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Unexpected enzyme-catalysed [4+2] cycloaddition and rearrangement in polyether antibiotic biosynthesis


Enzymes that catalyse remarkable Diels–Alder-like [4+2] cyclizations have been previously implicated in the biosynthesis of spirotetronate and spirotetramate antibiotics. Biosynthesis of the polyether antibiotic tetronasin is not expected to require such steps, yet the tetronasin gene cluster encodes enzymes Tsn11 and Tsn15, which are homologous to authentic [4+2] cyclases. Here, we show that deletion of Tsn11 led to accumulation of a late-stage intermediate, in which the two central rings of tetronasin and four of its twelve asymmetric centres remain unformed. In vitro reconstitution showed that Tsn11 catalyses an apparent inverse-electron-demand hetero-Diels–Alder-like [4+2] cyclization of this species to form an unexpected oxadecalin compound that is then rearranged by Tsn15 to form tetronasin. To gain structural and mechanistic insight into the activity of Tsn15, the crystal structure of a Tsn15-substrate complex has been solved at 1.7 Å resolution.

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Fig. 1: [4+2] cyclases in polyether tetronate biosynthesis.
Fig. 2: Functional characterization of the Diels-Alderase homologues Tsn11 and Tsn15 in tetronasin biosynthesis.
Fig. 3: The crystal structure of Tsn15.
Fig. 4: Structural homologues of Tsn15 and their respective reactions.
Fig. 5: Structure of Tsn15 and a Tsn15-substrate complex.
Fig. 6: The proposed mechanism for formation of the cyclohexane and tetrahydropyran rings of tetronasin.

Data availability

The tetronasin biosynthetic gene cluster sequence is available on GenBank (accession code: FJ462704). The crystal structure data is available on the PDB (accession codes: 6NOI (Tsn15) and 6NNW (Tsn15-substrate complex)). All other data that supports the findings of this study are available from the corresponding author on reasonable request.


  1. 1.

    Diels, O. & Alder, K. Synthesen in der hydroaromatischen Reihe. Justus Liebigs Ann. der Chem. 460, 98–122 (1928).

    CAS  Google Scholar 

  2. 2.

    Corey, E. J. Catalytic enantioselective Diels–Alder reactions: methods, mechanistic fundamentals, pathways, and applications. Angew. Chem. Int. Ed. Engl. 41, 1650–1667 (2002).

    CAS  PubMed  Google Scholar 

  3. 3.

    Lichman, B. R., O’Connor, S. E. & Kries, H. Biocatalytic strategies towards [4+ 2] cycloadditions. Chem. Eur. J. 25, 6864–6877 (2019).

    CAS  PubMed  Google Scholar 

  4. 4.

    Jeon, B., Wang, S.-A., Ruszczycky, M. W. & Liu, H. Natural [4+2]-cyclases. Chem. Rev. 117, 5367–5388 (2017).

    CAS  PubMed  Google Scholar 

  5. 5.

    Ma, S. M. et al. Complete reconstitution of a highly reducing iterative polyketide synthase. Science 326, 589–592 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Kasahara, K. et al. Solanapyrone synthase, a possible Diels–Alderase and iterative Type I polyketide synthase encoded in a biosynthetic gene cluster from Alternaria solani. ChemBioChem 11, 1245–1252 (2010).

    CAS  PubMed  Google Scholar 

  7. 7.

    Kim, H. J., Ruszczycky, M. W., Choi, S., Liu, Y. & Liu, H. Enzyme-catalysed [4+2] cycloaddition is a key step in the biosynthesis of Spinosyn A. Nature 473, 109–112 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Fage, C. D. et al. The structure of SpnF, a standalone enzyme that catalyzes [4+2] cycloaddition. Nat. Chem. Biol. 11, 256–258 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Hashimoto, T. et al. Biosynthesis of versipelostatin: identification of an enzyme-catalyzed [4+2]-cycloaddition required for macrocyclization of spirotetronate-containing polyketides. J. Am. Chem. Soc. 137, 572–575 (2015).

    CAS  PubMed  Google Scholar 

  10. 10.

    Byrne, M. J. et al. The catalytic mechanism of a natural Diels–Alderase revealed in molecular detail. J. Am. Chem. Soc. 138, 6095–6098 (2016).

    CAS  PubMed  Google Scholar 

  11. 11.

    Tian, Z. et al. An enzymatic [4+2] cyclization cascade creates the pentacyclic core of pyrroindomycins. Nat. Chem. Biol. 11, 259–265 (2015).

    CAS  PubMed  Google Scholar 

  12. 12.

