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Structure and biosynthesis of a macrocyclic peptide containing an unprecedented lysine-to-tryptophan crosslink

Nature Chemistry volume 7, pages 431–437 (2015)Cite this article

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Abstract

Streptococcal bacteria use peptide signals as a means of intraspecies communication. These peptides can contain unusual post-translational modifications, providing opportunities for expanding our understanding of nature's chemical and biosynthetic repertoires. Here, we have combined tools from natural products discovery and mechanistic enzymology to elucidate the structure and biosynthesis of streptide, a streptococcal macrocyclic peptide. We show that streptide bears an unprecedented post-translational modification involving a covalent linkage between two unactivated carbons within the side chains of lysine and tryptophan. The biosynthesis of streptide was addressed by genetic and biochemical studies. The former implicated a new SPASM-domain-containing radical SAM enzyme StrB, while the latter revealed that StrB contains two [4Fe–4S] clusters and installs the unusual lysine-to-tryptophan crosslink in a single step. By intramolecularly stitching together the side chains of lysine and tryptophan, StrB provides a new route for biosynthesizing macrocyclic peptides.

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Figure 1: Biosynthetic gene cluster and structure of streptide.
Figure 2: Proposed biosynthesis for streptide.
Figure 3: StrB contains two [4Fe–4S] clusters.
Figure 4: StrB catalyses Lys-to-Trp crosslink formation in StrA.
Figure 5: Mechanistic model for Lys-to-Trp crosslink formation catalysed by StrB.

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References

  1. Arnison, P. G. et al. Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature. Nat. Prod. Rep. 30, 108–160 (2013).

    Article  CAS  Google Scholar 

  2. Trauger, J. W., Kohli, R. M., Mootz, H. D., Marahiel, M. A. & Walsh, C. T. Peptide cyclization catalysed by the thioesterase domain of tyrocidine synthetase. Nature 407, 215–218 (2000).

    Article  CAS  Google Scholar 

  3. Duquesne, S. et al. Two enzymes catalyze the maturation of a lasso peptide in Escherichia coli. Chem. Biol. 14, 793–803 (2007).

    Article  CAS  Google Scholar 

  4. Dorenbos, R. et al. Thiol-disulfide oxidoreductases are essential for the production of the lantibiotic sublancin 168. J. Biol. Chem. 277, 16682–16688 (2002).

    Article  CAS  Google Scholar 

  5. Ziemert, N., Ishida, K., Liaimer, A., Hertweck, C. & Dittmann, E. Ribosomal synthesis of tricyclic depsipeptides in bloom-forming cyanobacteria. Angew. Chem. Int. Ed. 47, 7756–7759 (2008).

    Article  CAS  Google Scholar 

  6. Philmus, B., Christiansen, G., Yoshida, W. Y. & Hemscheidt, T. K. Post-translational modification in microviridin biosynthesis. ChemBioChem 9, 3066–3073 (2008).

    Article  CAS  Google Scholar 

  7. Ji, G., Beavis, R. & Novick, R. P. Bacterial interference caused by autoinducing peptide variants. Science 276, 2027–2030 (1997).

    Article  CAS  Google Scholar 

  8. Mayville, P. et al. Structure–activity analysis of synthetic autoinducing thiolactone peptides from Staphylococcus aureus responsible for virulence. Proc. Natl Acad. Sci. USA 96, 1218–1223 (1999).

    Article  CAS  Google Scholar 

  9. Li, B. et al. Structure and mechanism of the lantibiotic cyclase involved in nisin biosynthesis. Science 311, 1464–1467 (2006).

    Article  CAS  Google Scholar 

  10. Flühe, L. et al. The radical SAM enzyme AlbA catalyzes thioether bond formation in subtilosin A. Nature Chem. Biol. 8, 350–357 (2012).

    Article  Google Scholar 

  11. Müller, W. M., Schmiederer, T., Ensle, P. & Süssmuth, R. D. In vitro biosynthesis of the prepeptide of type-III lantibiotic labyrinthopeptin A2 including formation of a C–C bond as a post-translational modification. Angew. Chem. Int. Ed. 49, 2436–2440 (2010).

