Peptide Cyclization Catalyzed by Cyanobactin Macrocyclases

  • Wael E. Houssen
Part of the Methods in Molecular Biology book series (MIMB, volume 2012)


Cyclic peptides are an emerging class of therapeutics that can modulate targets not amenable to traditional small molecule intervention (e.g., protein–protein interactions). However, N-to-C macrocyclization of peptides is a challenging and often a low yielding chemical transformation. Several macrocyclases from cyanobactin biosynthetic clusters have been used to catalyze this reaction.

This chapter provides practical guidance to the processes of heterologous expression and purification of these enzymes as well as performing in vitro biochemical reactions. Finally, approaches to recover the final product from an enzymatic reaction mixture are also discussed.

Key words

Cyanobactins RiPPs Macrocyclases Ribosomal peptides Cyclic peptides Patellamides 


  1. 1.
    Villar EA, Beglov D, Chennamadhavuni S, Porco JA, Kozakov D, Vajda S et al (2014) How proteins bind macrocycles. Nat Chem Biol 10:723–731CrossRefGoogle Scholar
  2. 2.
    Gao M, Cheng K, Yin H (2015) Targeting protein-protein interfaces using macrocyclic peptides. Biopolymers 104:310–316CrossRefGoogle Scholar
  3. 3.
    Kotz J (2012) Bringing macrocycles full circle. SciBX 5(45):2–8Google Scholar
  4. 4.
    Driggers EM, Hale SP, Lee J, Terrett NK (2008) The exploration of macrocycles for drug discovery-an underexploited structural class. Nat Rev Drug Discov 7:608–624CrossRefGoogle Scholar
  5. 5.
    Zorzi A, Deyle K, Heinis C (2017) Cyclic peptide therapeutics: past, present and future. Curr Opin Chem Biol 38:24–29CrossRefGoogle Scholar
  6. 6.
    White CJ, Yudin AK (2011) Contemporary strategies for peptide macrocyclization. Nat Chem 3:509–524CrossRefGoogle Scholar
  7. 7.
    Martí-Centelles V, Pandey MD, Isabel Burguete M, Luis SV (2015) Macrocyclization reactions: the importance of conformational, configurational, and template-induced preorganization. Chem Rev 115:8736–8834CrossRefGoogle Scholar
  8. 8.
    Alexandru-Crivac C, Dalponte L, Houssen WE, Idress M, Jaspars M, Rickaby KA et al (2017) Cyclic peptides – a look to the future. In: Koehnke J, Naismith J, van der Donk WA (eds) Cyclic peptides: from bioorganic synthesis to applications. The Royal Society of Chemistry, LondonGoogle Scholar
  9. 9.
    Houssen WE, Jaspars M (2010) Azole-based cyclic peptides from the sea squirt Lissoclinum patella: old scaffolds, new avenues. ChemBioChem 11:1803–1815CrossRefGoogle Scholar
  10. 10.
    Arnison PG, Bibb MJ, Bierbaum G, Bowers AA, Bugni TS, Bulaj G et al (2013) Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature. Nat Prod Rep 30:108–160CrossRefGoogle Scholar
  11. 11.
    Leikoski N, Liu L, Jokela J, Wahlsten M, Gugger M, Calteau A et al (2013) Genome mining expands the chemical diversity of the cyanobactin family to include highly modified linear peptides. Chem Biol 20:1033–1043CrossRefGoogle Scholar
  12. 12.
    Schmidt EW, Nelson JT, Rasko DA, Sudek S, Eisen JA, Haygood MG et al (2005) Patellamide A and C biosynthesis by a microcin-like pathway in Prochloron didemni, the cyanobacterial symbiont of Lissoclinum patella. Proc Natl Acad Sci U S A 102:7315–7320CrossRefGoogle Scholar
  13. 13.
    Donia MS, Ravel J, Schmidt EW (2008) A global assembly line for cyanobactins. Nat Chem Biol 4:341–343CrossRefGoogle Scholar
  14. 14.
    Koehnke J, Bent A, Houssen WE, Zollman D, Morawitz F, Shirran S et al (2012) The mechanism of patellamide macrocyclization revealed by the characterization of the PatG macrocyclase domain. Nat Struct Mol Biol 19:767–772CrossRefGoogle Scholar
  15. 15.
