Skip to main content

Thank you for visiting 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.

Structural basis of the Cope rearrangement and cyclization in hapalindole biogenesis


Hapalindole alkaloids are a structurally diverse class of cyanobacterial natural products defined by their varied polycyclic ring systems and diverse biological activities. These complex metabolites are generated from a common biosynthetic intermediate by the Stig cyclases in three mechanistic steps: a rare Cope rearrangement, 6-exo-trig cyclization, and electrophilic aromatic substitution. Here we report the structure of HpiC1, a Stig cyclase that catalyzes the formation of 12-epi-hapalindole U in vitro. The 1.5-Å structure revealed a dimeric assembly with two calcium ions per monomer and with the active sites located at the distal ends of the protein dimer. Mutational analysis and computational methods uncovered key residues for an acid-catalyzed [3,3]-sigmatropic rearrangement, as well as specific determinants that control the position of terminal electrophilic aromatic substitution, leading to a switch from hapalindole to fischerindole alkaloids.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1: Biogenesis of hapalindole alkaloids.
Fig. 2: HpiC1 structural overview at 1.5 Å.
Fig. 3: Active site of SeMet HpiC1 W73M/K132M.
Fig. 4: In vitro characterization of HpiC1 mutants using 1 as substrate.
Fig. 5: Quantum mechanics analysis.
Fig. 6: Molecular dynamics simulations of the active site.


  1. 1.

    Bhat, V., Dave, A., MacKay, J. A. & Rawal, V. H. The chemistry of hapalindoles, fischerindoles, ambiguines, and welwitindolinones. Alkaloids Chem. Biol. 73, 65–160 (2014).

    Article  CAS  Google Scholar 

  2. 2.

    Asthana, R. K. et al. Identification of an antimicrobial entity from the cyanobacterium Fischerella sp. isolated from bark of Azadirachta indica (Neem) tree. J. Appl. Phycol. 18, 33–39 (2006).

    Article  CAS  Google Scholar 

  3. 3.

    Becher, P. G., Keller, S., Jung, G., Süssmuth, R. D. & Jüttner, F. Insecticidal activity of 12-epi-hapalindole J isonitrile. Phytochemistry 68, 2493–2497 (2007).

    Article  CAS  Google Scholar 

  4. 4.

    Cagide, E. et al. Hapalindoles from the cyanobacterium Fischerella: potential sodium channel modulators. Chem. Res. Toxicol. 27, 1696–1706 (2014).

    Article  CAS  Google Scholar 

  5. 5.

    Mo, S., Krunic, A., Chlipala, G. & Orjala, J. Antimicrobial ambiguine isonitriles from the cyanobacterium Fischerella ambigua. J. Nat. Prod. 72, 894–899 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Mo, S., Krunic, A., Santarsiero, B. D., Franzblau, S. G. & Orjala, J. Hapalindole-related alkaloids from the cultured cyanobacterium Fischerella ambigua. Phytochemistry 71, 2116–2123 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Zhang, X. & Smith, C. D. Microtubule effects of welwistatin, a cyanobacterial indolinone that circumvents multiple drug resistance. Mol. Pharmacol. 49, 288–294 (1996).

    CAS  Google Scholar 

  8. 8.

    Hillwig, M. L., Zhu, Q. & Liu, X. Biosynthesis of ambiguine indole alkaloids in cyanobacterium Fischerella ambigua. ACS Chem. Biol. 9, 372–377 (2014).

    Article  CAS  Google Scholar 

  9. 9.

    Raveh, A. & Carmeli, S. Antimicrobial ambiguines from the cyanobacterium Fischerella sp. collected in Israel. J. Nat. Prod. 70, 196–201 (2007).

    Article  CAS  Google Scholar 

  10. 10.

    Stratmann, K. et al. Welwitindolinones, unusual alkaloids from the blue-green algae Hapalosiphon welwitschii and Westiella intricata. Relationship to fischerindoles and hapalinodoles. J. Am. Chem. Soc. 116, 9935–9942 (1994).