    Article  Google Scholar 

  12. Zerbe, K. et al. An oxidative phenol coupling reaction catalyzed by OxyB, a cytochrome P450 from the vancomycin-producing microorganism. Angew. Chem. Int. Ed. 43, 6709–6713 (2004).

    Article  CAS  Google Scholar 

  13. Dunbar, K. L. & Mitchell, D. A. Revealing nature's synthetic potential through the study of ribosomal natural product biosynthesis. ACS Chem. Biol. 8, 473–487 (2013).

    Article  CAS  Google Scholar 

  14. Ibrahim, M. et al. Control of the transcription of a short gene encoding a cyclic peptide in Streptococcus thermophilus: a new quorum-sensing system? J. Bacteriol. 189, 8844–8854 (2007).

    Article  CAS  Google Scholar 

  15. Fleuchot, B. et al. Rgg proteins associated with internalized small hydrophobic peptides: a new quorum-sensing mechanism in streptococci. Mol. Microbiol. 80, 1102–1119 (2011).

    Article  CAS  Google Scholar 

  16. Gardan, R., Besset, C., Guillot, A., Gitton, C. & Monnet, V. The oligopeptide transport system is essential for the development of natural competence in Streptococcus thermophilus strain LMD-9. J. Bacteriol. 191, 4647–4655 (2009).

    Article  CAS  Google Scholar 

  17. Lyon, W. R., Gibson, C. M. & Caparon, M. G. A role for trigger factor and an rgg-like regulator in the transcription, secretion and processing of the cysteine proteinase of Streptococcus pyogenes. EMBO J. 17, 6263–6275 (1998).

    Article  CAS  Google Scholar 

  18. Chaussee, M. S., Ajdic, D. & Ferretti, J. J. The rgg gene of Streptococcus pyogenes NZ131 positively influences extracellular SPE B production. Infect. Immun. 67, 1715–1722 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Mashburn-Warren, L., Morrison, D. A. & Federle, M. J. A novel double-tryptophan peptide pheromone controls competence in Streptococcus spp. via an Rgg regulator. Mol. Microbiol. 78, 589–606 (2010).

    Article  CAS  Google Scholar 

  20. Fernandez, A., Borges, F., Gintz, B., Decaris, B. & Leblond-Bourget, N. The rggC locus, with a frameshift mutation, is involved in oxidative stress response by Streptococcus thermophilus. Arch. Microbiol. 186, 161–169 (2006).

    Article  CAS  Google Scholar 

  21. Qi, F., Chen, P. & Caufield, P. W. Functional analyses of the promoters in the lantibiotic mutacin II biosynthetic locus in Streptococcus mutans. Appl. Environ. Microbiol. 65, 652–658 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Kawulka, K. E. et al. Structure of subtilosin A, a cyclic antimicrobial peptide from Bacillus subtilis with unusual sulfur to alpha-carbon cross-links: formation and reduction of alpha-thio-alpha-amino acid derivatives. Biochemistry 43, 3385–3395 (2004).

    Article  CAS  Google Scholar 

  23. Rea, M. C. et al. Thuricin CD, a posttranslationally modified bacteriocin with a narrow spectrum of activity against Clostridium difficile. Proc. Natl Acad. Sci. USA 107, 9352–9357 (2010).

    Article  CAS  Google Scholar 

  24. Sit, C. S., McKay, R. T., Hill, C., Ross, R. P. & Vederas, J. C. The 3D structure of thuricin CD, a two-component bacteriocin with cysteine sulfur to α-carbon cross-links. J. Am. Chem. Soc. 133, 7680–7683 (2011).

    Article  CAS  Google Scholar 

  25. Sit, C. S., van Belkum, M. J., McKay, R. T., Worobo, R. W. & Vederas, J. C. The 3D solution structure of thurincin H, a bacteriocin with four sulfur to α-carbon crosslinks. Angew. Chem. Int. Ed. 50, 8718–8721 (2011).

