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FAD-dependent enzyme-catalysed intermolecular [4+2] cycloaddition in natural product biosynthesis

Nature Chemistry volume 12, pages 620–628 (2020)Cite this article

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Abstract

The Diels–Alder reaction is one of the most powerful and widely used methods in synthetic chemistry for the stereospecific construction of carbon–carbon bonds. Despite the importance of Diels–Alder reactions in the biosynthesis of numerous secondary metabolites, no naturally occurring stand-alone Diels–Alderase has been demonstrated to catalyse intermolecular Diels–Alder transformations. Here we report a flavin adenine dinucleotide-dependent enzyme, Morus alba Diels–Alderase (MaDA), from Morus cell cultures, that catalyses an intermolecular [4+2] cycloaddition to produce the natural isoprenylated flavonoid chalcomoracin with a high efficiency and enantioselectivity. Density functional theory calculations and preliminary measurements of the kinetic isotope effects establish a concerted but asynchronous pericyclic pathway. Structure-guided mutagenesis and docking studies demonstrate the interactions of MaDA with the diene and dienophile to catalyse the [4+2] cycloaddition. MaDA exhibits a substrate promiscuity towards both dienes and dienophiles, which enables the expedient syntheses of structurally diverse natural products. We also report a biosynthetic intermediate probe (BIP)-based target identification strategy used to discover MaDA.

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Fig. 1: Proposed biosynthetic pathway for plant-derived D–A-type natural products and our strategy to identify the hypothetical intermolecular [4+2] pericyclase.
Fig. 2: Identification of the intermolecular [4+2] pericyclase using activity-based protein purification and BIP-based target identification.
Fig. 3: Functional and biochemical characterization of MaMO and MaDA.
Fig. 4: Substrate scope of MaDA and the chemoenzymatic synthesis of D–A natural products.
Fig. 5: DFT calculations and KIE results for the intermolecular [4+2] cycloaddition reaction.
Fig. 6: Structure, molecular dynamics (MD) simulation and site-directed mutagenesis characterization of MaDA.

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Data availability

The data that support the findings of this study are available with this article and its Supplementary Information, or are available from the corresponding authors upon reasonable request. The gene sequences of MaDA and MaMO as amplified from cell cultures of M. alba are deposited in GenBank, accession no. MK573629 and no. MK573628, respectively. The structural factor and coordinate of MaDA are deposited in the Protein Data Bank under ID 6JQH.

References

  1. Nicolaou, K. C., Snyder, S. A., Montagnon, T. & Vassilikogiannakis, G. The Diels–Alder reaction in total synthesis. Angew. Chem. Int. Ed. 41, 1668–1698 (2002).

    CAS  Google Scholar 

  2. Oikawa, H. & Tokiwano, T. Enzymatic catalysis of the Diels–Alder reaction in the biosynthesis of natural products. Nat. Prod. Rep. 21, 321–352 (2004).

    CAS  PubMed  Google Scholar 

  3. Stocking, E. M. & Williams, R. M. Chemistry and biology of biosynthetic Diels–Alder reactions. Angew. Chem. Int. Ed. 42, 3078–3115 (2003).

    CAS  Google Scholar 

  4. Minami, A. & Oikawa, H. Recent advances of Diels–Alderases involved in natural product biosynthesis. J. Antibiot. 69, 500–506 (2016).

    CAS  Google Scholar 

  5. Klas, K., Tsukamoto, S., Sherman, D. H. & Williams, R. M. Natural Diels–Alderases: elusive and irresistable. J. Org. Chem. 80, 11672–11685 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Jeon, B. S., Wang, S.-A., Ruszczycky, M. W. & Liu, H. W. Natural [4+2]-cyclases. Chem. Rev. 117, 5367–5388 (2016).

    PubMed  PubMed Central  Google Scholar 

  7. Jamieson, C. S., Ohashi, M., Liu, F., Tang, Y. & Houk, K. N. The expanding world of biosynthetic pericyclase: cooperation of experiment and theory for discovery. Nat. Prod. Rep. 36, 698–713 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Kasahara, K. et al. Solanapyrone synthase, a possible Diels–Alderase and iterative type I polyketide synthase encoded in a biosynthetic gene cluster from Alternaria solani. ChemBioChem 11, 1245–1252 (2010).

