Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Peptide cyclization catalysed by the thioesterase domain of tyrocidine synthetase

Nature volume 407, pages 215–218 (2000)Cite this article

Abstract

In the biosynthesis of many macrocyclic natural products by multidomain megasynthases, a carboxy-terminal thioesterase (TE) domain is involved in cyclization and product release1,2; however, it has not been determined whether TE domains can catalyse macrocyclization (and elongation in the case of symmetric cyclic peptides) independently of upstream domains. The inability to decouple the TE cyclization step from earlier chain assembly steps has precluded determination of TE substrate specificity, which is important for the engineered biosynthesis of new compounds1. Here we report that the excised TE domain from tyrocidine synthetase efficiently catalyses cyclization of a decapeptide-thioester to form the antibiotic tyrocidine A, and can catalyse pentapeptide-thioester dimerization followed by cyclization to form the antibiotic gramicidin S. By systematically varying the decapeptide-thioester substrate and comparing cyclization rates, we also show that only two residues (one near each end of the decapeptide) are critical for cyclization. This specificity profile indicates that the tyrocidine synthetase TE, and by analogy many other TE domains, will be able to cyclize and release a broad range of new substrates and products produced by engineered enzymatic assembly lines.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Role of the thioesterase (TE) domain in biosynthesis of the cyclic decapeptide antibiotic tyrocidine A.
Figure 2: Cyclization activity and substrate specificity of the TycC TE domain.
Figure 3: Role of the TE domain in biosynthesis of the cyclic decapeptide antibiotic gramicidin S.
Figure 4: The tyrocidine synthetase thioesterase domain (TycC TE) catalyses dimerization of the pentapeptide-SNAC GLP5 and cyclization of the resulting decapeptide-SNAC to form gramicidin S.

Similar content being viewed by others

References

  1. Cane, D. E., Walsh, C. T. & Khosla, C. Harnessing the biosynthetic code: combinations, permutations and mutations. Science 282, 63– 68 (1998).

    Article  CAS  Google Scholar 

  2. Marahiel, M. A., Stachelhaus, T. & Mootz, H. D. Modular peptide synthetases involved in nonribosomal peptide synthesis. Chem. Rev. 97, 2651– 2674 (1997).

    Article  CAS  Google Scholar 

  3. Lambalot, R. H. et al. A new enzyme superfamily–the phosphopantetheinyl-transferases. Chem. Biol. 3, 923–936 (1996).

    Article  CAS  Google Scholar 

  4. Lawson, D. M. et al. Structure of a myristoyl-ACP-specific thioesterase from Vibrio harveyi. Biochemistry 33, 9382 –9388 (1994).

    Article  CAS  Google Scholar 

  5. Li, J., Szittner, R., Derewenda, Z. S. & Meighen, E. A. Conversion of serine-114 to cysteine-114 and the role of the active site nucleophile in acyl transfer by myristoyl-ACP thioesterase from Vibrio harveyi. Biochemistry 35, 9967– 9973 (1996).

    Article  CAS  Google Scholar 

  6. Shaw-Reid, C. A. et al. Assembly line enzymology by multimodular nonribosomal peptide synthetases: the thioesterase domain of E. coli EntF catalyses both elongation and cyclolactonization. Chem. Biol. 6, 385–400 (1999).

    Article  CAS  Google Scholar 

  7. Aggarwal, R., Caffrey, P., Leadlay, P. F., Smith, C. J. & Staunton, J. The thioesterase domain of the erythromycin-producing polyketide synthase: mechanistic studies in vitro to investigate its mode of action and substrate specificity. J. Chem. Soc. Chem. Comm. 15, 1519– 1520 (1995).

    Article  Google Scholar 

  8. Gokhale, R. S., Hunziker, D., Cane, D. E. & Khosla, C. Mechanism and specificity of the terminal thioesterase domain from the erythromycin polyketide synthase. Chem. Biol. 6, 117– 125 (1998).

    Article  Google Scholar 

  9. Cortes, J. et al. Repositioning of a domain in a modular polyketide synthase to promote specific chain cleavage. Science 268, 1487–1489 (1995).

    Article  ADS  CAS  Google Scholar 

  10. Kao, C. M. et al. Gain of function mutagenesis of the erythromycin polyketide synthase. 2. Engineered biosynthesis of an eight-membered ring tetraketide lactone. J. Am. Chem. Soc. 119, 11339– 11340 (1997).

