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An alternative route to cyclic terpenes by reductive cyclization in iridoid biosynthesis

Nature volume 492, pages 138–142 (2012)Cite this article

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Abstract

The iridoids comprise a large family of distinctive bicyclic monoterpenes that possess a wide range of pharmacological activities, including anticancer, anti-inflammatory, antifungal and antibacterial activities1,2,3,4. Additionally, certain iridoids are used as sex pheromones in agriculturally important species of aphids, a fact that has underpinned innovative and integrated pest management strategies5. To harness the biotechnological potential of this natural product class, the enzymes involved in the biosynthetic pathway must be elucidated. Here we report the discovery of iridoid synthase, a plant-derived enzyme that generates the iridoid ring scaffold, as evidenced by biochemical assays, gene silencing, co-expression analysis and localization studies. In contrast to all known monoterpene cyclases, which use geranyl diphosphate as substrate and invoke a cationic intermediate, iridoid synthase uses the linear monoterpene 10-oxogeranial as substrate and probably couples an initial NAD(P)H-dependent reduction step with a subsequent cyclization step via a Diels–Alder cycloaddition or a Michael addition. Our results illustrate how a short-chain reductase was recruited as cyclase for the production of iridoids in medicinal plants. Furthermore, we highlight the prospects of using unrelated reductases to generate artificial cyclic scaffolds. Beyond the recognition of an alternative biochemical mechanism for the biosynthesis of cyclic terpenes, we anticipate that our work will enable the large-scale heterologous production of iridoids in plants and microorganisms for agricultural5,6,7,8 and pharmaceutical1,2,3,4,9 applications.

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Figure 1: Identification of the iridoid synthase from C. roseus.
Figure 2: In vivo studies of iridoid synthase.
Figure 3: Substrate specificity and mechanistic considerations.
Figure 4: An alternative cyclization catalysed by the fungal reductase EasA to generate the new artificial cyclopentanoid, named ‘diquatdial’ here.

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Accession codes

Accessions

DDBJ/GenBank/EMBL

Sequence Read Archive

Data deposits

The sequence of iridoid synthase has been deposited in GenBank/EMBL/DDBJ with accession no. JX974564 and the short sequence reads are available in the NCBI SRA database under accession number SRP005953.

Change history

  • 05 December 2012

    The two stereoisomers in Fig. 3a were corrected.

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Acknowledgements

The present study was funded by the Danish Council for Independent Research (Natural Sciences) through Postdoctoral Fellowship 10-082858 granted to F.G.-F. Additional funding was received from the NIH (GM074820), the j004561SRC (BB/J004561/1 and BB/J009091/1) and the John Innes Foundation. This work was conducted under Defra Plant Health Licence PHSI 449/6612(07/2011). W.S.G. gratefully acknowledges a National Science Foundation Predoctoral Fellowship. Y.C. would like to thank the NSF for grant DMS-1209112. K. R. Chauhan is acknowledged for providing initial nepetalactol samples. We thank S. Fairhurst, L. Hill and A. Jones for NMR, LC–MS and GC–MS assistance, respectively. J. Ward and N. Hawkins are acknowledged for the GC–MS-based high-resolution mass spectra (HRMS) of synthesized compounds. R. Buell and E. Góngora-Castillo provided many helpful discussions on the transcriptome data. The purified EasA protein was provided J. Z. Cheng, to whom we are deeply indebted. S.E.O. receives salary support through a synergy initiative between the John Innes Centre and the University of East Anglia.

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Author notes

  1. Ezekiel Nims

    Present address: Present address: Ra Pharmaceuticals, Inc., Cambridge, Massachusetts 02139, USA.,

Authors and Affiliations

  1. Department of Biological Chemistry, John Innes Centre, Norwich NR4 7UH, UK,

    Fernando Geu-Flores, Nathaniel H. Sherden, Weslee S. Glenn & Sarah E. O’Connor

  2. Université François-Rabelais de Tours, EA2106, Biomolécules et Biotechnologies Végétales, 37200 Tours, France,

    Vincent Courdavault

  3. Université de Toulouse, UPS, UMR 5546, Laboratoire de Recherche en Sciences Végétales, BP 42617 Auzeville, F-31326 Castanet-Tolosan, France,

    Vincent Burlat

  4. CNRS, UMR 5546, BP 42617, F-31326 Castanet-Tolosan, France,

    Vincent Burlat

  5. Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA,

    Weslee S. Glenn & Ezekiel Nims

  6. Department of Statistics and Probability, Michigan State University, East Lansing, Michigan 48824, USA,

    Cen Wu & Yuehua Cui

  7. School of Chemistry, University of East Anglia, Norwich NR4 7TJ, UK

    Sarah E. O’Connor

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Contributions

F.G.-F. and S.E.O. conceived the project; C.W., Y.C. and F.G.-F. constructed the G10H mutual ranking list; E.N. cloned the iridoid synthase from cDNA; F.G.-F. and W.S.G. expressed the recombinant protein; N.H.S. performed all chemical synthesis; F.G.-F. carried out all enzyme assays, enzyme-based synthesis, VIGS experiments, and feeding experiments; V.C. performed the sub-cellular localization studies; V.B. carried out the cellular localization studies; F.G.-F. and S.E.O. drafted the manuscript; and S.E.O. acted as overall study director.

Corresponding author

Correspondence to Sarah E. O’Connor.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-8, Supplementary Tables 1-2, Supplementary Text, which explains the methods used to synthesize the chemicals used in the main paper and additional references. (PDF 2146 kb)

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Geu-Flores, F., Sherden, N., Courdavault, V. et al. An alternative route to cyclic terpenes by reductive cyclization in iridoid biosynthesis. Nature 492, 138–142 (2012). https://doi.org/10.1038/nature11692

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