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.

  • Article
  • Published:

A dual transacylation mechanism for polyketide synthase chain release in enacyloxin antibiotic biosynthesis

Nature Chemistry volume 11, pages 906–912 (2019)Cite this article

Subjects

Abstract

Polyketide synthases assemble diverse natural products with numerous important applications. The thioester intermediates in polyketide assembly are covalently tethered to acyl carrier protein domains of the synthase. Several mechanisms for polyketide chain release are known, contributing to natural product structural diversification. Here, we report a dual transacylation mechanism for chain release from the enacyloxin polyketide synthase, which assembles an antibiotic with promising activity against Acinetobacter baumannii. A non-elongating ketosynthase domain transfers the polyketide chain from the final acyl carrier protein domain of the synthase to a separate carrier protein, and a non-ribosomal peptide synthetase condensation domain condenses it with (1S,3R,4S)-3,4-dihydroxycyclohexane carboxylic acid. Molecular dissection of this process reveals that non-elongating ketosynthase domain-mediated transacylation circumvents the inability of the condensation domain to recognize the acyl carrier protein domain. Several 3,4-dihydroxycyclohexane carboxylic acid analogues can be employed for chain release, suggesting a promising strategy for producing enacyloxin analogues.

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

Fig. 1: Proposed mechanism for chain release from the enacyloxin PKS and confirmation that Bamb_5915 and Bamb_5917 are involved in enacyloxin biosynthesis.
Fig. 2: Acyl transfer and active site acylation assays reveal that the KS0 domain of Bamb_5919 functions as a transacylase.
Fig. 3: Functional characterization of Bamb_5915.
Fig. 4: In vitro reconstitution of chain release from the enacyloxin PKS.
Fig. 5: Bamb_5915 tolerates a wide range of acyl acceptors.
Fig. 6: Examples of trans-AT PKS architectures containing KS0 domains.

Similar content being viewed by others

Data availability

The genome sequence of B. ambifaria BCC0203 has been deposited in the European Nucleotide Archive (accession no. ERS782625). All other data are available from the authors upon request.

References

  1. Hertweck, C. The biosynthetic logic of polyketide diversity. Angew. Chem. Int. Ed. 48, 4688–4716 (2009).

    Article  CAS  Google Scholar 

  2. Fischbach, M. A. & Walsh, C. T. Assembly-line enzymology for polyketide and nonribosomal peptide antibiotics: logic, machinery and mechanisms. Chem. Rev. 106, 3468–3496 (2006).

    Article  CAS  Google Scholar 

  3. Du, L. & Lou, L. PKS and NRPS release mechanisms. Nat. Prod. Rep. 27, 255–278 (2010).

    Article  CAS  Google Scholar 

  4. Tang, G.-L., Cheng, Y.-Q. & Shen, B. Leinamycin biosynthesis revealing unprecedented architectural complexity for a hybrid polyketide synthase and nonribosomal peptide synthetase. Chem. Biol. 11, 33–45 (2004).

    Article  CAS  Google Scholar 

  5. Awodi, U. R., Ronan, J. L., Masschelein, J., de Los Santos, E. L. C. & Challis, G. L. Thioester reduction and aldehyde transamination are universal steps in actinobacterial polyketide alkaloid biosynthesis. Chem. Sci. 8, 411–415 (2017).

    Article  CAS  Google Scholar 

  6. Mullowney, M. W., McClure, R. A., Robey, M. T., Kelleher, N. L. & Thomson, R. J. Natural products from thioester reductase containing biosynthetic pathways. Nat. Prod. Rep. 35, 847–878 (2018).

    Article  CAS  Google Scholar 

  7. Mo, S. J. et al. Elucidation of the Streptomyces coelicolor pathway to 2-undecylpyrrole, a key intermediate in undecylprodiginine and streptorubin B biosynthesis. Chem. Biol. 15, 137–148 (2008).

    Article  CAS  Google Scholar 

  8. Hu, D. X., Withall, D. M., Challis, G. L. & Thomson, R. J. Structure, chemical synthesis and biosynthesis of prodiginine natural products. Chem. Rev. 116, 7818–7853 (2016).