    CAS  PubMed  Google Scholar 

  9. 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  Google Scholar 

  10. Ose, T. et al. Insight into a natural Diels–Alder reaction from the structure of macrophomate synthase. Nature 422, 185–189 (2003).

    CAS  PubMed  Google Scholar 

  11. Hudson, G. A., Zhang, Z., Tietz, J. I., Mitchell, D. A. & van der Donk, W. A. In vitro biosynthesis of the core scaffold of the thiopeptide thiomuracin. J. Am. Chem. Soc. 137, 16012–16015 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Kim, H. J., Ruszczycky, M. W., Choi, S. H., Liu, Y. N. & Liu, H. W. Enzyme-catalysed [4+2] cycloaddition is a key step in the biosynthesis of spinosyn A. Nature 473, 109–112 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Hashimoto, T. et al. Biosynthesis of versipelostatin: identification of an enzyme-catalyzed [4+2]-cycloaddition required for macrocyclization of spirotetronate-containing polyketides. J. Am. Chem. Soc. 137, 572–575 (2015).

    CAS  PubMed  Google Scholar 

  14. Byrne, M. J. et al. The catalytic mechanism of a natural Diels–Alderase revealed in molecular detail. J. Am. Chem. Soc. 138, 6095–6098 (2016).

    CAS  PubMed  Google Scholar 

  15. Tian, Z. et al. An enzymatic [4+2] cyclization cascade creates the pentacyclic core of pyrroindomycins. Nat. Chem. Biol. 11, 259–265 (2015).

    CAS  PubMed  Google Scholar 

  16. Li, L. et al. Biochemical characterization of a eukaryotic decalin-forming Diels–Alderase. J. Am. Chem. Soc. 138, 15837–15840 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Li, L. et al. Genome mining and assembly-line biosynthesis of the UCS1025A pyrrolizidinone family of fungal alkaloids. J. Am. Chem. Soc. 140, 2067–2071 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Kato, N. et al. Control of the stereochemical course of [4+2] cycloaddition during trans-decalin formation by Fsa2-family enzymes. Angew. Chem. Int. Ed. 57, 9754–9758 (2018).

    CAS  Google Scholar 

  19. Tan, D. et al. Genome-mined Diels–Alderase catalyzes formation of the cis-octahydrodecalins of varicidin A and B. J. Am. Chem. Soc. 141, 769–773 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Zhang, Z. et al. Enzyme-catalyzed inverse-electron demand Diels–Alder reaction in the biosynthesis of antifungal ilicicolin H. J. Am. Chem. Soc. 141, 5659–5663 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Ohashi, M. et al. SAM-dependent enzyme-catalysed pericyclic reactions in natural product biosynthesis. Nature 549, 502–506 (2017).

    PubMed  PubMed Central  Google Scholar 

  22. Zhang, B. et al. Enzyme-catalysed [6+4] cycloadditions in the biosynthesis of natural products. Nature 568, 122–126 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Siegel, J. B. et al. Computational design of an enzyme catalyst for a stereoselective bimolecular Diels–Alder reaction. Science 329, 309–313 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Preiswerk, N. et al. Impact of scaffold rigidity on the design and evolution of an artificial Diels–Alderase. Proc. Natl Acad. Sci. USA 111, 8013–8018 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  Google Scholar 

  26. Nomura, T., Hano, Y. & Fukai, T. Chemistry and biosynthesis of isoprenylated flavonoids from Japanese mulberry tree. Proc. Jpn Acad. B 85, 391–408 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Nomura, T. & Hano, Y. Isoprenoid-substituted phenolic compounds of moraceous plants. Nat. Prod. Rep. 11, 205–218 (1994).

    CAS  PubMed  Google Scholar 

  28. Dat, N. T. et al. Hypoxia-inducible factor-1 inhibitory benzofurans and chalcone-derived Diels–Alder adducts from Morus species. J. Nat. Prod. 72, 39–43 (2009).

