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Uncoupled activation and cyclization in catmint reductive terpenoid biosynthesis

Nature Chemical Biology volume 15, pages 71–79 (2019)Cite this article

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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.

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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.

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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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  1. Benjamin R. Lichman

    Present address: Department of Biology, University of York, York, UK

Authors and Affiliations

  1. The John Innes Centre, Department of Biological Chemistry, Norwich Research Park, Norwich, UK

    Benjamin R. Lichman, Mohamed O. Kamileen, Gabriel R. Titchiner, Gerhard Saalbach, Clare E. M. Stevenson, David M. Lawson & Sarah E. O’Connor

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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.

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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

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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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