    Article  CAS  Google Scholar 

  11. 11.

    Li, S. et al. Hapalindole/ambiguine biogenesis is mediated by a Cope rearrangement, C‒C bond-forming cascade. J. Am. Chem. Soc. 137, 15366–15369 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Cope, A. C. & Hardy, E. M., The introduction of substituted vinyl groups. V. A rearrangement involving the migration of an allyl group in a three-carbon system. J. Am. Chem. Soc. 62, 441–444 (1940).

    Article  CAS  Google Scholar 

  13. 13.

    Ilardi, E. A., Stivala, C. E. & Zakarian, A. [3,3]-Sigmatropic rearrangements: recent applications in the total synthesis of natural products. Chem. Soc. Rev. 38, 3133–3148 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. 14.

    DeClue, M. S., Baldridge, K. K., Künzler, D. E., Kast, P. & Hilvert, D. Isochorismate pyruvate lyase: a pericyclic reaction mechanism? J. Am. Chem. Soc. 127, 15002–15003 (2005).

    Article  CAS  Google Scholar 

  15. 15.

    Luk, L. Y., Qian, Q. & Tanner, M. E. A Cope rearrangement in the reaction catalyzed by dimethylallyltryptophan synthase? J. Am. Chem. Soc. 133, 12342–12345 (2011).

    Article  CAS  Google Scholar 

  16. 16.

    Tanner, M. E. Mechanistic studies on the indole prenyltransferases. Nat. Prod. Rep. 32, 88–101 (2015).

    Article  CAS  Google Scholar 

  17. 17.

    Li, S. et al. Decoding cyclase-dependent assembly of hapalindole and fischerindole alkaloids. Nat. Chem. Biol. 13, 467–469 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Zhu, Q. & Liu, X. Discovery of a calcium-dependent enzymatic cascade for the selective assembly of hapalindole-type alkaloids: On the biosynthetic origin of hapalindole U. Angew. Chem. Int. Edn Engl. 56, 9062–9066 (2017).

    Article  CAS  Google Scholar 

  19. 19.

    Zhu, Q. & Liu, X. Molecular and genetic basis for early stage structural diversifications in hapalindole-type alkaloid biogenesis. Chem. Commun. (Camb.) 53, 2826–2829 (2017).

    Article  CAS  Google Scholar 

  20. 20.

    Krissinel, E. & Henrick, K. Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774–797 (2007).

    Article  CAS  Google Scholar 

  21. 21.

    Holm, L. & Rosenström, P. Dali server: conservation mapping in 3D. Nucleic Acids Res. 38, W545–W549 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. 22.

    von Schantz, L. et al. Structural basis for carbohydrate-binding specificity‐‐a comparative assessment of two engineered carbohydrate-binding modules. Glycobiology 22, 948–961 (2012).

    Article  CAS  Google Scholar 

  23. 23.

    Trott, O. & Olson, A. J. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J. Comput. Chem. 31, 455–461 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Lutz, R. P. Catalysis of the Cope and Claisen rearrangements. Chem. Rev. 84, 205–247 (1984).

    Article  CAS  Google Scholar 

  25. 25.

    Wendt, K. U., Poralla, K. & Schulz, G. E. Structure and function of a squalene cyclase. Science 277, 1811–1815 (1997).

    Article  CAS  Google Scholar 

  26. 26.

    Starks, C. M., Back, K., Chappell, J. & Noel, J. P. Structural basis for cyclic terpene biosynthesis by tobacco 5-epi-aristolochene synthase. Science 277, 1815–1820 (1997).

    Article  CAS  Google Scholar 

  27. 27.

    Lesburg, C. A., Zhai, G., Cane, D. E. & Christianson, D. W. Crystal structure of pentalenene synthase: mechanistic insights on terpenoid cyclization reactions in biology. Science 277, 1820–1824 (1997).