    CAS  PubMed  Google Scholar 

  29. Wang, M. et al. Diels–Alder adducts with PTP1B inhibition from Morus notabilis. Phytochemistry 109, 140–146 (2015).

    CAS  PubMed  Google Scholar 

  30. Fukai, T., Kaitou, K. & Terada, S. Antimicrobial activity of 2-arylbenofurans from Morus species against methicillin-resistant Staphylococcus aureus. Fitoterapia 76, 708–711 (2005).

    CAS  PubMed  Google Scholar 

  31. Esposito, F. et al. Kuwanon-L as a new allosteric HIV-1 integrase inhibitor: molecular modeling and biological evaluation. ChemBioChem 16, 2507–2512 (2015).

    CAS  PubMed  Google Scholar 

  32. Ueda, S., Nomura, T., Fukai, T. & Matsumoto, J. Kuwanon J, a new Diels–Alder adduct and chalcomoracin from callus culture of Morus alba L. Chem. Pharm. Bull. 30, 3042–3045 (1982).

    CAS  Google Scholar 

  33. Hano, Y., Nomura, T. & Ueda, S. Biosynthesis of optically active Diels–Alder type adducts revealed by an aberrant metabolism of O-methylated precursors in Morus alba cell cultures. J. Chem. Soc. Chem. Commun. 8, 610–613 (1990).

    Google Scholar 

  34. Hano, Y., Nomura, T. & Ueda, S. Biosynthesis of chalcomoracin and kuwanon J, the Diels–Alder type adducts, in Morus alba L. cell cultures. Chem. Pharm. Bull. 37, 554–556 (1989).

    CAS  Google Scholar 

  35. Han, J. et al. Enantioselective biomimetic total syntheses of kuwanons I and J and brosimones A and B. Angew. Chem. Int. Ed. 53, 9257–9261 (2014).

    CAS  Google Scholar 

  36. Qi, C. et al. Biomimetic dehydrogenative Diels–Alder cycloadditions: total syntheses of brosimones A and B. Angew. Chem. Int. Ed. 52, 8345–8348 (2013).

    CAS  Google Scholar 

  37. Gunawan, C. & Rizzacasa, M. Mulberry Diels–Alder adducts: synthesis of chalcomoracin and mulberrofuran C methyl ethers. Org. Lett. 12, 1388–1391 (2010).

    CAS  PubMed  Google Scholar 

  38. Field, B. & Osbourn, A. E. Metabolic diversification-independent assembly of operon-like gene clusters in different plants. Science 320, 543–547 (2008).

    CAS  PubMed  Google Scholar 

  39. Geu-Flores, F. et al. An alternative route to cyclic terpenes by reductive cyclization in iridoid biosynthesis. Nature 492, 138–142 (2012).

    CAS  PubMed  Google Scholar 

  40. Chen, X. et al. A pathogenesis-related 10 protein catalyzes the final step in thebaine biosynthesis. Nat. Chem. Biol. 14, 738–743 (2018).

    CAS  PubMed  Google Scholar 

  41. Medema, M. H. & Osbourn, A. Computational genomic identification and functional reconstitution of plant natural product biosynthetic pathways. Nat. Prod. Rep. 33, 951–962 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Zhang, D.-W., Tao, X.-Y., Yu, L.-Y. & Dai, J.-G. New 2-arylbenzofuran metabolite from cell cultures of Morus alba. J. Asian Nat. Prod. Res. 17, 683–688 (2015).

    CAS  PubMed  Google Scholar 

  43. Zou, Y. et al. Tandem prenyltransferases catalyze isoprenoid elongation and complexity generation in biosynthesis of quinolone alkaloids. J. Am. Chem. Soc. 137, 4980–4983 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Shoyama, Y. et al. Structure and function of ∆1-tetrahydrocannabinolic acid (THCA) synthase, the enzyme controlling the psychoactivity of Cannabis sativa. J. Mol. Biol. 423, 96–105 (2012).