    Lee J, McIntosh J, Hathaway BJ, Schmidt EW (2009) Using marine natural products to discover a protease that catalyzes peptide macrocyclization of diverse substrates. J Am Chem Soc 131:2122–2124CrossRefGoogle Scholar
  16. 16.
    Koehnke J, Bent AF, Zollman D, Smith K, Houssen WE, Zhu X et al (2013) The cyanobactin heterocyclase enzyme: a processive adenylase that operates with a defined order of reaction. Angew Chem Int Ed 52:13991–13396CrossRefGoogle Scholar
  17. 17.
    Bent AF, Mann G, Houssen WE, Mykhaylyk V, Duman R, Thomas L et al (2016) Structure of the cyanobactin oxidase ThcOx from Cyanothece sp. PCC 7425, the first structure to be solved at Diamond Light Source beamline I23 by means of S-SAD. Acta Crystallogr D Struct Biol 72:1174–1180CrossRefGoogle Scholar
  18. 18.
    Sardar D, Hao Y, Lin Z, Morita M, Nair SK, Schmidt EW (2017) Enzymatic N- and C-protection in cyanobactin RiPP natural products. J Am Chem Soc 139:2884–2887CrossRefGoogle Scholar
  19. 19.
    Parajuli A, Kwak DH, Dalponte L, Leikoski N, Galica T, Umeobika U et al (2016) A unique tryptophan C-prenyltransferase from the kawaguchipeptin biosynthetic pathway. Angew Chem Int Ed 55:3596–3599CrossRefGoogle Scholar
  20. 20.
    McIntosh JA, Donia MS, Nair SK, Schmidt EW (2011) Enzymatic basis of ribosomal peptide prenylation in cyanobacteria. J Am Chem Soc 133:13698–13705CrossRefGoogle Scholar
  21. 21.
    Donia MS, Schmidt EW (2011) Linking chemistry and genetics in the growing cyanobactin natural products family. Chem Biol 18:508–519CrossRefGoogle Scholar
  22. 22.
    Houssen WE, Bent AF, McEwan AR, Pieiller N, Tabudravu J, Koehnke J et al (2014) An efficient method for the in vitro production of azol(in)e-based cyclic peptides. Angew Chem Int Ed 53:14171–14174CrossRefGoogle Scholar
  23. 23.
    Ruffner DE, Schmidt EW, Heemstra JR (2015) Assessing the combinatorial potential of the RiPP cyanobactin tru pathway. ACS Synth Biol 4:482–492CrossRefGoogle Scholar
  24. 24.
    Koehnke J, Mann G, Bent AF, Ludewig H, Shirran S, Botting C et al (2015) Structural analysis of leader peptide binding enables leader-free cyanobactin processing. Nat Chem Biol 11:558–563CrossRefGoogle Scholar
  25. 25.
    Houssen WE, Koehnke J, Zollman D, Vendome J, Raab A, Smith MCM et al (2012) The discovery of new cyanobactins from Cyanothece PCC 7425 defines a new signature for processing of patellamides. ChemBioChem 13:2683–2689CrossRefGoogle Scholar
  26. 26.
    Agarwal V, Pierce E, McIntosh J, Schmidt EW, Nair SK (2012) Structures of cyanobactin maturation enzymes define a family of transamidating proteases. Chem Biol 19:1411–1422CrossRefGoogle Scholar
  27. 27.
    McIntosh JA, Donia MS, Schmidt EW (2010) Insights into heterocyclization from two highly similar enzymes. J Am Chem Soc 132:4089–4091CrossRefGoogle Scholar
  28. 28.
    Bent AF, Koehnke J, Houssen WE, Smith MCM, Jaspars M, Naismith JH (2013) Structure of PatF from Prochloron didemni. Acta Crystallogr F Struct Biol Commun 69:618–623CrossRefGoogle Scholar
  29. 29.
    Mann G, Koehnke J, Bent AF, Graham R, Houssen W, Jaspars M et al (2014) The structure of the cyanobactin domain of unknown function from PatG in the patellamide gene cluster. Acta Crystallogr F Struct Biol Commun 70:1597–1603CrossRefGoogle Scholar
  30. 30.
    Koehnke J, Bent AF, Houssen WE, Mann G, Jaspars M, Naismith JH (2014) The structural biology of patellamide biosynthesis. Curr Opin Struct Biol 29:112–121CrossRefGoogle Scholar
  31. 31.
    Donia MS, Hathaway BJ, Sudek S, Haygood MG, Rosovitz MJ, Ravel J et al (2006) Natural combinatorial peptide libraries in cyanobacterial symbionts of marine ascidians. Nat Chem Biol 2:729–735CrossRefGoogle Scholar
  32. 32.
