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.
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Carruthers, W. Cycloaddition Reactions in Organic Synthesis (Pergamon, Oxford, 1990)
Desimoni, G., Tacconi, G., Barco, A. & Pollini, G. P. Natural Products Synthesis through Percyclic Reactions (American Chemical Society, Washington, DC 1983)
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)
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)
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)
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)
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)
Ichihara, A. & Oikawa, H. in Comprehensive Natural Products Chemistry (eds Barton, D., Nakanishi, K. & Meth-Cohn, O.) Vol. 1, 367–408 (Elsevier, Amsterdam, 1999)
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)
Oikawa, H. et al. Macrophomate synthase: Unusual enzyme catalyzing multiple reactions from pyrones to benzoates. Tetrahedr. Lett. 40, 6983–6986 (1999)
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)
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)
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)
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)
Ichihara, A., Murakami, K. & Sakamura, S. Synthesis of pyrenocines A, B and pyrenocheatic acid A. Tetrahedron 43, 5245–5250 (1987)
Afarinkia, K., Vinader, V., Nelson, T. D. & Posner, G. H. Diels–Alder cycloadditions of 2-pyrones and 2-pyridones. Tetrahedron 48, 9111–9171 (1992)
Sauer, J. & Sustmann, R. Mechanic aspects of Diels–Alder reactions: A critical survey. Angew. Chem. Int. Edn Engl. 19, 779–807 (1980)
Blake, J. F. & Jorgensen, W. L. Solvent effects on a Diels–Alder reaction from computer simulations. J. Am. Chem. Soc. 113, 7430–7432 (1991)
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)
Heine, A. et al. An antibody exo Diels–Alderase inhibitor complex at 1.95 angstrom resolution. Science 179, 1934–1940 (1998)
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)
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)
Braisted, A. C. & Schultz, P. G. An antibody-catalyzed biomolecular Diels–Alder reaction. J. Am. Chem. Soc. 112, 7430–7431 (1991)
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)
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)
Xu, J. et al. Evolution of shape complementarity and catalytic efficiency from a primordial antibody template. Science 286, 2345–2348 (1999)
Collaborative Computational Project, Number 4. The CCP4 suite: Programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994)
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)
Brünger, A. T. et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998)
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.
The authors declare that they have no competing financial interests.
About this article
Cite this article
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
Nature Chemistry (2020)
Discovering Biomolecules with Huisgenase Activity: Designed Repeat Proteins as Biocatalysts for (3 + 2) Cycloadditions
Journal of the American Chemical Society (2020)
Chemistry - A European Journal (2019)
Trends in Chemistry (2019)
The expanding world of biosynthetic pericyclases: cooperation of experiment and theory for discovery
Natural Product Reports (2019)