    CAS  PubMed  Google Scholar 

  45. Yang, Z. et al. Relationships between product ratios in ambimodal pericyclic reactions and bond lengths in transition structures. J. Am. Chem. Soc. 140, 3061–3067 (2018).

    CAS  PubMed  Google Scholar 

  46. Farrow, S. C. et al. Biosynthesis of an anti-addiction agent from the iboga plant. J. Am. Chem. Soc. 141, 12979–12983 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Chen, Q. et al. Enzymatic intermolecular hetero-Diels–Alder reaction in the biosynthesis of tropolonic sesquiterpenes. J. Am. Chem. Soc. 141, 14052–14056 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. He, N. et al. Draft genome sequence of the mulberry tree Morus notabilis. Nat. Commun. 4, 2445 (2013).

    PubMed  Google Scholar 

  49. Frisch, M. J. et al. Gaussian 09 (Gaussian Inc., 2009).

  50. Chai, J. D. & Head-Gordon, M. Long-range corrected hybrid density functionals with damped atom–atom dispersion corrections. Phys. Chem. Chem. Phys. 10, 6615–6620 (2008).

    CAS  PubMed  Google Scholar 

  51. Bootsma, A. N. & Wheeler, S. Popular integration grids can result in large errors in DFT-computed free energies. Preprint at https://doi.org/10.26434/chemrxiv.8864204.v5 (2019).

  52. Grimme, S. Supramolecular binding thermodynamics by dispersion-corrected density functional theory. Chem. Eur. J. 18, 9955–9964 (2012).

    CAS  PubMed  Google Scholar 

  53. Takano, Y. & Houk, K. N. Benchmarking the conductor-like polarizable continuum model (CPCM) for aqueous solvation-free energies of neutral and ionic organic molecules. J. Chem. Theory Comput. 1, 70–77 (2005).

    PubMed  Google Scholar 

  54. Legault, C. Y. CYLview 1.0b (University of Sherbrooke, 2009); http://www.cylview.org

  55. Jeona, B.-S. et al. Investigation of the mechanism of the SpnF-catalyzed [4+2]-cycloaddition reaction in the biosynthesis of spinosyn A. Proc. Natl Acad. Sci. USA 114, 10408–10413 (2017).

    Google Scholar 

  56. Wilde, T. C., Blotny, G. & Pollack, R. M. Experimental evidence for enzyme-enhanced coupled motion/quantum mechanical hydrogen tunneling by ketosteroid isomerase. J. Am. Chem. Soc. 130, 6577–6585 (2008).

    CAS  PubMed  Google Scholar 

  57. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Cryst. 40, 658–674 (2007).

    CAS  Google Scholar 

  58. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Cryst. D 66, 486–501 (2010).

    CAS  Google Scholar 

  59. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Cryst. D 66, 213–221 (2010).

    CAS  Google Scholar 

  60. Durrant, J. D., Votapka, L., Sørensen, J. & Amaro, R. E. POVME 2.0: an enhanced tool for determining pocket shape and volume characteristics. J. Chem. Theory Comput. 10, 5047–5056 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  61. Coleman, R. G., Carchia, M., Sterling, T., Irwin, J. J. & Shoichet, B. K. Ligand pose and orientational sampling in molecular docking. PLoS ONE 8, e75992 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  62. Peng, S.-M., Zhou, Y. & Huang, N. Improving the accuracy of pose prediction in molecular docking via structural filtering and conformational clustering. Chin. Chem. Lett. 24, 1001–1004 (2013).

    CAS  Google Scholar 

  63. Jacobson, M. P. et al. A hierarchical approach to all-atom protein loop prediction. Proteins 55, 351–367 (2004).

    CAS  PubMed  Google Scholar 

  64. Bowers, K. J et al. Scalable algorithms for molecular dynamics simulations on commodity clusters. In Proc. ACM/IEEE Conference on Supercomputing (SC06) 84 (ACM, 2006).

  65. Banks, J. L. et al. Integrated modeling program, applied chemical theory (IMPACT). J. Comput. Chem. 26, 1752–1780 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  66. Jorgensen, W. L., Chandrasekhar, J., Madura, J. D., Impey, R. W. & Klein, M. L. Comparison of simple potential functions for simulating liquid water. J. Chem. Phys. 79, 926–935 (1983).

