Insight into a natural Diels–Alder reaction from the structure of macrophomate synthase

Abstract

The Diels–Alder reaction, which forms a six-membered ring from an alkene (dienophile) and a 1,3-diene, is synthetically very useful for construction of cyclic products with high regio- and stereoselectivity under mild conditions1. It has been applied to the synthesis of complex pharmaceutical and biologically active compounds2. Although evidence3,4,5,6,7 on natural Diels–Alderases has been accumulated in the biosynthesis of secondary metabolites8, there has been no report on the structural details of the natural Diels–Alderases. The function and catalytic mechanism of the natural Diels–Alderase are of great interest owing to the diversity of molecular skeletons in natural Diels–Alder adducts8. Here we present the 1.70 Å resolution crystal structure of the natural Diels–Alderase, fungal macrophomate synthase (MPS)3, in complex with pyruvate. The active site of the enzyme is large and hydrophobic, contributing amino acid residues that can hydrogen-bond to the substrate 2-pyrone. These data provide information on the catalytic mechanism of MPS, and suggest that the reaction proceeds via a large-scale structural reorganization of the product.

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Figure 1: Details of individual reaction steps with macrophomate synthase.
Figure 2: Overall structure of MPS.
Figure 3: Active site view showing Mg2+ coordination, the electron density map, and the proposed model of the early transition state.
Figure 4: Comparison of Diels–Alderases.

References

  1. 1

    Carruthers, W. Cycloaddition Reactions in Organic Synthesis (Pergamon, Oxford, 1990)

    Google Scholar 

  2. 2

    Desimoni, G., Tacconi, G., Barco, A. & Pollini, G. P. Natural Products Synthesis through Percyclic Reactions (American Chemical Society, Washington, DC 1983)

    Google Scholar 

  3. 3

    Watanabe, K., Mie, T., Ichihara, A., Oikawa, H. & Honma, M. Detailed reaction mechanism of macrophomate synthase. Extraordinary enzyme catalyzing five-step transformation from 2-pyrones to benzoates. J. Biol. Chem. 275, 38393–38401 (2000)

    CAS  Article  Google Scholar 

  4. 4

    Oikawa, H., Katayama, K., Suzuki, Y. & Ichihara, A. Enzymatic activity catalysing exo-selective Diels–Alder reaction in solanapyrone biosynthesis. J. Chem. Soc. Chem. Commun. 1321–1322 (1995)

  5. 5

    Katayama, K., Kobayashi, T., Oikawa, H., Honma, M. & Ichihara, A. Enzymatic activity and partial purification of solanapyrone synthase: First enzyme catalyzing Diels–Alder reaction. Biochim. Biophys. Acta 1384, 387–395 (1998)

    CAS  Article  Google Scholar 

  6. 6

    Oikawa, H., Kobayashi, T., Katayama, K., Suzuki, Y. & Ichihara, A. Total synthesis of (- )-solanapyrone a via enzymatic Diels–Alder reaction of prosolanapyrone. J. Org. Chem. 63, 8748–8756 (1998)

    CAS  Article  Google Scholar 

  7. 7

    Auclair, K. et al. Lovastatin nonaketide synthase catalyzes an intramolecular Diels–Alder reaction of a substrate analogue. J. Am. Chem. Soc. 122, 11519–11520 (2000)

    CAS  Article  Google Scholar 

  8. 8

    Ichihara, A. & Oikawa, H. in Comprehensive Natural Products Chemistry (eds Barton, D., Nakanishi, K. & Meth-Cohn, O.) Vol. 1, 367–408 (Elsevier, Amsterdam, 1999)

    Google Scholar 

  9. 9

    Sakurai, I., Miyajima, H., Akiyama, K., Shimizu, S. & Yamamoto, Y. Studies on metabolites of Macrophoma commelinae. IV. Substrate specificity in the biotransformation of 2-pyrones to substituted benzoic acids. Chem. Pharm. Bull. 36, 2003–2011 (1988)

    CAS  Article  Google Scholar 

  10. 10

    Oikawa, H. et al. Macrophomate synthase: Unusual enzyme catalyzing multiple reactions from pyrones to benzoates. Tetrahedr. Lett. 40, 6983–6986 (1999)

    CAS  Article  Google Scholar 

  11. 11

    Watanabe, K. et al. Macrophomate synthase: Characterization, sequence, and expression in Escherichia coli of the novel enzyme catalyzing unusual multistep transformation from 2-pyrones to benzoates. J. Biochem. 127, 467–473 (2000)

    CAS  Article  Google Scholar 

  12. 12

    Izard, T. & Blackwell, N. C. Crystal structures of the metal-dependent 2-dehydro-3-deoxy-galactarate aldolase suggest a novel reaction mechanism. EMBO J. 19, 3849–3856 (2000)

