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Fragmentation and [4 + 3] cycloaddition in sodorifen biosynthesis

Nature Chemistry volume 15, pages 1164–1171 (2023)Cite this article

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Abstract

Terpenes constitute the largest class of natural products. Their skeletons are formed by terpene cyclases (TCs) from acyclic oligoprenyl diphosphates through sophisticated enzymatic conversions. These enzyme reactions start with substrate ionization through diphosphate abstraction, followed by a cascade reaction via cationic intermediates. Based on isotopic-labelling experiments in combination with a computational study, the cyclization mechanism for sodorifen, a highly methylated sesquiterpene from the soil bacterium Serratia plymuthica, was resolved. A peculiar problem in its biosynthesis lies in the formation of several methyl groups from chain methylene carbons. The underlying mechanism involves a methyltransferase-mediated cyclization and unprecedented ring contraction with carbon extrusion from the chain to form a methyl group. A terpene cyclase subsequently catalyses a fragmentation into two reactive intermediates, followed by hydrogen transfers between them and recombination of the fragments by [4 + 3] cycloaddition. This study solves the intricate mechanistic problem of extra methyl group formation in sodorifen biosynthesis.

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Fig. 1: Biosynthesis of sodorifen (1).
Fig. 2: Cyclization mechanism from FPP to presodorifen diphosphate (2).
Fig. 3: Cyclization mechanism from presodorifen diphosphate (2) to sodorifen (1).
Fig. 4: The absolute configuration of 2.
Fig. 5: The absolute configuration of 2.

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Data availability

The authors declare that the main data supporting the findings of this study are available within the Article, Supplementary Videos, Supplementary Data, and Supplementary Information. Crystallographic data for the structure reported in this Article have been deposited at the Cambridge Crystallographic Data Centre, under deposition number CCDC 2213524. Copies of the data can be obtained free of charge via https://www.ccdc.cam.ac.uk/structures/. Data and plasmids described in this study can be obtained from the corresponding author on reasonable request. Source data are provided with this paper.

References

  1. Ryu, C.-M. et al. Bacterial volatiles promote growth in arabidopsis. Proc. Natl Acad. Sci. USA 100, 4927–4932 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Lee, H. H., Molla, M. N., Cantor, C. R. & Collins, J. J. Bacterial charity work leads to population-wide resistance. Nature 467, 82–86 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Chernin, L. et al. Quorum-sensing quenching by rhizobacterial volatiles. Environ. Microbiol. Rep. 3, 698–704 (2011).

    CAS  PubMed  Google Scholar 

  4. Vespermann, A., Kai, M. & Piechulla, B. Rhizobacterial volatiles affect the growth of fungi and Arabidopsis thaliana. Appl. Environ. Microbiol. 73, 5639–5641 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. von Reuss, S. H., Kai, M., Piechulla, B. & Francke, W. Octamethylbicyclo[3.2.1]octadienes from the rhizobacterium Serratia odorifera. Angew. Chem. Int. Ed. 49, 2009–2010 (2010).

    Google Scholar 

  6. Domik, D., Magnus, N. & Piechulla, B. Analysis of a new cluster of genes involved in the synthesis of the unique volatile organic compound sodorifen of Serratia plymuthica 4Rx13. FEMS Microbiol. Lett. 363, fnw139 (2016).

    PubMed  Google Scholar 

  7. Domik, D. et al. A terpene synthase is involved in the synthesis of the volatile organic compound sodorifen of Serratia plymuthica 4Rx13. Front. Microbiol. 7, 737 (2016).

    PubMed  PubMed Central  Google Scholar 

  8. von Reuss, S. et al. Sodorifen biosynthesis in the rhizobacterium Serratia plymuthica involves methylation and cyclization of MEP-derived farnesyl pyrophosphate by a SAM-dependent C-methyltransferase. J. Am. Chem. Soc. 140, 11855–11862 (2018).

    Google Scholar 

  9. Duell, E. R. et al. Direct pathway cloning of the sodorifen biosynthetic gene cluster and recombinant generation of its product in E. coli. Microb. Cell. Fact. 18, 32 (2019).

    PubMed  PubMed Central  Google Scholar 

  10. Rabe, P. et al. Conformational analysis, thermal rearrangement and EI-MS-fragmentation mechanism of (1(10)E,4E,6S,7R)-germacradien-6-ol by 13C-labeling experiments. Angew. Chem. Int. Ed. 54, 13448–13451 (2015).

    CAS  Google Scholar 

  11. Eustaquio, A. S., Pojer, F., Noel, J. P. & Moore, B. S. Discovery and characterization of a marine bacterial SAM-dependent chlorinase. Nat. Chem. Biol. 4, 69–74 (2008).

    CAS  PubMed  Google Scholar 

  12. Rabe, P. et al. Terpene cyclases from social amoebae. Angew. Chem. Int. Ed. 55, 15420–15423 (2016).

    CAS  Google Scholar 

  13. Rabe, P. et al. Mechanistic investigantions on two bacterial diterpene cyclases: spiroviolene synthase and tsukubadiene synthase. Angew. Chem. Int. Ed. 56, 2776–2779 (2017).

    CAS  Google Scholar 

  14. Rinkel, J. & Dickschat, J. S. Addressing the chemistry of germacrene A by isotope labeling experiments. Org. Lett. 21, 2426–2429 (2019).

    CAS  PubMed  Google Scholar 

  15. Rinkel, J., Lauterbach, L., Rabe, P. & Dickschat, J. S. Two diterpene synthases for spiroalbatene and cembrene A from Allokutzneria albata. Angew. Chem. Int. Ed. 57, 3238–3241 (2018).

    CAS  Google Scholar 

  16. Cornforth, J. W., Cornforth, R. H., Donninger, C. & Popjak, G. Studies on the biosynthesis of cholesterol XIX. Steric course of hydrogen eliminations and of C–C bond formations in squalene biosynthesis. Proc. R. Soc. London, Ser. B 163, 492–514 (1966).

    CAS  Google Scholar 

  17. Rinkel, J. et al. Mechanisms of the diterpene cyclases β-pinacene synthase from Dictyostelium discoideum and hydropyrene synthase from Streptomyces clavuligerus. Chem. Eur. J. 23, 10501–10505 (2017).

    CAS  PubMed  Google Scholar 

  18. Reetz, M. T. Dytropic rearrangements, a new class of orbital-symmetry controlled reactions. Type I. Angew. Chem. Int. Ed. 11, 129–130 (1972).

    CAS  Google Scholar 

  19. Hugelshofer, C. L. & Magauer, T. Dyotropic rearrangements in natural product total synthesis and biosynthesis. Nat. Prod. Rep. 34, 228–234 (2017).

    CAS  PubMed  Google Scholar 

  20. Gutierrez, O. & Tantillo, D. J. Analogies between synthetic and biosynthetic reactions in which [1,2]-alkyl shifts are combined with other events: dyotropic, Schmidt, and carbocation rearrangements. J. Org. Chem. 77, 8845–8850 (2012).

    CAS  PubMed  Google Scholar 

  21. Li, H. & Dickschat, J. S. Isotopic labelling experiments and enzymatic preparation of iso-casbenes with casbene synthase from Ricinus communis. Org. Chem. Front. 9, 795–801 (2022).

    CAS  Google Scholar 

  22. Mitsuhashi, T., Rinkel, J., Okada, M., Abe, I. & Dickschat, J. S. Mechanistic characterization of two chimeric sesterterpene synthases from Penicillium. Chem. Eur. J. 23, 10053–10057 (2017).