    CAS  Google Scholar 

  67. Kräutler, V., Van Gunsteren, W. F. & Hünenberger, P. H. A fast SHAKE algorithm to solve distance constraint equations for small molecules in molecular dynamics simulations. J. Comput. Chem. 22, 501–508 (2001).

    Google Scholar 

  68. Darden, T., York, D. & Pedersen, L. Particle mesh Ewald: An Nlog(N) method for Ewald sums in large systems. J. Chem. Phys. 98, 10089–10092 (1993).

    CAS  Google Scholar 

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Acknowledgements

We thank X.-F. Fu (Peking University) for providing the crucial instrument and technical support to us; L. Li (NIBS) and W. Zhou (Peking University) for assistance in LC–MS/MS proteomics analysis; T. Cai (NIBS) for assistance in the transcriptome analysis; J. Y. Xiao (Peking University) for assistance in the expression of MaDA in insect cells using a baculovirus expression system. We also thank H. C. Lam and J. H. Snyder for proofreading the manuscript. We are grateful to the staff members of Shanghai Synchrotron Radiation Facility (beamline BL17U) for their support during X-ray data collection. This work was financially supported by National Natural Science Foundation of China grants (21625201, 21661140001, 21961142010, 91853202 and 21521003 to X.L.); the National Key Research and Development Program of China (2017YFA0505200 to X.L.); the Beijing Outstanding Young Scientist Program (BJJWZYJH01201910001001 to X.L.); CAMS Innovation Fund for Medical Sciences (CIFMS-2016-I2M-3-012 and 2019-I2M-1-005 to J.D.); the Drug Innovation Major Project (2018ZX09711001-001-006 to J.D.); Key Project at Central Government Level for the Ability Establishment of Sustainable Use for Valuable Chinese Medicine Resources (2060302 to J.D.); JSPS A3 Foresight Program to H.O. and the Fundamental Research Funds for the Central Universities (2017PT35001 to J.D.). Computational support was provided by the Special Program for Applied Research on Super Computation of the NSFC-Guangdong Joint Fund (the second phase) under grant no. U1501501. This work was also inspired by the international and interdisciplinary environment of the JSPS Core-to-Core Program ‘Asian Chemical Biology Initiative’. K.N.H. is grateful to the National Science Foundation (grant no. CHE-1764328) for financial support. Calculations were performed on the Hoffman2 cluster at the University of California, Los Angeles, and the Extreme Science and Engineering Discovery Environment (XSEDE), which is supported by the National Science Foundation (grant OCI-1053575). L.G. is supported in part by a Postdoctoral Fellowship of Peking-Tsinghua Center for Life Sciences.

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Author notes

  1. These authors contributed equally: Lei Gao, Cong Su, Xiaoxia Du, Ruishan Wang.

Authors and Affiliations

  1. State Key Laboratory of Bioactive Substance and Function of Natural Medicines; CAMS Key Laboratory of Enzyme and Biocatalysis of Natural Drugs, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China

    Lei Gao, Cong Su, Kebo Xie & Jungui Dai

  2. Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Department of Chemical Biology, College of Chemistry and Molecular Engineering, Synthetic and Functional Biomolecules Center, and Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China

    Lei Gao, Xiaoxia Du, Xiaojing Liu, Runze Tian, Liyun Zhang, Qianqian Guo & Xiaoguang Lei

  3. State Key Laboratory Breeding Base of Dao-di Herbs, National Resources Center of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China

    Ruishan Wang, Lanping Guo & Luqi Huang

  4. Department of Chemistry and Biochemistry, University of California, Los Angeles, CA, USA

    Shuming Chen & K. N. Houk

  5. National Institute of Biological Sciences (NIBS), Beijing, China

    Yu Zhou, She Chen & Niu Huang

  6. Division of Chemistry, Graduate School of Science, Hokkaido University, Sapporo, Japan

    Chengwei Liu, Atsushi Minami & Hideaki Oikawa

  7. Faculty of Pharmaceutical Sciences, Teikyo Heisei University, Tokyo, Japan

    Yoshio Hano & Manabu Shimazaki

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  1. Lei Gao
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Contributions

