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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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.
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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.
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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.
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Supplementary information
Supplementary Information
Supplementary Figs. 1–73, Tables 1–6, Schemes 1–8, and experimental and synthetic procedures.
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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DOI: https://doi.org/10.1038/s41557-023-01223-z
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