    Zhang, Z. et al. Enzyme-catalyzed inverse-electron demand Diels–Alder reaction in the biosynthesis of antifungal ilicicolin H. J. Am. Chem. Soc. 141, 5659–5663 (2019).

    CAS  PubMed  Google Scholar 

  13. 13.

    Cogan, D. P. et al. Structural insights into enzymatic [4+2] aza-cycloaddition in thiopeptide antibiotic biosynthesis. Proc. Natl Acad. Sci. USA 114, 12928–12933 (2017).

    CAS  PubMed  Google Scholar 

  14. 14.

    Ohashi, M. et al. SAM-dependent enzyme-catalysed pericyclic reactions in natural product biosynthesis. Nature 549, 502–506 (2017).

    PubMed  PubMed Central  Google Scholar 

  15. 15.

    Demetriadou, A. K. et al. Biosynthesis of the polyketide polyether Antibiotic ICI 139603 in Streptomyces longisporoflavus from 18O-labelled acetate and propionate. J. Chem. Soc., Chem. Commun. 19, 408–410 (1985).

    Google Scholar 

  16. 16.

    Demydchuk, Y. et al. Analysis of the tetronomycin gene cluster: insights into the biosynthesis of a polyether tetronate antibiotic. ChemBioChem 9, 1136–1145 (2008).

    CAS  PubMed  Google Scholar 

  17. 17.

    Sun, Y., Hong, H., Gillies, F., Spencer, J. B. & Leadlay, P. F. Glyceryl-S-Acyl carrier protein as an intermediate in the biosynthesis of tetronate antibiotics. ChemBioChem 9, 150–156 (2008).

    CAS  PubMed  Google Scholar 

  18. 18.

    Hailes, H. C., Jackson, C. M., Leadlay, P. F., Ley, S. V. & Staunton, J. Biosynthesis of tetronasin: part 1 introduction and investigation of the diketide and triketide intermediates bound to the polyketide synthase. Tetrahedron Lett. 35, 307–310 (1994).

    CAS  Google Scholar 

  19. 19.

    Boons, G.-J. et al. Novel polyene cyclisation routes to the acyl tetronic acid ionophore tetronasin (ICI M139603). Tetrahedron Lett. 35, 323–326 (1994).

    CAS  Google Scholar 

  20. 20.

    Riva, E. et al. Chemical probes for the functionalization of polyketide intermediates. Angew. Chem. Int. Ed. 53, 11944–11949 (2014).

    CAS  Google Scholar 

  21. 21.

    Tosin, M., Smith, L. & Leadlay, P. F. Insights into lasalocid A ring formation by chemical chain termination in vivo. Angew. Chem. Int. Ed. Engl. 50, 11930–11933 (2011).

    CAS  PubMed  Google Scholar 

  22. 22.

    Zheng, Q. et al. Structural insights into a flavin-dependent [4+2] cyclase that catalyzes trans-decalin formation in pyrroindomycin biosynthesis. Cell Chem. Biol. 25, 718–727 (2018).

    CAS  PubMed  Google Scholar 

  23. 23.

    Bosserman, M. A., Downey, T., Noinaj, N., Buchanan, S. K. & Rohr, J. Molecular insight into substrate recognition and catalysis of Baeyer–Villiger monooxygenase MtmOIV, the key frame-modifying enzyme in the biosynthesis of anticancer agent mithramycin. ACS Chem. Biol. 8, 2466–2477 (2013).

    CAS  PubMed  Google Scholar 

  24. 24.

    Vieweg, L., Reichau, S., Schobert, R., Leadlay, P. F. & Süssmuth, R. D. Recent advances in the field of bioactive tetronates. Nat. Prod. Rep. 31, 1554–1584 (2014).

    CAS  PubMed  Google Scholar 

  25. 25.

    Jiang, X. & Wang, R. Recent developments in catalytic asymmetric inverse-electron-demand Diels–Alder reaction. Chem. Rev. 113, 5515–5546 (2013).

    CAS  PubMed  Google Scholar 

  26. 26.

    Pałasz, A. Recent advances in inverse-electron-demand hetero-Diels–Alder reactions of 1-oxa-1,3-butadienes. Top. Curr. Chem. 374, 24 (2016).

    Google Scholar 

  27. 27.

    Zheng, Q. et al. Enzyme-dependent [4+2] cycloaddition depends on lid-like interaction of the N-terminal sequence with the catalytic core in PyrI4. Cell Chem. Biol. 23, 352–360 (2016).

    CAS  PubMed  Google Scholar 

  28. 28.