    CAS  Article  Google Scholar 

  13. 13

    Watanabe, K., Mie, T., Ichihara, A., Oikawa, H. & Honma, M. Substrate diversity of macrophomate synthase catalyzing an unusual multistep transformation from 2-pyrones to benzoates. Biosci. Biotechnol. Biochem. 64, 530–538 (2000)

    CAS  Article  Google Scholar 

  14. 14

    Watanabe, K., Mie, T., Ichihara, A., Oikawa, H. & Honma, M. Reaction mechanism of the macrophomate synthase: Experimental evidence on intermediacy of a bicyclic compound. Tetrahedr. Lett. 41, 1443–1446 (2000)

    CAS  Article  Google Scholar 

  15. 15

    Ichihara, A., Murakami, K. & Sakamura, S. Synthesis of pyrenocines A, B and pyrenocheatic acid A. Tetrahedron 43, 5245–5250 (1987)

    CAS  Article  Google Scholar 

  16. 16

    Afarinkia, K., Vinader, V., Nelson, T. D. & Posner, G. H. Diels–Alder cycloadditions of 2-pyrones and 2-pyridones. Tetrahedron 48, 9111–9171 (1992)

    CAS  Article  Google Scholar 

  17. 17

    Sauer, J. & Sustmann, R. Mechanic aspects of Diels–Alder reactions: A critical survey. Angew. Chem. Int. Edn Engl. 19, 779–807 (1980)

    Article  Google Scholar 

  18. 18

    Blake, J. F. & Jorgensen, W. L. Solvent effects on a Diels–Alder reaction from computer simulations. J. Am. Chem. Soc. 113, 7430–7432 (1991)

    CAS  Article  Google Scholar 

  19. 19

    Romesberg, F. E., Spiller, B., Schultz, P. G. & Stevens, R. C. Immunological origins of binding and catalysis in a Diels–Alderase antibody. Science 279, 1929–1933 (1998)

    ADS  CAS  Article  Google Scholar 

  20. 20

    Heine, A. et al. An antibody exo Diels–Alderase inhibitor complex at 1.95 angstrom resolution. Science 179, 1934–1940 (1998)

    ADS  Article  Google Scholar 

  21. 21

    Chen, J., Deng, Q., Wang, R., Houk, K. & Hilvert, D. Shape complementarity, binding-site dynamics, and transition state stabilization: A theoretical study of Diels–Alder catalysis by antibody 1E9. Chembiochem 1, 255–261 (2000)

    CAS  Article  Google Scholar 

  22. 22

    Hilvert, D., Hill, K. W., Nared, K. D. & Auditor, M-T. Antibody catalysis of a Diels–Alder reaction. J. Am. Chem. Soc. 111, 9261–9262 (1989)

    CAS  Article  Google Scholar 

  23. 23

    Braisted, A. C. & Schultz, P. G. An antibody-catalyzed biomolecular Diels–Alder reaction. J. Am. Chem. Soc. 112, 7430–7431 (1991)

    Article  Google Scholar 

  24. 24

    Gouverneur, V. E. et al. Control of the exo and endo pathways of the Diels–Alder reaction by antibody catalysis. Science 262, 204–208 (1993)

    ADS  CAS  Article  Google Scholar 

  25. 25

    Ylikauhaluoma, J. T. et al. Anti-metallocene antibodies — a new approach to enantioselective catalysis of the Diels–Alder reaction. J. Am. Chem. Soc. 117, 7041–7047 (1995)

    CAS  Article  Google Scholar 

  26. 26

    Xu, J. et al. Evolution of shape complementarity and catalytic efficiency from a primordial antibody template. Science 286, 2345–2348 (1999)

    CAS  Article  Google Scholar 

  27. 27

    Collaborative Computational Project, Number 4. The CCP4 suite: Programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994)

    Article  Google Scholar 

  28. 28

    de La Fortelle, E. & Bricogne, G. Maximum-likelihood heavy-atom parameter refinement for multiple isomorphous replacement and multiwavelength anomalous diffraction methods. Methods Enzymol. 276, 472–494 (1997)

    CAS  Article  Google Scholar 

  29. 29

    Brünger, A. T. et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998)

    Article  Google Scholar 

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Acknowledgements

We thank S. Wakatsuki, M. Suzuki and N. Igarashi of the Photon Factory, Japan, for help in data collection at beamline BL18B. This work was supported in part by National Project on Protein Structural and Functional Analyses from the Ministry of Education, Science, Sports and Culture of Japan.

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Correspondence to Hideaki Oikawa or Isao Tanaka.

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Ose, T., Watanabe, K., Mie, T. et al. Insight into a natural Diels–Alder reaction from the structure of macrophomate synthase. Nature 422, 185–189 (2003). https://doi.org/10.1038/nature01454

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