X.L., J.D. and L.G. initiated the project. X.L., J.D., L.G., C.S., X.D. and R.W. conceived and designed the experiments, analysed the data and prepared the manuscript, with input from all the authors. L.G., X.L. and R.T. conducted the chemical synthesis. L.G. and She Chen performed the pull-down assay and LC–MS/MS analysis. C.S. and K.X. conducted the activity-based protein purification and the enzymology experiments. X.D. performed the X-ray crystallization and data analysis of MaDA. R.W. cloned the genes of MaDA and MaMO. Y.Z. and N.H. performed docking and MD studies. L.G., C.S., X.D. and Q.G. performed the protein mutagenesis and expression. C.L., A.M. and H.O. helped the transcriptome analysis. C.S. cultivated the cell cultures. Y.H. and M.S. provided important information from the enzymatic assay. Shuming Chen performed DFT calculations under the guidance of K.N.H. L.G. and L.Z. performed the KIE experiments. L.G., C.S., X.D. and R.W. contributed equally to this work. X.L., J.D. and L.H. managed the whole project.

Corresponding authors

Correspondence to Luqi Huang, Jungui Dai or Xiaoguang Lei.

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Extended data

Extended Data Fig. 1 The time dependent assay using SEC fraction of crude enzyme.

The reaction mixture containing 20 mM Tris-HCl, pH 7.5, 100 mM morachalcone A (2), 100 μM moracin C (3) as substrates and 9.8 μg of crude cell lysate in a final volume of 100 μL was incubated at 30 °C for 1 h. The reactions were terminated by the addition of 200 μL of ice-cold MeOH and were centrifuged at 15,000 g for 30 min. The supernatants were analysed by the LC/MS. The experiments were repeated three times independently with similar results.

Extended Data Fig. 2 The evidence suggesting BBE-like enzyme as the putative oxidase.

a, The proposed mechanism for the formation of diene 4 catalysed by BBE-like enzyme. b, In vitro reaction analysis of 2 and 3 with or without dioxygen using SEC fraction: i) without SEC fraction (negative control), ii) with SEC fraction and dioxygen (positive control), iii) with SEC fraction but without dioxygen. The reaction buffer containing 2 and 3 was degassed in −78 °C and charged with argon, and then SEC fraction was added under argon atmosphere at room temperature. The experiments were repeated two times independently with similar results. c, Detection of hydrogen peroxide using Hydrogen Peroxide Assay Kit (Beyotime, S0038). The purple color indicated the existence of hydrogen peroxide in the reaction buffer. The experiments were repeated three times independently with similar results.

Extended Data Fig. 3 The activities of the probe 8 and its analogues and the silver staining of pull-down assay using 8 as the photoaffinity probe.

a, The structures of morachalcone A derivatives and the corresponding chalcomoracin derivatives. b, In vitro analysis using AS fraction (0.25 mg/mL)with different morachalcone A derivatives (100 μM): i) 2 and 3 without the AS fraction, ii) 2 and 3 with the AS fraction, iii) 5 and 3 with the AS fraction, iv) 6 and 3 with the AS fraction, v) 7 and 3 with the AS fraction, vi) 8 and 3 with the AS fraction. The experiments were repeated three times independently with similar results. c, The SEC fraction was incubated on ice with or without BIP 8 under irradiation of 365 nm UV light or without UV irradiation for 1 h. The following lysates were used for streptavidin-agarose pull-down assays, and the precipitates were resolved by 8% SDS-PAGE, followed by silver staining. The indicated bands were excised and subjected to LC-MS/MS proteomics analysis. The experiments were repeated two times independently with similar results.

Extended Data Fig. 4 In vitro reaction analysis of MaMO and MaDA.