    Article  CAS  Google Scholar 

  9. Sun, Y. et al. In vitro reconstruction of tetronate RK-682 biosynthesis. Nat. Chem. Biol. 6, 99–101 (2010).

    Article  CAS  Google Scholar 

  10. Watanabe, T., Izaki, K. & Takahashi, H. New polyenic antibiotics active against Gram-positive and -negative bacteria. I. Isolation and purification of antibiotics produced by Gluconobacter sp. W-315. J. Antibiot. ( Tokyo ) 35, 1141–1147 (1982).

    Article  CAS  Google Scholar 

  11. Watanabe, T. et al. New polyenic antibiotics active against Gram-positive and Gram-negative bacteria. IV. Structural elucidation of enacyloxin IIa. J. Antibiot. ( Tokyo ) 45, 470–475 (1992).

    Article  CAS  Google Scholar 

  12. Mahenthiralingam, E. et al. Enacyloxins are products of an unusual hybrid modular polyketide synthase encoded by a cryptic Burkholderia ambifaria genomic island. Chem. Biol. 18, 665–677 (2011).

    Article  CAS  Google Scholar 

  13. Cetin, R. et al. Enacyloxin IIa, an inhibitor of protein biosynthesis that acts on elongation factor Tu and the ribosome. EMBO J. 15, 2604–2611 (1996).

    Article  CAS  Google Scholar 

  14. Parmeggiani, A. et al. Enacyloxin IIa pinpoints a binding pocket of elongation factor Tu for development of novel antibiotics. J. Biol. Chem. 281, 2893–2900 (2006).

    Article  CAS  Google Scholar 

  15. Zuurmond, A. M., Olsthoorn-Tieleman, L. N., Martien de Graaf, J., Parmeggiani, A. & Kraal, B. Mutant EF-Tu species reveal novel features of the enacyloxin IIa inhibition mechanism on the ribosome. J. Mol. Biol. 294, 627–637 (1999).

    Article  CAS  Google Scholar 

  16. Helfrich, E. J. N. & Piel, J. Biosynthesis of polyketides by trans-AT polyketide synthases. Nat. Prod. Rep. 33, 231–316 (2016).

    Article  CAS  Google Scholar 

  17. Huang, Y., Tang, G.-L., Pan, G., Chang, C.-Y. & Shen, B. Characterization of the ketosynthase and acyl carrier protein domains at the LnmI nonribosomal peptide synthetase–polyketide synthase interface for leinamycin biosynthesis. Org. Lett. 18, 4288–4291 (2016).

    Article  CAS  Google Scholar 

  18. He, H.-Y., Tang, M.-C., Zhang, F. & Tang, G.-L. Cis-double bond formation by thioesterase and transfer by ketosynthase in FR901464 biosynthesis. J. Am. Chem. Soc. 136, 4488–4491 (2014).

    Article  CAS  Google Scholar 

  19. Flannagan, R. S., Linn, T. & Valvano, M. A. A system for the construction of targeted unmarked gene deletions in the genus Burkholderia. Environ. Microbiol. 10, 1652–1660 (2008).

    Article  CAS  Google Scholar 

  20. Li, W. et al. Broad spectrum antibiotic activity and disease suppression by the potential biocontrol agent Burkholderia ambifaria BC-F. Crop Prot. 21, 129–135 (2002).

    Article  Google Scholar 

  21. Thomas, I., Martin, C. J., Wilkinson, C. J., Staunton, J. & Leadlay, P. F. Skipping in a hybrid polyketide synthase: evidence for ACP-to-ACP chain transfer. Chem. Biol. 9, 781–787 (2002).

    Article  CAS  Google Scholar 

  22. Lin, S., Van Lanen, S. G. & Shen, B. A free-standing condensation enzyme catalyzing ester bond formation in C-1027 biosynthesis. Proc. Natl Acad. Sci. U.S.A. 106, 4183–4188 (2009).

    Article  CAS  Google Scholar 

  23. Kosol, S. et al. Structural basis for chain release from the enacyloxin polyketide synthase. Nat. Chem. https://doi.org/10.1038/s41557-019-0335-5 (2019).

  24. Ye, Z., Musiol, E. M., Weber, T. & Williams, G. J. Reprogramming acyl carrier protein interactions of an acyl-CoA promiscuous trans-acyltransferase. Cell Chem. Biol. 21, 636–646 (2014).