    CAS  PubMed  Google Scholar 

  23. Cornforth, J. W., Cornforth, R. H., Popjak, G. & Yengoyan, L. Studies on the biosynthesis of cholesterol XX. Steric course of decarboxylation of 5-pyrophosphomevalonate and of the carbon to carbon bond formation in the biosynthesis of farnesyl pyrophosphate. J. Biol. Chem. 241, 3970–3987 (1966).

    CAS  PubMed  Google Scholar 

  24. Adamo, C. & Barone, V. Exchange functionals with improved long-range behavior and adiabatic connection methods without adjustable parameters: the mPW and mPW1PW models. J. Chem. Phys. 108, 664–675 (1998).

    CAS  Google Scholar 

  25. Matsuda, S. P. T., Wilson, W. K. & Xiong, Q. Mechanistic insights into triterpene synthesis from quantum mechanical calculations. Detection of systematic errors in B3LYP cyclization energies. Org. Biomol. Chem. 4, 530–543 (2006).

    CAS  PubMed  Google Scholar 

  26. Hong, Y. J. & Tantillo, D. J. A maze of dyotropic rearrangements and triple shifts: carbocation rearrangements connecting stemarene, stemodene, betaerdene, aphidicolene, and scopadulanol. J. Org. Chem. 83, 3780–3793 (2018).

    CAS  PubMed  Google Scholar 

  27. Quan, Z., Hou, A., Goldfuss, B. & Dickschat, J. S. Mechanism of the bifunctional multiple product sesterterpene synthase AcAS from Aspergillus calidoustus. Angew. Chem. Int. Ed. 61, e202117273 (2022).

    CAS  Google Scholar 

  28. Lemfack, M. C. et al. Reaction mechanism of the farnesyl pyrophosphate C-methyltransferase towards the biosynthesis of pre-sodorifen pyrophosphate by Serratia plymuthica 4Rx13. Sci. Rep. 11, 3182 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Burger, U., Delay, A. & Mazenod, F. 1,2,3,4,5-Pentamethyl-5-acetyl-cyclopentadien-1,3, ein ungewöhnliches Keton. Helv. Chim. Acta 57, 2106–2111 (1974).

    CAS  Google Scholar 

  30. Pace, V. et al. Efficient access to all-carbon quaternary and tertiary α-functionalized homoallyl-type aldehydes from ketones. Angew. Chem. Int. Ed. 56, 12677–12682 (2017).

    CAS  Google Scholar 

  31. Dess, D. B. & Martin, J. C. Readily accessible 12-I-51 oxidant for the conversion of primary and secondary alcohols to aldehydes and ketones. J. Org. Chem. 48, 4155–4156 (1983).

    CAS  Google Scholar 

  32. Horner, L., Hoffmann, H., Wippel, H. G. & Klahre, G. Phosphinoxyde als olefiniemgsreagenzien. Chem. Ber. 92, 2499–2505 (1959).

    CAS  Google Scholar 

  33. Wadsworth, W. S. & Emmons, W. D. The utility of phosphonate carbanions in olefin synthesis. J. Am. Chem. Soc. 83, 1733–1738 (1961).

    CAS  Google Scholar 

  34. Wise, M. L., Savage, T. J., Katahira, E. & Croteau, R. Monoterpene synthases from common sage (Salvia officinalis): cDNA Isolation, characterization, and functional expression of (+)-sabinene synthase, 1,8-cineol synthase, and (+)-bornyl diphosphate synthase. J. Biol. Chem. 273, 14891–14899 (1998).

    CAS  PubMed  Google Scholar 

  35. Nakano, C., Kim, H.-K. & Ohnishi, Y. Identification of the first bacterial monoterpene cyclase, a 1,8-cineole synthase, that catalyzes the direct conversion of geranyl diphosphate. ChemBioChem 12, 1988–1991 (2011).