    Brás NF, Ferreira P, Calixto AR, Jaspars M, Houssen W, Naismith JH et al (2016) The catalytic mechanism of the marine-derived macrocyclase PatGmac. Chem A Eur J 22:13089–13097CrossRefGoogle Scholar
  33. 33.
    Schechter I, Berger A (1967) On the size of the active site in proteases. I. Papain. Biochem Biophys Res Commun 27:157–162CrossRefGoogle Scholar
  34. 34.
    Booth J, Alexandru-Crivac C, Rickaby K, Nneoyiegbe A, Umeobika C, McEwan AR et al (2017) A blind test of computational technique for predicting the likelihood of peptide sequences to cyclize. J Phys Chem Lett 8:2310–2315CrossRefGoogle Scholar
  35. 35.
    Sivonen K, Leikoski N, Fewer DP, Jokela J (2010) Cyanobactins—ribosomal cyclic peptides produced by cyanobacteria. Appl Microbiol Biotechnol 86:1213–1225CrossRefGoogle Scholar
  36. 36.
    McIntosh JA, Robertson CR, Agarwal V, Nair SK, Bulaj GW, Schmidt EW (2010) Circular logic: nonribosomal peptide-like macrocyclization with a ribosomal peptide catalyst. J Am Chem Soc 132:15499–15501CrossRefGoogle Scholar
  37. 37.
    Oueis E, Jaspars M, Westwood NJ, Naismith JH (2016) Enzymatic macrocyclization of 1,2,3-triazole peptide mimetics. Angew Chem Int Ed 55:5842–5845CrossRefGoogle Scholar
  38. 38.
    Koehnke J, Morawitz F, Bent AF, Houssen WE, Shirran SL, Fuszard MA et al (2013) An enzymatic route to selenazolines. ChemBioChem 14:564–567CrossRefGoogle Scholar
  39. 39.
    Oueis E, Nardone B, Jaspars M, Westwood NJ, Naismith JH (2017) Synthesis of hybrid cyclopeptides via enzymatic macrocyclization. Chemistry Open 6:11–14Google Scholar
  40. 40.
    Oueis E, Stevenson H, Jaspars M, Westwood NJ, Naismith JH (2017) Bypassing the proline/thiazoline requirement of the macrocyclase PatG. Chem Commun 53:12274–12277CrossRefGoogle Scholar
  41. 41.
    Ziemert N, Ishida K, Quillardet P, Bouchier C, Hertweck C, de Marsac NT et al (2008) Microcyclamide biosynthesis in two strains of Microcystis aeruginosa: from structure to genes and vice versa. Appl Environ Microbiol 74:1791–1797CrossRefGoogle Scholar
  42. 42.
    Alexandru-Crivac C, Umeobika C, Leikoski N, Jokela J, Rickabi K, Grilo AM et al (2017) Cyclic peptide production using a macrocyclase with enhanced substrate promiscuity and relaxed recognition determinants. Chem Commun 53:10656–10659CrossRefGoogle Scholar
  43. 43.
    Falorni M, Conti S, Giacomelli G, Cossu S, Soccolini F (1995) Optically active 4-oxaproline derivatives: new useful chiral synthons derived from serine and threonine. Tetrahedron Asymmetry 6:287–294CrossRefGoogle Scholar
  44. 44.
    Weinstein AB, Schuman DP, Tan ZX, Stahl SS (2013) Synthesis of vicinal aminoalcohols by stereoselective aza-wacker cyclizations: access to (-)-acosamine by redox relay. Angew Chem Int Ed 52:11867–11870CrossRefGoogle Scholar
  45. 45.
    Liu H, Naismith JH (2009) A simple and efficient expression and purification system using two newly constructed vectors. Protein Expr Purif 63:102–111CrossRefGoogle Scholar
  46. 46.
    Studier FW (2005) Protein production by auto-induction in high-density shaking cultures. Protein Expr Purif 41:207–234CrossRefGoogle Scholar
  47. 47.
    Dalponte L, Parajuli A, Younger E, Mattila A, Jokela J, Wahlsten M, Leikoski N, Sivonen K, Jarmusch SA, Houssen WE, Fewer DP (2018) N-prenylation of tryptophan by an aromatic prenyltransferase from the cyanobactin biosynthetic pathway. Biochemistry 57(50):6860–6867CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Wael E. Houssen
    • 1
    • 2
    • 3
  1. 1.Marine Biodiscovery Centre, Chemistry DepartmentUniversity of AberdeenAberdeenUK
  2. 2.Institute of Medical SciencesUniversity of AberdeenAberdeenUK
  3. 3.Pharmacognosy Department, Faculty of PharmacyMansoura UniversityMansouraEgypt

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