    Hofmann, E., Zerbe, P. & Schaller, F. The crystal structure of Arabidopsis thaliana allene oxide cyclase: insights into the oxylipin cyclization reaction. Plant Cell 18, 3201–3217 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Yoeun, S., Cho, K. & Han, O. Structural evidence for the substrate channeling of rice allene oxide cyclase in biologically analogous Nazarov reaction. Front. Chem. 6, 500 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Costa, K. C., Glasser, N. R., Conway, S. J. & Newman, D. K. Pyocyanin degradation by a tautomerizing demethylase inhibits Pseudomonas aeruginosa biofilms. Science 355, 170–173 (2017).

    CAS  PubMed  Google Scholar 

  31. 31.

    Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345 (2009).

    CAS  PubMed  Google Scholar 

  32. 32.

    MacNeil, D. J. et al. Analysis of Streptomyces avermitilis genes required for avermectin biosynthesis utilizing a novel integration vector. Gene 111, 61–68 (1992).

    CAS  PubMed  Google Scholar 

  33. 33.

    Wilkinson, C. J. et al. Increasing the efficiency of heterologous promoters in actinomycetes. J. Mol. Microbiol. Biotechnol. 4, 417–426 (2002).

    CAS  PubMed  Google Scholar 

  34. 34.

    Harding S. E. & Rowe S. E. Analytical Ultracentrifugation in Biochemistry and Polymer Science (Royal Society of Chemistry, 1992).

  35. 35.

    Schuck, P. Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and lamm equation modeling. Biophys. J. 78, 1606–1619 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Kabsch, W. XDS. Acta Crystallogr. D 66, 125–132 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Evans, P. R. & Murshudov, G. N. How good are my data and what is the resolution? Acta Crystallogr. D 69, 1204–1214 (2013).

    CAS  PubMed  Google Scholar 

  38. 38.

    Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D 67, 235–242 (2011).

    CAS  PubMed  Google Scholar 

  39. 39.

    Terwilliger, T. C. et al. Decision-making in structure solution using Bayesian estimates of map quality: the PHENIX AutoSol wizard. Acta Crystallogr. D 65, 582–601 (2009).

    CAS  PubMed  Google Scholar 

  40. 40.

    Adams, P. D. et al. The Phenix software for automated determination of macromolecular structures. Methods 55, 94–106 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Terwilliger, T. C. et al. Iterative model building, structure refinement and density modification with the PHENIX AutoBuild wizard. Acta Crystallogr. D 64, 61–69 (2008).

    CAS  PubMed  Google Scholar 

  42. 42.

    McCoy, A. J. Solving structures of protein complexes by molecular replacement with phaser. Acta Crystallogr. D 63, 32–41 (2007).

    CAS  PubMed  Google Scholar 

  43. 43.

    Afonine, P. V. et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D 68, 352–367 (2012).

    CAS  PubMed  Google Scholar 

  44. 44.

    Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004).

    PubMed  Google Scholar 

  45. 45.

    Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010).

    CAS  PubMed  Google Scholar 

  46. 46.

    DeLano, W. L. The PyMOL Molecular Graphics System (Delano Scientific, 2002).

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R.L was supported by the Woolf Fisher Trust and the Cambridge Commonwealth European and International Trust. M.V.B.D was supported by the São Paulo Research Foundation under grant nos 2015/09188-8, 2017/50140-4 and 2018/00351-1. F.C.R.P was supported by a CNPq (National Council for Scientific and Technological Development) fellowship (141090/2016-2). M.T and R.J gratefully acknowledge EPSRC (DTA PhD studentship to R.J.); BBSRC (project grant no. BB/J007250/1 to M.T.) and the FAPESP–Warwick Joint Fund (for a SPRINT award to M.V.B.D. and M.T.).

Author information




R.L., P.F.L., F.J.L., M.T. and M.V.B.D. developed the hypothesis and designed the study. Y.D., Y.S. and M.S. cloned, sequenced and analysed the gene clusters. R.L., Y.S. and H.H. performed gene deletions, whereas M.T. and R.J. performed and analysed the experiments with chain-terminating probes. R.L. performed protein expressions and purifications, in vitro experiments and compound isolations. R.L. and F.J.L. performed compound characterizations. F.C.R.P., R.L. and M.V.B.D. solved the crystal structures. All authors analysed and discussed the results. P.F.L., R.L. and F.C.R.P. prepared the manuscript.

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Correspondence to Peter F. Leadlay.

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Supplementary Figs. 1–35, Supplementary Tables 1–6 and Supplementary Notes 1 and 2

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Little, R., Paiva, F.C.R., Jenkins, R. et al. Unexpected enzyme-catalysed [4+2] cycloaddition and rearrangement in polyether antibiotic biosynthesis. Nat Catal 2, 1045–1054 (2019).

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