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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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.
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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.
The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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.
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.
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.
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.
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.
Data collection and refinement statistics of MaDA-FAD.
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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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