    CAS  Google Scholar 

Download references

Acknowledgements

This research was supported by grants from the BBSRC (BB/L021692/1 to G.L.C., E.M. and J.P. and BB/K002341/1 to G.L.C.), the European Commission (through a Marie Sklodowska-Curie Fellowship to J.M., contract no. 656067) and the Research Foundation Flanders (to J.M.). Support from the University of Warwick through a fellowship from the Institute of Advanced Study (to P.K.S.) and a PhD studentship (to C.H.) is acknowledged. The Bruker maXis Impact and maXis II UHPLC-ESI-Q-TOF-MS systems used in this research were funded by the BBSRC (BB/K002341/1 and BB/M017982/1, respectively). G.L.C. is the recipient of a Wolfson Research Merit Award from the Royal Society (WM130033). Z.L.Y. was funded by a Cardiff University Undergraduate Research Opportunities Programme (CUROP) award to C.J. and E.M. The authors thank M. Tosin for providing the Sfp, PanK, PPAT and DPCK expression vectors. Correspondence and requests for materials should be addressed to G.L.C.

Author information

Author notes

  1. Joleen Masschelein

    Present address: Rega Institute for Medical Research, KU Leuven, Leuven, Belgium

  2. Paulina K. Sydor

    Present address: Pure Biologics S.A., Wrocław, Poland

  3. Cerith Jones

    Present address: Faculty of Computing, Engineering and Science, University of South Wales, Pontypridd, UK

  4. These authors contributed equally: Joleen Masschelein and Paulina K. Sydor.

Authors and Affiliations

  1. Department of Chemistry, University of Warwick, Coventry, UK

    Joleen Masschelein, Paulina K. Sydor, Christian Hobson, Rhiannon Howe, Douglas M. Roberts & Gregory L. Challis

  2. Cardiff School of Biosciences, Cardiff University, Cardiff, UK

    Cerith Jones, Zhong Ling Yap & Eshwar Mahenthiralingam

  3. Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, UK

    Julian Parkhill

  4. Warwick Integrative Synthetic Biology Centre, University of Warwick, Coventry, UK

    Gregory L. Challis

  5. Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia

    Gregory L. Challis

Authors
  1. Joleen Masschelein
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Paulina K. Sydor
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Christian Hobson
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Rhiannon Howe
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Cerith Jones
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Douglas M. Roberts
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Zhong Ling Yap
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Julian Parkhill
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Eshwar Mahenthiralingam
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Gregory L. Challis
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.M., P.K.S. and G.L.C. designed the experiments. P.K.S. cloned the B. ambifaria genes into pET151 and created the site-directed mutants. J.M., P.K.S. and C.H. carried out the in vitro biochemical experiments. C.J., Z.L.Y. and E.M. identified strain BCC0203 as being amenable to genetic manipulation, established selection conditions and constructed and complemented the B. ambifaria gene deletion mutants. J.P. supervised sequencing and assembly of the B. ambifaria BCC0203 genome. C.J. analysed the genome sequence of strain BCC0203 and made the initial comparisons to the genome of the AMMD strain. C.H., R.H., D.M.R., P.K.S. and J.M. synthesized substrates. J.M. and G.L.C. wrote the manuscript with input from the other authors.

Corresponding author

Correspondence to Gregory L. Challis.

Ethics declarations

Competing interests

The broad substrate specificity of Bamb_5915, exploited for the production of enacyloxin analogues, is the subject of International (PCT) Patent Application no. PCT/GB2018/051058 (applicant, University of Warwick; inventors, G.L.C., J.M., C.H., X. Jian; status of application, filed 23 April 2018).

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

The Supplementary Information file contains details of the methods used, and Supplementary Figs. 1–15 and Supplementary Tables 1–2

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Masschelein, J., Sydor, P.K., Hobson, C. et al. A dual transacylation mechanism for polyketide synthase chain release in enacyloxin antibiotic biosynthesis. Nat. Chem. 11, 906–912 (2019). https://doi.org/10.1038/s41557-019-0309-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41557-019-0309-7

This article is cited by

Search

Advanced search

Quick links

Nature Briefing Microbiology

Sign up for the Nature Briefing: Microbiology newsletter — what matters in microbiology research, free to your inbox weekly.

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