    Article  CAS  Google Scholar 

  11. Jacobsen, J. R., Hutchinson, C. R., Cane, D. E. & Khosla, C. Precursor-directed biosynthesis of erythromycin analogs by an engineered polyketide synthase. Science 277, 367– 369 (1997).

    Article  CAS  Google Scholar 

  12. McDaniel, R. et al. Multiple genetic modifications of the erythromycin polyketide synthase to produce a library of novel “unnatural” natural products. Proc. Natl Acad. Sci. USA 96, 1846– 1851 (1999).

    Article  ADS  CAS  Google Scholar 

  13. Tang, L., Fu, H. & McDaniel, R. Formation of functional heterologous complexes using subunits from the picromycin, erythromycin and oleandomycin polyketide synthases. Chem. Biol. 7, 77–84 ( 2000).

    Article  CAS  Google Scholar 

  14. Xue, Y. & Sherman, D. H. Alternative modular polyketide synthase expression controls macrolactone structure. Nature 403, 571–575 (2000).

    Article  ADS  CAS  Google Scholar 

  15. Stachelhaus, T., Schneider, A. & Marahiel, M. A. Rational design of peptide antibiotics by targeted replacement of bacterial and fungal domains. Science 269, 69–72 (1995).

    Article  ADS  CAS  Google Scholar 

  16. de Ferra, F., Rodriguez, F., Tortora, O., Tosi, C. & Grandi, G. Engineering of peptide synthetases. J. Biol. Chem 272, 25304– 25309 (1997).

    Article  CAS  Google Scholar 

  17. Mootz, H. D., Schwarzer, D. & Marahiel, M. A. Construction of hybrid peptide synthetases by module and domain fusions. Proc. Natl Acad. Sci. USA 97, 5848–5853 (2000).

    Article  ADS  CAS  Google Scholar 

  18. Belshaw, P. J., Walsh, C. T. & Stachelhaus, T. Aminoacyl-CoAs as probes of condensation domain selectivity in nonribosomal peptide synthesis. Science 284, 486–489 (1999).

    Article  ADS  CAS  Google Scholar 

  19. Mootz, H. D. & Marahiel, M. A. The tyrocidine biosynthesis operon of Bacillus brevis: complete nucleotide sequence and biochemical characterization of functional internal adenylation domains. J. Bacteriol. 179, 6843–6850 ( 1997).

    Article  CAS  Google Scholar 

  20. Roskoski, R., Kleinkauf, H., Gevers, W. & Lipmann, F. Isolation of enzyme-bound peptide intermediates in tyrocidine biosynthesis. Biochemistry 9, 4846–4851 (1970).

    Article  Google Scholar 

  21. Hori, K. et al. Molecular cloning and nucleotide sequence of the gramicidin S synthetase 1 gene. J. Biochem. (Tokyo) 106, 639–645 (1989).

    Article  CAS  Google Scholar 

  22. Turgay, K., Krause, M. & Marahiel, M. A. Four homologous domains in the primary structure of GrsB are related to domains in a superfamily of adenylate-forming enzymes. Mol. Microbiol. 6, 529– 546 (1992).

    Article  CAS  Google Scholar 

  23. Boger, D. L. & Ichikawa, S. Total syntheses of thiocoraline and BE-22179: establishment of relative and absolute stereochemistry. J. Am. Chem. Soc. 122, 2956–2957 (2000).

    Article  CAS  Google Scholar 

  24. Jackson, D. Y. et al. A designed peptide ligase for total synthesis of ribonuclease A with unnatural catalytic residues. Science 266, 243–247 (1994).

    Article  ADS  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by grants from the NIH (to C.T.W.), the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie (to M.A.M.), a NIH postdoctoral fellowship (to J.W.T.) and a PhD fellowship of the Stiftung Stipendien-Fonds des Verbandes der Chemischen Industrie (to H.D.M.).

Author information

Authors and Affiliations

  1. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, 02115, Massachusetts, USA

    John W. Trauger, Rahul M. Kohli & Christopher T. Walsh

  2. Biochemie/Fachbereich Chemie, Philipps-Universität Marburg, Hans-Meerwein-Strasse, Marburg, 35032, Germany

    Henning D. Mootz & Mohamed A. Marahiel

Authors
  1. John W. Trauger
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rahul M. Kohli
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Henning D. Mootz
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Mohamed A. Marahiel
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Christopher T. Walsh
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Christopher T. Walsh.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Trauger, J., Kohli, R., Mootz, H. et al. Peptide cyclization catalysed by the thioesterase domain of tyrocidine synthetase. Nature 407, 215–218 (2000). https://doi.org/10.1038/35025116

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/35025116

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing