Uncoupled activation and cyclization in catmint reductive terpenoid biosynthesis

Abstract

Terpene synthases typically form complex molecular scaffolds by concerted activation and cyclization of linear starting materials in a single enzyme active site. Here we show that iridoid synthase, an atypical reductive terpene synthase, catalyzes the activation of its substrate 8-oxogeranial into a reactive enol intermediate, but does not catalyze the subsequent cyclization into nepetalactol. This discovery led us to identify a class of nepetalactol-related short-chain dehydrogenase enzymes (NEPS) from catmint (Nepeta mussinii) that capture this reactive intermediate and catalyze the stereoselective cyclisation into distinct nepetalactol stereoisomers. Subsequent oxidation of nepetalactols by NEPS1 provides nepetalactones, metabolites that are well known for both insect-repellent activity and euphoric effects in cats. Structural characterization of the NEPS3 cyclase reveals that it binds to NAD+ yet does not utilize it chemically for a non-oxidoreductive formal [4 + 2] cyclization. These discoveries will complement metabolic reconstructions of iridoid and monoterpene indole alkaloid biosynthesis.

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Fig. 1: Nepetalactones and terpenoid biosynthesis.
Fig. 2: Iridoid synthase (ISY) reaction mechanism.
Fig. 3: Formation of nepetalactones by NEPS enzymes.
Fig. 4: NEPS activities explored with (S)-8-oxocitronellal (6).
Fig. 5: Structure of NEPS enzymes.
Fig. 6: NEPS variants.

Data availability

The sequences of N. mussinii NEPS enzyme have been deposited in GenBank/EMBL/DDBJ with the accession codes MG677124 (NmNEPS1), MG677125 (NmNEPS2) and MG677126 (NmNEPS3). The NAD+ bound NmNEPS3 (7S-cis-cis-nepetalactol cyclase) X-ray structure has been deposited in the PDB with the accession code 6F9Q. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD008704. Detailed experimental procedures and can be found in the Supplementary Information. The authors declare that all other data supporting the findings of this study are available within this article and its Supplementary Information or from the authors upon reasonable request.

References

  1. 1.

    McElvain, S. M., Bright, R. D. & Johnson, P. R. The constituents of the volatile oil of catnip. I. Nepetalic acid, nepetalactone and related compounds. J. Am. Chem. Soc. 63, 1558–1563 (1941).

    CAS  Article  Google Scholar 

  2. 2.

    Formisano, C., Rigano, D. & Senatore, F. Chemical constituents and biological activities of Nepeta species. Chem. Biodivers. 8, 1783–1818 (2011).

    CAS  Article  PubMed  Google Scholar 

  3. 3.

    Todd, N. B. Inheritance of the catnip response in domestic cats. J. Hered. 53, 54–56 (1962).

    CAS  Article  PubMed  Google Scholar 

  4. 4.

    Bates, R. B. & Sigel, C. W. Terpenoids. cis-trans- and trans-cis- nepetalactones. Experientia 19, 564–565 (1963).

    CAS  Article  Google Scholar 

  5. 5.

    Waller, G. R., Price, G. H. & Mitchell, E. D. Feline attractant, cis,trans-nepetalactone: metabolism in the domestic cat. Science 164, 1281–1282 (1969).

    CAS  Article  PubMed  Google Scholar 

  6. 6.

    Dawson, G. et al. Identification of an aphid sex pheromone. Nature 325, 614–616 (1987).

    CAS  Article  Google Scholar 

  7. 7.

    Clark, L. J., Hamilton, J. G. C., Chapman, J. V., Rhodes, M. J. C. & Hallahan, D. L. Analysis of monoterpenoids in glandular trichomes of the catmint Nepeta racemosa. Plant J. 11, 1387–1393 (1997).

    CAS  Article  Google Scholar 

  8. 8.

    Eisner, T. Catnip: its raison d’etre. Science 146, 1318–1320 (1964).

    CAS  Article  PubMed  Google Scholar 

  9. 9.

    Birkett, M. A., Hassanali, A., Hoglund, S., Pettersson, J. & Pickett, J. A. Repellent activity of catmint, Nepeta cataria, and iridoid nepetalactone isomers against Afro-tropical mosquitoes, ixodid ticks and red poultry mites. Phytochemistry 72, 109–114 (2011).