    Article  CAS  Google Scholar 

  26. Güntert, P., Mumenthaler, C. & Wüthrich, K. Torsion angle dynamics for NMR structure calculation with the new program DYANA. J. Mol. Biol. 273, 283–298 (1997).

    Article  Google Scholar 

  27. Herrmann, T., Güntert, P. & Wüthrich, K. Protein NMR structure determination with automated NOE-identification in the NOESY spectra using the new software ATNOS. J. Biomol. NMR 24, 171–189 (2002).

    Article  CAS  Google Scholar 

  28. Haft, D. H. & Basu, M. K. Biological systems discovery in silico: radical S-adenosylmethionine protein families and their target peptides for posttranslational modification. J. Bacteriol. 193, 2745–2755 (2011).

    Article  CAS  Google Scholar 

  29. Haft, D. H. Bioinformatic evidence for a widely distributed, ribosomally produced electron carrier precursor, its maturation proteins, and its nicotinoprotein redox partners. BMC Genomics 12, 21–34 (2011).

    Article  CAS  Google Scholar 

  30. Frey, P. A. & Booker, S. J. Radical mechanisms of S-adenosylmethionine-dependent enzymes. Adv. Protein Chem. 58, 1–45 (2001).

    Article  CAS  Google Scholar 

  31. Booker, S. J. Anaerobic functionalization of unactivated C–H bonds. Curr. Opin. Chem. Biol. 13, 58–73 (2009).

    Article  CAS  Google Scholar 

  32. Broderick, J. B., Duffus, B. R., Duschene, K. S. & Shepard, E. M. Radical S-adenosylmethionine enzymes. Chem. Rev. 114, 4229–4317 (2014).

    Article  CAS  Google Scholar 

  33. Fang, Q., Peng, J. & Dierks, T. Post-translational formylglycine modification of bacterial sulfatases by the radical S-adenoyslmethionine protein AtsB. J. Biol. Chem. 279, 14570–14578 (2004).

    Article  CAS  Google Scholar 

  34. Berteau, O., Guillot, A., Benjdia, A. & Rabot, S. A new type of bacterial sulfatase reveals a novel maturation pathway in prokaryotes. J. Biol. Chem. 281, 22464–22470 (2006).

    Article  CAS  Google Scholar 

  35. Benjdia, A. et al. Anaerobic sulfatase-maturating enzymes: radical SAM enzymes able to catalyze in vitro sulfatase post-translational modification. J. Am. Chem. Soc. 129, 3462–3463 (2007).

    Article  CAS  Google Scholar 

  36. Grove, T. L., Lee, K. H., St Clair, J., Krebs, C. & Booker, S. J. In vitro characterization of AtsB, a radical SAM formylglycine-generating enzyme that contains three [4Fe–4S] clusters. Biochemistry 47, 7523–7538 (2008).

    Article  CAS  Google Scholar 

  37. Grove, T. L. et al. Further characterization of Cys-type and Ser-type anaerobic sulfatase maturating enzymes suggests a commonality in the mechanism of catalysis. Biochemistry 52, 2874–2887 (2013).

    Article  CAS  Google Scholar 

  38. Goldman, P. J. et al. X-ray structure of an AdoMet radical activase reveals an anaerobic solution for formylglycine posttranslational modification. Proc. Natl Acad. Sci. USA 110, 8519–8524 (2013).

    Article  CAS  Google Scholar 

  39. Lanz, N. D. et al. RlmN and AtsB as models for the overproduction and characterization of radical SAM proteins. Methods Enzymol. 516, 125–152 (2012).

    Article  CAS  Google Scholar 

  40. Johnson, D. C., Unciuleac, M. C. & Dean, D. R. Controlled expression and functional analysis of iron–sulfur cluster biosynthetic components within Azotobacter vinelandii. J. Bacteriol. 188, 7551–7561 (2006).

    Article  CAS  Google Scholar 

  41. Wecksler, S. R. et al. Pyrroloquinoline quinon biogenesis: demonstration that PqqE from Klebsiella pneumoniae is a radical S-adenosyl-L-methionine enzyme. Biochemistry 48, 10151–10161 (2009).

