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Enzyme-catalysed [4+2] cycloaddition is a key step in the biosynthesis of spinosyn A

Abstract

The Diels–Alder reaction is a [4+2] cycloaddition reaction in which a cyclohexene ring is formed between a 1,3-diene and an electron-deficient alkene via a single pericyclic transition state1. This reaction has been proposed as a key transformation in the biosynthesis of many cyclohexene-containing secondary metabolites2,3,4,5. However, only four purified enzymes have thus far been implicated in biotransformations that are consistent with a Diels–Alder reaction, namely solanapyrone synthase6, LovB7,8, macrophomate synthase9,10, and riboflavin synthase11,12. Although the stereochemical outcomes of these reactions indicate that the product formation could be enzyme-guided in each case, these enzymes typically demonstrate more than one catalytic activity, leaving their specific influence on the cycloaddition step uncertain. In our studies of the biosynthesis of spinosyn A, a tetracyclic polyketide-derived insecticide from Saccharopolyspora spinosa13,14, we identified a cyclase, SpnF, that catalyses a transannular [4+2] cycloaddition to form the cyclohexene ring in spinosyn A. Kinetic analysis demonstrates that SpnF specifically accelerates the ring formation reaction with an estimated 500-fold rate enhancement. A second enzyme, SpnL, was also identified as responsible for the final cross-bridging step that completes the tetracyclic core of spinosyn A in a manner consistent with a Rauhut–Currier reaction15. This work is significant because SpnF represents the first example characterized in vitro of a stand-alone enzyme solely committed to the catalysis of a [4+2] cycloaddition reaction. In addition, the mode of formation of the complex perhydro-as-indacene moiety in spinosyn A is now fully established.

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Figure 1
Figure 2: HPLC analysis showing the reactions catalysed by SpnF, SpnL and SpnM.
Figure 3: Kinetic analysis demonstrating that SpnM and SpnF, respectively, catalyse the dehydration and cyclization steps of macrolactone 3.
Figure 4: The spinosyn aglycone biosynthetic pathway.

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Acknowledgements

We thank C. Whitman for review of the manuscript, L. Hong for carrying out the early cloning work, B. Shoulders and S. Sorey for assistance with the interpretation of the NMR spectra, and S. Mansoorabadi and E. Isiorho for discussions on the reaction mechanisms and structural modelling of SpnF. This work is supported in part by grants from the National Institutes of Health (GM035906, F32AI082906), the Texas Higher Education Coordination Board (ARP-003658-0093-2007), and the Welch Foundation (F-1511).

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Authors

Contributions

H.-w.L. provided the scientific direction and the overall experimental design for the studies. H.J.K. designed and performed most of the experiments. S.-h.C. participated in the chemical synthesis of the substrate for SpnJ (2) and the characterization of the structures of the enzyme reaction products. M.W.R. analysed the kinetic experiment data. Y.-n.L. carried out the mutation studies of SpnF. H.J.K., M.W.R. and H.-w.L. wrote the manuscript.

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Correspondence to Hung-wen Liu.

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The authors declare no competing financial interests.

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The file contains Supplementary Text, Supplementary Figures 1-8 with legends, Supplementary Table 1, additional references and spectral data. (PDF 2728 kb)

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Kim, H., Ruszczycky, M., Choi, Sh. et al. Enzyme-catalysed [4+2] cycloaddition is a key step in the biosynthesis of spinosyn A. Nature 473, 109–112 (2011). https://doi.org/10.1038/nature09981

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