    CAS  Article  PubMed  Google Scholar 

  10. 10.

    Rajaonarivony, J. I. M., Gershenzon, J. & Croteau, R. Characterization and mechanism of (4 S)-limonene synthase, a monoterpene cyclase from the glandular trichomes of peppermint (Mentha x piperita). Arch. Biochem. Biophys. 296, 49–57 (1992).

    CAS  Article  PubMed  Google Scholar 

  11. 11.

    Poulter, C. D., Argyle, J. C. & Mash, E. A. Letter: Prenyltransferase. New evidence for an ionization-condensation-elimination mechanism with 2-fluorogeranyl pyrophosphate. J. Am. Chem. Soc. 99, 957–959 (1977).

    CAS  Article  PubMed  Google Scholar 

  12. 12.

    Baunach, M., Franke, J. & Hertweck, C. Terpenoid biosynthesis off the beaten track: unconventional cyclases and their impact on biomimetic synthesis. Angew. Chem. Int. Ed. Engl. 54, 2604–2626 (2015).

    CAS  Article  PubMed  Google Scholar 

  13. 13.

    Tantillo, D. J. Importance of inherent substrate reactivity in enzyme-promoted carbocation cyclization/rearrangements. Angew. Chem. Int. Ed. Engl. 56, 10040–10045 (2017).

    CAS  Article  PubMed  Google Scholar 

  14. 14.

    Miettinen, K. et al. The seco-iridoid pathway from Catharanthus roseus. Nat. Commun. 5, 3606 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Geu-Flores, F. et al. An alternative route to cyclic terpenes by reductive cyclization in iridoid biosynthesis. Nature 492, 138–142 (2012).

    CAS  Article  PubMed  Google Scholar 

  16. 16.

    O’Connor, S. E. & Maresh, J. J. Chemistry and biology of monoterpene indole alkaloid biosynthesis. Nat. Prod. Rep. 23, 532–547 (2006).

    Article  CAS  PubMed  Google Scholar 

  17. 17.

    Alagna, F. et al. Identification and characterization of the iridoid synthase involved in oleuropein biosynthesis in olive (Olea europaea) fruits. J. Biol. Chem. 291, 5542–5554 (2016).

    CAS  Article  PubMed  Google Scholar 

  18. 18.

    Kries, H., Kellner, F., Kamileen, M. O. & O’Connor, S. E. Inverted stereocontrol of iridoid synthase in snapdragon. J. Biol. Chem. 292, 14659–14667 (2017).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Sherden, N. H. et al. Identification of iridoid synthases from Nepeta species: Iridoid cyclization does not determine nepetalactone stereochemistry. Phytochemistry 145, 48–56 (2018).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Kries, H. et al. Structural determinants of reductive terpene cyclization in iridoid biosynthesis. Nat. Chem. Biol. 12, 6–8 (2016).

    CAS  Article  PubMed  Google Scholar 

  21. 21.

    Hu, Y. et al. Structures of iridoid synthase from Cantharanthus roseus with bound NAD+, NADPH, or NAD+/10-oxogeranial: reaction mechanisms. Angew. Chem. Int. Ed. Engl. 54, 15478–15482 (2015).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Qin, L. et al. Structure of iridoid synthase in complex with NADP+/8-oxogeranial reveals the structural basis of its substrate specificity. J. Struct. Biol. 194, 224–230 (2016).

    CAS  Article  PubMed  Google Scholar 

  23. 23.

    Dawson, G. W., Pickett, J. A. & Smiley, D. W. M. The aphid sex pheromone cyclopentanoids: synthesis in the elucidation of structure and biosynthetic pathways. Bioorg. Med. Chem. 4, 351–361 (1996).

    CAS  Article  PubMed  Google Scholar 

  24. 24.

    Liblikas, I. et al. Simplified isolation procedure and interconversion of the diastereomers of nepetalactone and nepetalactol.J. Nat. Prod. 68, 886–890 (2005).

    CAS  Article  PubMed  Google Scholar 

  25. 25.

    Cucinotta, C. S., Ruini, A., Catellani, A. & Stirling, A. Ab initio molecular dynamics study of the keto-enol tautomerism of acetone in solution. Chemphyschem 7, 1229–1234 (2006).