    Article  CAS  Google Scholar 

  42. Goldman, P. J., Grove, T. L., Booker, S. J. & Drennan, C. L. X-ray analysis of butirosin biosynthetic enzyme BtrN redefines structural motifs for AdoMet radical chemistry. Proc. Natl Acad. Sci. USA 110, 15949–15954 (2013).

    Article  CAS  Google Scholar 

  43. Flühe, L. et al. Two [4Fe–4S] clusters containing radical SAM enzyme SkfB catalzye thioether bond formation during the maturation of the sporulation killing factor. J. Am. Chem. Soc. 135, 959–962 (2013).

    Article  Google Scholar 

  44. Oman, T. J. & van der Donk, W. A. Follow the leader: the use of leader peptides to guide natural product biosynthesis. Nature Chem. Biol. 6, 9–18 (2010).

    Article  CAS  Google Scholar 

  45. Wu, W., Lieder, K. W., Reed, G. H. & Frey, P. A. Observation of a second substrate radical intermediate in the reaction of lysine 2,3-aminomutase: a radical centered on the beta-carbon of the alternative substrate, 4-thia-L-lysine. Biochemistry 34, 10532–10537 (1995).

    Article  CAS  Google Scholar 

  46. Ruszczycky, M. W., Choi, S. H. & Liu, H. W. Stoichiometry of the redox neutral deamination and oxidative dehydrogenation reactions catalzyed by the radical SAM enzyme DesII. J. Am. Chem. Soc. 132, 2359–2369 (2010).

    Article  CAS  Google Scholar 

  47. Grove, T. L., Ahlum, J. H., Sharma, P., Krebs, C. & Booker, S. J. A consensus mechanism for radical SAM-dependent dehydrogenation? BtrN contains two [4Fe–4S] clusters. Biochemistry 49, 3783–3785 (2010).

    Article  CAS  Google Scholar 

  48. Mitchell, J. Streptococcus mitis: walking the line between commensalism and pathogenesis. Mol. Oral Microbiol. 26, 89–98 (2011).

    Article  CAS  Google Scholar 

  49. Le Doare, K. & Heath, P. T. An overview of global GBS epidemiology. Vaccine 31, D7–D12 (2013).

    Article  Google Scholar 

  50. Fittipaldi, N., Segura, M., Grenier, D. & Gottschalk, M. Virulence factors involved in the pathogenesis of the infection caused by the swine pathogen and zoonotic agent Streptococcus suis. Future Microbiol. 7, 259–279 (2012).

    Article  CAS  Google Scholar 

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Acknowledgements

The authors thank P. Güntert and H. Arthanari for invaluable advice on CYANA, I. Pelczer and K. Connover at the Princeton Chemistry NMR facility for assistance with NMR data acquisition, S. Booker for the gift of pDB1282, K. Shatalin for the gift of pKS1, and Z. Brown for advice regarding peptide synthesis. This work was supported by the National Institutes of Health (grant no. GM098299, to M.R.S.) and by Princeton University start-up funds.

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  1. Kelsey R. Schramma and Leah B. Bushin: These authors contributed equally to this work

Authors and Affiliations

  1. Department of Chemistry, Princeton University, Princeton, New Jersey, USA

    Kelsey R. Schramma, Leah B. Bushin & Mohammad R. Seyedsayamdost

  2. Department of Molecular Biology, Princeton University, Princeton, New Jersey, USA

    Mohammad R. Seyedsayamdost

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K.R.S., L.B.B. and M.R.S. designed and executed experiments, and analysed data. M.R.S. conceived of the project and wrote the manuscript.

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Correspondence to Mohammad R. Seyedsayamdost.

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Schramma, K., Bushin, L. & Seyedsayamdost, M. Structure and biosynthesis of a macrocyclic peptide containing an unprecedented lysine-to-tryptophan crosslink. Nature Chem 7, 431–437 (2015). https://doi.org/10.1038/nchem.2237

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