    CAS  PubMed  Google Scholar 

  36. Jamieson, C. S., Ohashi, M., Liu, F., Tang, Y. & Houk, K. N. The expanding world of biosynthetic pericyclases: cooperation of experiment and theory for discovery. Nat. Prod. Rep. 36, 698–713 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Zhang, B. et al. Enzyme-catalysed [6 + 4] cycloadditions in the biosynthesis of natural products. Nature 568, 122–126 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Harmata, M. The (4 + 3)-cycloaddition reaction: simple allylic cations as dienophiles. Chem. Commun. 46, 8886–8903 (2010).

    CAS  Google Scholar 

  39. Giets, R. D. & Schiestl, R. H. High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat. Protoc. 2, 31–34 (2007).

    Google Scholar 

  40. Dickschat, J. S., Pahirulzaman, K. A. K., Rabe, P. & Klapschinski, T. A. An improved technique for the rapid chemical characterisation of bacterial terpene cyclases. ChemBioChem 15, 810–814 (2014).

    CAS  PubMed  Google Scholar 

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

    CAS  Google Scholar 

  42. Frisch, M. J. et al. Gaussian 16, revision B.01 (Gaussian, 2016).

  43. Lauterbach, L., Goldfuss, B. & Dickschat, J. S. Two diterpene synthases from Chryseobacterium: chryseodiene synthase and wanjudiene synthase. Angew. Chem. Int. Ed. 59, 11943–11947 (2020).

    CAS  Google Scholar 

  44. Grimme, S. Supramolecular binding thermodynamics by dispersion-corrected density functional theory. Chem. Eur. J. 18, 9955–9964 (2012).

    CAS  PubMed  Google Scholar 

  45. Luchini, G., Alegre-Requena, J. V., Guan, Y., Funes-Ardoiz, I. & Paton, R. S. GoodVibes v.3.0.1 (2019); https://patonlab.com/robert-paton/

Download references

Acknowledgements

This work was funded by the Deutsche Forschungsgemeinschaft (DFG) (DI1536/11-1, project number 469042295) and supported by the computing centre of the University of Cologne (RRZK), providing CPU time on the DFG-funded supercomputer CHEOPS. We thank P. Garbeva (Wageningen) for strain S. plymuthica PRI-2C, B. Piechulla (Rostock) and S. von Reuss (Neuchatel) for discussions, and Andreas Schneider (Bonn) for HPLC separations.

Author information

Authors and Affiliations

  1. Kekulé-Institut für Organische Chemie und Biochemie, Rheinische Friedrich-Wilhelms-Universität Bonn, Bonn, Germany

    Houchao Xu, Lukas Lauterbach & Jeroen S. Dickschat

  2. Institut für Organische Chemie, Universität zu Köln, Köln, Germany

    Bernd Goldfuss

  3. Institut für Anorganische Chemie, Rheinische Friedrich-Wilhelms-Universität Bonn, Bonn, Germany

    Gregor Schnakenburg

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Contributions

H.X. and L.L. performed syntheses and labelling experiments. B.G. performed density functional theory computations. G.S. solved the X-ray structure of (4S,5S)-12. J.S.D. designed and supervised research and wrote the manuscript with additions by all other authors.

Corresponding author

Correspondence to Jeroen S. Dickschat.

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Competing interests

The authors declare no competing interests.

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Peer review information

Nature Chemistry thanks the anonymous reviewer(s) for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–73, Tables 1–6, Schemes 1–8, and experimental and synthetic procedures.

Reporting Summary

Supplementary Data 1

Computed structure of P1* in Supplementary Fig. 42.

Supplementary Data 2

Computed structure of P1*-P3*-TS in Supplementary Fig. 42.

Supplementary Data 3

Computed structure of P3* in Supplementary Fig. 42.

Supplementary Data 4

Computed structure of P3*-TS in Supplementary Fig. 42.

Supplementary Data 5

Computed structure of P4* in Supplementary Fig. 42.

Supplementary Data 6

Computed structure of P4*-TS in Supplementary Fig. 42.

Supplementary Data 7

Computed structure of P5* in Supplementary Fig. 42.