    CAS  Article  PubMed  Google Scholar 

  26. 26.

    Alagona, G., Ghio, C. & Nagy, P. I. The catalytic effect of water on the keto-enol tautomerism. Pyruvate and acetylacetone: a computational challenge. Phys. Chem. Chem. Phys. 12, 10173–10188 (2010).

    CAS  Article  PubMed  Google Scholar 

  27. 27.

    Schreiber, S. L., Meyers, H. V. & Wiberg, K. B. Stereochemistry of the intramolecular enamine/enal (enone) cycloaddition reaction and subsequent Transformations. J. Am. Chem. Soc. 108, 8274–8277 (1986).

    CAS  Article  Google Scholar 

  28. 28.

    Hallahan, D. L., West, J. M., Smiley, D. W. M. & Pickett, J. A. Nepetalactol oxidoreductase in trichomes of the catmint Nepeta racemosa. Phytochemistry 48, 421–427 (1998).

    CAS  Article  Google Scholar 

  29. 29.

    Moummou, H., Kallberg, Y., Tonfack, L. B., Persson, B. & van der Rest, B. The plant short-chain dehydrogenase (SDR) superfamily: genome-wide inventory and diversification patterns. BMC Plant Biol. 12, 219 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Weng, J. K. & Noel, J. P. The remarkable pliability and promiscuity of specialized metabolism. Cold Spring Harb. Symp. Quant. Biol. 77, 309–320 (2012).

    Article  PubMed  Google Scholar 

  31. 31.

    Tatsis, E. C. et al. A three enzyme system to generate the Strychnos alkaloid scaffold from a central biosynthetic intermediate. Nat. Commun. 8, 316 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Davin, L. B. et al. Stereoselective bimolecular phenoxy radical coupling by an auxiliary (dirigent) protein without an active center. Science 275, 362–366 (1997).

    CAS  Article  PubMed  Google Scholar 

  33. 33.

    Pickel, B. et al. An enantiocomplementary dirigent protein for the enantioselective laccase-catalyzed oxidative coupling of phenols. Angew. Chem. Int. Ed. Engl. 49, 202–204 (2010).

    CAS  Article  PubMed  Google Scholar 

  34. 34.

    Brown, S., Clastre, M., Courdavault, V. & O’Connor, S. E. De novo production of the plant-derived alkaloid strictosidine in yeast. Proc. Natl Acad. Sci. U.S.A. 112, 3205–3210 (2015).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Campbell, A. et al. Engineering of a nepetalactol-producing platform strain of Saccharomyces cerevisiae for the production of plant seco-iridoids. ACS Synth. Biol. 5, 405–414 (2016).

    CAS  Article  PubMed  Google Scholar 

  36. 36.

    Billingsley, J. M. et al. Engineering the biocatalytic selectivity of iridoid production in Saccharomyces cerevisiae. Metab. Eng. 44, 117–125 (2017).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Tyanova, S., Temu, T. & Cox, J. The MaxQuant computational platform for mass spectrometry-based shotgun proteomics. Nat. Protoc. 11, 2301–2319 (2016).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  38. 38.

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

  39. 39.

    Winter, G. Xia2: An expert system for macromolecular crystallography data reduction. J. Appl. Crystallogr. 43, 186–190 (2010).

    CAS  Article  Google Scholar 

  40. 40.

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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  41. 41.

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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Youn, B., Moinuddin, S. G. A., Davin, L. B., Lewis, N. G. & Kang, C. Crystal structures of apo-form and binary/ternary complexes of Podophyllum secoisolariciresinol dehydrogenase, an enzyme involved in formation of health-protecting and plant defense lignans. J. Biol. Chem. 280, 12917–12926 (2005).

    CAS  Article  PubMed  Google Scholar 

  43. 43.

    Bunkóczi, G. & Read, R. J. Improvement of molecular-replacement models with Sculptor. Acta Crystallogr. D. Biol. Crystallogr. 67, 303–312 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. 44.

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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Cowtan, K. The Buccaneer software for automated model building. 1. Tracing protein chains. Acta Crystallogr. D. Biol. Crystallogr. 62, 1002–1011 (2006).

    Article  CAS  PubMed  Google Scholar 

  46. 46.

