Structural basis of the Cope rearrangement and cyclization in hapalindole biogenesis

Published online:


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.

  • Subscribe to Nature Chemical Biology for full access:



Additional access options:

Already a subscriber?  Log in  now or  Register  for online access.

Additional information

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


  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).

  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).

  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).

  4. 4.

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

  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).

  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).

  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).

  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).

  9. 9.

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

  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).

  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).

  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).

  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).

  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).

  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).

  16. 16.

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

  17. 17.

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

  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).

  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).

  20. 20.

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

  21. 21.

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

  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).

  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).

  24. 24.

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

  25. 25.

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

  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).

  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).

  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).

  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).

  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).

  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).

  32. 32.

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

  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).

  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).

  35. 35.

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

  36. 36.

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

  37. 37.

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

  38. 38.

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

  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).

  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).

  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).

  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).

  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).

  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).

  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).

  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).

  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).

  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).

  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).

  50. 50.

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

  51. 51.

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

  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).

  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).

  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).

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


  1. Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA

    • Sean A. Newmister
    • , Shasha Li
    • , Andrew N. Lowell
    • , Fengan Yu
    • , Janet L. Smith
    •  & David H. Sherman
  2. Department of Medicinal Chemistry, University of Michigan, Ann Arbor, MI, USA

    • Shasha Li
    •  & David H. Sherman
  3. Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, CA, USA

    • Marc Garcia-Borràs
    • , Jacob N. Sanders
    • , Song Yang
    •  & K. N. Houk
  4. Department of Biological Chemistry, University of Michigan, Ann Arbor, MI, USA

    • Janet L. Smith
  5. Department of Chemistry, Colorado State University, Fort Collins, CO, USA

    • Robert M. Williams
  6. University of Colorado Cancer Center, Aurora, CO, USA

    • Robert M. Williams
  7. Department of Chemistry, University of Michigan, Ann Arbor, MI, USA

    • David H. Sherman
  8. Department of Microbiology and Immunology, University of Michigan, Ann Arbor, MI, USA

    • David H. Sherman


  1. Search for Sean A. Newmister in:

  2. Search for Shasha Li in:

  3. Search for Marc Garcia-Borràs in:

  4. Search for Jacob N. Sanders in:

  5. Search for Song Yang in:

  6. Search for Andrew N. Lowell in:

  7. Search for Fengan Yu in:

  8. Search for Janet L. Smith in:

  9. Search for Robert M. Williams in:

  10. Search for K. N. Houk in:

  11. Search for David H. Sherman in:


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.

Competing interests

The authors declare no competing interests.

Corresponding authors

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

Supplementary information

  1. Supplementary Text and Figures

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

  2. Life Sciences Reporting Summary