—a comprehensive resource for the genome mining of biosynthetic gene clusters. Nucleic Acids Res 43:W237–W243. CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    Wever WJ, Bogart JW, Baccile JA, Chan AN, Schroeder FC, Bowers AA (2015) Chemoenzymatic synthesis of thiazolyl peptide natural products featuring an enzyme-catalyzed formal [4+2] cycloaddition. J Am Chem Soc 137:3494–3497. CrossRefPubMedPubMedCentralGoogle Scholar
  51. 51.
    Wiederanders B (2003) Structure-function relationships in class CA1 cysteine peptidase propeptides. Acta Biochim Pol 50:691–713PubMedGoogle Scholar
  52. 52.
    Wieland Brown LC, Acker MG, Clardy J, Walsh CT, Fischbach MA (2009) Thirteen posttranslational modifications convert a 14-residue peptide into the antibiotic thiocillin. Proc Natl Acad Sci U S A 106:2549–2553. CrossRefPubMedPubMedCentralGoogle Scholar
  53. 53.
    Willey JM, van der Donk WA (2007) Lantibiotics: peptides of diverse structure and function. Annu Rev Microbiol 61:477–501. CrossRefPubMedGoogle Scholar
  54. 54.
    Wilson MC, Mori T, Rückert C, Uria AR, Helf MJ, Takada K, Gernert C, Steffens UAE, Heycke N, Schmitt S, Rinke C, Helfrich EJN, Brachmann AO, Gurgui C, Wakimoto T, Kracht M, Crüsemann M, Hentschel U, Abe I, Matsunaga S, Kalinowski J, Takeyama H, Piel J (2014) An environmental bacterial taxon with a large and distinct metabolic repertoire. Nature 506:58–62. CrossRefPubMedGoogle Scholar

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

Personalised recommendations