    Article  CAS  Google Scholar 

  28. 28.

    Jenson, C. & Jorgensen, W. L. Computational investigations of carbenium ion reactions relevant to sterol biosynthesis. J. Am. Chem. Soc. 119, 10846–10854 (1997).

    Article  CAS  Google Scholar 

  29. 29.

    Abou-Hachem, M. et al. Calcium binding and thermostability of carbohydrate binding module CBM4-2 of Xyn10A from Rhodothermus marinus. Biochemistry 41, 5720–5729 (2002).

    Article  CAS  Google Scholar 

  30. 30.

    Montanier, C. Y. et al. A novel, noncatalytic carbohydrate-binding module displays specificity for galactose-containing polysaccharides through calcium-mediated oligomerization. J. Biol. Chem. 286, 2499–22509 (2011).

    Article  CAS  Google Scholar 

  31. 31.

    Van Duyne, G. D., Standaert, R. F., Karplus, P. A., Schreiber, S. L. & Clardy, J. Atomic structures of the human immunophilin FKBP-12 complexes with FK506 and rapamycin. J. Mol. Biol. 229, 105–124 (1993).

    Article  Google Scholar 

  32. 32.

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Murshudov, G. N. et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D Biol. Crystallogr. 67, 355–367 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. 36.

    McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. 37.

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

    Article  CAS  Google Scholar 

  38. 38.

    Becke, A. D. Density‐functional thermochemistry. III. The role of exact exchange. J. Chem. Phys. 98, 5648–5652 (1993).

    Article  CAS  Google Scholar 

  39. 39.

    Lee, C., Yang, W. & Parr, R. G. Development of the Colle-Salvetti correlation-energy formula into a functional of the electron density. Phys. Rev. B Condens. Matter 37, 785–789 (1988).

    Article  CAS  Google Scholar 

  40. 40.

    Grimme, S., Antony, J., Ehrlich, S. & Krieg, H. A consistent and accurate ab initio parametrization of density functional dispersion correction (DFT-D) for the 94 elements H-Pu. J. Chem. Phys. 132, 154104 (2010).

    Article  CAS  Google Scholar 

  41. 41.

    Grimme, S., Ehrlich, S. & Goerigk, L. Effect of the damping function in dispersion corrected density functional theory. J. Comput. Chem. 32, 1456–1465 (2011).

    Article  CAS  Google Scholar 

  42. 42.

    Miertuš, S., Scrocco, E. & Tomasi, J. Electrostatic interaction of a solute with a continuum. A direct utilizaion of ab initio molecular potentials for the prevision of solvent effects. Chem. Phys. 55, 117–129 (1981).

    Article  Google Scholar 

  43. 43.

    Miertus, S. & Tomasi, J. Approximate evaluations of the electrostatic free-energy and internal energy changes in solution processes. Chem. Phys. 65, 239–245 (1982).

    Article  CAS  Google Scholar 

  44. 44.

    Pascual-ahuir, J. L., Silla, E. & Tuñon, I. GEPOL: An improved description of molecular surfaces. III. A new algorithm for the computation of a solvent-excluding surface. J. Comput. Chem. 15, 1127–1138 (1994).

    Article  CAS  Google Scholar 

  45. 45.

    Li, L., Li, C., Zhang, Z. & Alexov, E. On the dielectric “constant” of proteins: smooth dielectric function for macromolecular modeling and its implementation in DelPhi. J. Chem. Theory Comput. 9, 2126–2136 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. 46.

    Schutz, C. N. & Warshel, A. What are the dielectric “constants” of proteins and how to validate electrostatic models? Proteins 44, 400–417 (2001).

    Article  CAS  Google Scholar 

  47. 47.

    Salomon-Ferrer, R., Götz, A. W., Poole, D., Le Grand, S. & Walker, R. C. Routine microsecond molecular dynamics simulations with AMBER on GPUs. 2. Explicit solvent particle mesh Ewald. J. Chem. Theory Comput. 9, 3878–3888 (2013).