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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  47. 47.

    Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D. Biol. Crystallogr. 53, 240–255 (1997).

    CAS  Article  PubMed  Google Scholar 

  48. 48.

    Davis, I. W. et al. MolProbity: all-atom contacts and structure validation for proteins and nucleic acids. Nucleic Acids Res. 35, W375–W383 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  49. 49.

    Zhang, Y. I-TASSER server for protein 3D structure prediction. BMC Bioinformatics 9, 40 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. 50.

    Krieger, E. et al. Improving physical realism, stereochemistry, and side-chain accuracy in homology modeling: four approaches that performed well in CASP8. Proteins 77, 114–122 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  51. 51.

    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 

  52. 52.

    Larkin, M. A. et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948 (2007).

    CAS  Article  Google Scholar 

  53. 53.

    Robert, X. & Gouet, P. Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res. 42, W320–W324 (2014).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  54. 54.

    Mint Evolutionary Genomics Consortium. Phylogenomic mining of the mints reveals multiple mechanisms contributing to the evolution of chemical diversity in Lamiaceae. Mol. Plant 11, 1084–1096 (2018).

  55. 55.

    Ringer, K. L., Davis, E. M. & Croteau, R. Monoterpene metabolism. Cloning, expression, and characterization of (-)-isopiperitenol/(-)-carveol dehydrogenase of peppermint and spearmint. Plant Physiol. 137, 863–872 (2005).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  56. 56.

    Edgar, R. C. Muscle: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics 5, 113 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. 57.

    Nguyen, L. T., Schmidt, H. A., von Haeseler, A. & Minh, B. Q. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol. Biol. Evol. 32, 268–274 (2015).

    CAS  Article  PubMed  Google Scholar 

  58. 58.

    Kalyaanamoorthy, S., Minh, B. Q., Wong, T. K. F., von Haeseler, A. & Jermiin, L. S. ModelFinder: fast model selection for accurate phylogenetic estimates. Nat. Methods 14, 587–589 (2017).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  59. 59.

    Minh, B. Q., Nguyen, M. A. T. & von Haeseler, A. Ultrafast approximation for phylogenetic bootstrap. Mol. Biol. Evol. 30, 1188–1195 (2013).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We acknowledge funding from UK Biotechnological and Biological Sciences Research Council (BBSRC) and Engineering and Physical Sciences Research Council (EPSRC) joint-funded OpenPlant Synthetic Biology Research Centre (BB/L014130/1) and from the National Science Foundation Plant Genome Research Program (IOS- 1444499). For the X-ray data collection, we acknowledge Diamond Light Source for access to beamline I03 under proposal MX13467, with support from the European Community’s Seventh Framework Program (FP7/2007–2013) under grant agreement 283570 (BioStruct-X). We are grateful to: P. Brett (John Innes Centre) for assistance with GC–MS analysis and M. Vigoroux (John Innes Centre) for assistance with proteome annotations. We also thank K. Houk, J. Fell, H. Kries and D. Whitaker for discussions concerning the iridoid synthase and cyclization mechanisms.

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Contributions

S.E.O.’C. designed and supervised the project; B.R.L. performed molecular cloning, protein purification, enzyme assays, trichome isolation, chemical synthesis, phylogenetic analysis, homology modeling and computational docking; G.S. performed proteome analysis; M.O.K. assisted with protein purification, compound isolation and chemical synthesis; B.R.L., G.R.T. and C.E.M.S. performed crystallization trials and obtained crystals; B.R.L., G.R.T. and D.M.L. refined structures; B.R.L. and S.E.O.’C. wrote the manuscript.

Corresponding author

Correspondence to Sarah E. O’Connor.

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A UK patent application has been submitted based on the work reported here (GB1808663.7).

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Supplementary information

Supplementary Information

Supplementary Tables 1–4, Supplementary Figures 1–17

Reporting Summary

Supplementary Note 1

Synthetic Procedures

Supplementary Data Set 1

Proteomic Data

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Lichman, B.R., Kamileen, M.O., Titchiner, G.R. et al. Uncoupled activation and cyclization in catmint reductive terpenoid biosynthesis. Nat Chem Biol 15, 71–79 (2019). https://doi.org/10.1038/s41589-018-0185-2

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