When MaDA was incubated with 3 alone, no new peak appeared, which revealed the MaDA did not have the oxidative function. In contract, when MaMO was used, diene 4 was formed. On the other hand, when MaMO was incubated with 2 and 3, 3 was completely transformed to diene 4, but no chalcomoracin or oxidative product of 2 was observed which indicated MaMO neither oxidized 2 nor catalysed the [4 + 2] cycloaddition between 2 and 4. The experiments were repeated three times independently with similar results.

Extended Data Fig. 5 Effects of pH, temperature and divalent metal ions on the activity of MaMO and MaDA.

a, Effect of temperature on MaMO’s activity. b, Effect of divalent metal ions on MaMO’s activity. c, Effect of pH value on MaMO’s activity. d, Effect of temperature on MaDA’s activity. e, Effect of divalent metal ions on MaDA’s activity. f, Effect of pH value on MaDA’s activity. CK means ‘control check’. Enzyme activity values represent mean ± standard deviation (s.d.) of three independent replicates.

Extended Data Fig. 6 The time course for the incubation of morachalcone A (2) and 4 without MaDA.

The reaction mixture containing 20 mM Tris-HCl, pH 8.0, 100 μM morachalcone A (2), 100 μM diene (4) as substrates without MaDA in a final volume of 100 μL was incubated at 50 °C for 100 min. The reactions were terminated by the addition of 200 μL of ice-cold MeOH and were centrifuged at 15,000 g for 30 min. The supernatants were analysed by the LC/MS. The experiments were repeated three times independently with similar results.

Extended Data Fig. 7 Determination of the kinetic parameters of MaDA using stable diene 10 and morachalcone A (2).

a, Enzymatic assay of 2 and 10 with MaDA. Compounds 2 and 10 were incubated with 270 nM MaDA or boiled MaDA for 5 min. The experiments were repeated three times independently with similar results. b, Kinetic parameters of MaDA for 2 (5–300 μΜ) using 10 (1.5 mM) as diene. c, Kinetic parameters of MaDA for 10 (5–800 μΜ) using 2 (4 mM) as dienophile. KM, kcat and kcat/KM values represent mean ± standard deviation (s.d.) of three independent replicates.

Extended Data Fig. 8 Calculated transition states of the Diels-Alder reaction between dienophile 2 and dienes 4 or 10.

a, This data shows the calculated Diels-Alder transition states of Diels-Alder reaction between 2 and 4 leading to the four possible product regio- and stereoisomers. The computed barriers (all with respect to isolated reactants) show that TS-1 is the favored TS, suggesting that the reaction displays intrinsic regio- and stereoselectivity for the experimentally observed product isomer under enzyme catalysis. b, The DFT calculation supports an endo transition state and a concerted but asynchronous mechanism of the Diels-Alder reaction between 2 and 10. C–H hydrogen atoms are omitted for clarity. Interatomic distances are in Å. Energies are in kcal/mol.

Extended Data Fig. 9 Different absorbance data of MaDA at 450 nm at different conditions.

a, The absorbance of MaDA (34 μM) at 450 nm was detected every 5 min with or without adding 6.6 mM sodium dithionite (about 200 eq), indicating the reduced form of MaDA is stable under excess of sodium dithionite for at least 30 minutes. The experiments were repeated three times independently with similar results. b, The absorbance of 450 nm of MaDAwt, MaDAH116A and MaDAH116A-FAD were detected at the same protein concentration (34 μM). The MaDAH116A was dialysed in 1 M KBr solution to remove the non-covalent FAD. Absorbance values of 450 nm represent mean ± standard deviation (s.d.) of three independent replicates.

Extended Data Fig. 10

Data collection and refinement statistics of MaDA-FAD.

Supplementary Information

Supplementary Information

Supplementary Figs. 1–12, Tables 1–6, chemical synthesis, MaDA-mediated enzymatic synthesis, Cartesian coordinates of computed structures, NMR spectra and references.

Reporting summary

Supplementary Data 1

X-ray structure of MaDA.

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Gao, L., Su, C., Du, X. et al. FAD-dependent enzyme-catalysed intermolecular [4+2] cycloaddition in natural product biosynthesis. Nat. Chem. 12, 620–628 (2020). https://doi.org/10.1038/s41557-020-0467-7

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