    Article  CAS  Google Scholar 

  48. 48.

    Wang, J., Wolf, R. M., Caldwell, J. W., Kollman, P. A. & Case, D. A. Development and testing of a general amber force field. J. Comput. Chem. 25, 1157–1174 (2004).

    Article  CAS  Google Scholar 

  49. 49.

    Bayly, C. I., Cieplak, P., Cornell, W. D. & Kollman, P. A. A well-behaved electrostatic potential based method using charge restraints for deriving atomic charges - the RESP model. J. Phys. Chem. 97, 10269–10280 (1993).

    Article  CAS  Google Scholar 

  50. 50.

    Besler, B. H., Merz, K. M. & Kollman, P. A. Atomic charges derived from semiempirical methods. J. Comput. Chem. 11, 431–439 (1990).

    Article  CAS  Google Scholar 

  51. 51.

    Singh, U. C. & Kollman, P. A. An approach to computing electrostatic charges for molecules. J. Comput. Chem. 5, 129–145 (1984).

    Article  CAS  Google Scholar 

  52. 52.

    Jorgensen, W. L., Chandrasekhar, J., Madura, J. D., Impey, R. W. & Klein, M. L. Comparison of simple potential functions for simulating liquid water. J. Chem. Phys. 79, 926–935 (1983).

    Article  CAS  Google Scholar 

  53. 53.

    Maier, J. A. et al. Ff14sb: improving the accuracy of protein side chain and backbone parameters from ff99sb. J. Chem. Theory Comput. 11, 3696–3713 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. 54.

    Darden, T., York, D. & Pedersen, L. Particle mesh Ewald: An N log(N) method for Ewald sums in large systems. J. Chem. Phys. 98, 10089–10092 (1993).

    Article  CAS  Google Scholar 

Download references


The authors thank the National Science Foundation under the CCI Center for Selective C-H Functionalization (CHE-1700982), the National Institutes of Health (CA70375 to R.M.W. and D.H.S.), R35 GM118101, and the Hans W. Vahlteich Professorship (to D.H.S.) for financial support. M.G.-B. thanks the Ramón Areces Foundation for a postdoctoral fellowship. J.N.S. acknowledges the support of the National Institute of General Medical Sciences of the National Institutes of Health under Award Number F32GM122218. Computational resources were provided by the UCLA Institute for Digital Research and Education (IDRE) and the Extreme Science and Engineering Discovery Environment (XSEDE), which is supported by the NSF (OCI-1053575). The content does not necessarily represent the official views of the National Institutes of Health. Anton 2 computer time was provided by the Pittsburgh Supercomputing Center (PSC) through Grant R01GM116961 from the National Institutes of Health. The Anton 2 machine at PSC was generously made available by D.E. Shaw Research.

Author information




S.A.N. conducted protein preparation and crystallography. S.L. cloned the genes and assayed the enzymes. M.G.-B. conducted molecular dynamics simulations. J.N.S. conducted density functional theory calculations. A.N.L. synthesized substrates. F.Y. performed bioinformatics analyses. S.A.N., S.L., M.G.-B., J.N.S., S.Y., J.L.S., R.M.W., K.N.H. and D.H.S. designed research and conducted data analysis and interpretation. S.A.N., S.L., M.G.-B., J.N.S., J.L.S., K.N.H. and D.H.S. wrote the manuscript.

Corresponding authors

Correspondence to Robert M. Williams or K. N. Houk or David H. Sherman.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

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

Supplementary information

Supplementary Text and Figures

Supplementary Tables 12, Supplementary Figures 1–30 and Supplementary Notes


Life Sciences Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Newmister, S.A., Li, S., Garcia-Borràs, M. et al. Structural basis of the Cope rearrangement and cyclization in hapalindole biogenesis. Nat Chem Biol 14, 345–351 (2018).

Download citation

Further reading


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