Supplementary Data 8

Computed structure of P1*-NH3 in Supplementary Fig. 42.

Supplementary Data 9

Computed structure of P1*-NH3_TS in Supplementary Fig. 42.

Supplementary Data 10

Computed structure of P2*-NH3 in Supplementary Fig. 42.

Supplementary Data 11

Computed structure of P2*-NH3-TS in Supplementary Fig. 42.

Supplementary Data 12

Computed structure of P3*-NH3 in Supplementary Fig. 42.

Supplementary Data 13

Computed structure of S1 in Supplementary Fig. 43.

Supplementary Data 14

Computed structure of S1-TS in Supplementary Fig. 43.

Supplementary Data 15

Computed structure of S2 in Supplementary Fig. 43.

Supplementary Data 16

Computed structure of S2-TS in Supplementary Fig. 43.

Supplementary Data 17

Computed structure of S3 in Supplementary Fig. 43.

Supplementary Data 18

Computed structure of S3-TS in Supplementary Fig. 43.

Supplementary Data 19

Computed structure of S4 in Supplementary Fig. 43.

Supplementary Data 20

Computed structure of S4-TS in Supplementary Fig. 43.

Supplementary Data 21

Computed structure of S5a in Supplementary Fig. 43.

Supplementary Data 22

Computed structure of S5b in Supplementary Fig. 43.

Supplementary Data 23

Computed structure of S5-TS in Supplementary Fig. 43.

Supplementary Data 24

Computed structure of S6 in Supplementary Fig. 43.

Supplementary Data 25

Computed structure of S7a in Supplementary Fig. 43.

Supplementary Data 26

Computed structure of S7b in Supplementary Fig. 43.

Supplementary Data 27

Computed structure of S7-TS2 in Supplementary Fig. 43.

Supplementary Data 28

Computed structure of S7-TS1 in Supplementary Fig. 43.

Supplementary Data 29

Computed structure of S8 in Supplementary Fig. 43.

Supplementary Data 30

Computed structure of S8-TS in Supplementary Fig. 43.

Supplementary Data 31

Computed structure of S9 in Supplementary Fig. 43.

Supplementary Data 32

CIF file for the X-ray structure of (4S,5S)-12.

Supplementary Data 33

checkCIF reports for the X-ray structure of (4S,5S)-12.

Supplementary Video 1

Video of the transition state P1*-P3*-TS in Supplementary Fig. 42.

Supplementary Video 2

Video of the transition state P3*-TS in Supplementary Fig. 42.

Supplementary Video 3

Video of the transition state P4*-TS in Supplementary Fig. 42.

Supplementary Video 4

Video of the transition state P1*-NH3-TS in Supplementary Fig. 42.

Supplementary Video 5

Video of the transition state P2*-NH3-TS in Supplementary Fig. 42.

Supplementary Video 6

Video of the transition state S1-TS in Supplementary Fig. 43.

Supplementary Video 7

Video of the transition state S2-TS in Supplementary Fig. 43.

Supplementary Video 8

Video of the transition state S3-TS in Supplementary Fig. 43.

Supplementary Video 9

Video of the transition state S4-TS in Supplementary Fig. 43.

Supplementary Video 10

Video of the transition state S5-TS in Supplementary Fig. 43.

Supplementary Video 11

Video of the transition state S7-TS2 in Supplementary Fig. 43.

Supplementary Video 12

Video of the transition state St-TS1 in Supplementary Fig. 43.

Supplementary Video 13

Video of the transition state S8-TS in Supplementary Fig. 43.

Source data

Source Data Fig. 1

Unprocessed SDS-PAGE gel of MT and TC

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Xu, H., Lauterbach, L., Goldfuss, B. et al. Fragmentation and [4 + 3] cycloaddition in sodorifen biosynthesis. Nat. Chem. 15, 1164–1171 (2023). https://doi.org/10.1038/s41557-023-01223-z

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