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

Nat Chem. 2020 Jul;12(7):620-628. doi: 10.1038/s41557-020-0467-7. Epub 2020 May 25.
  • PMID: 32451436
  • DOI: 10.1038/s41557-020-0467-7


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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Benzofurans / chemical synthesis*
  • Benzofurans / chemistry
  • Biocatalysis
  • Biological Products / chemical synthesis*
  • Biological Products / chemistry
  • Cyclization
  • Cycloaddition Reaction / methods*
  • Lyases / chemistry*
  • Lyases / metabolism
  • Molecular Structure
  • Morus / chemistry
  • Morus / enzymology*
  • Oxidoreductases / chemistry*
  • Oxidoreductases / metabolism
  • Stereoisomerism
  • Substrate Specificity


  • Benzofurans
  • Biological Products
  • chalcomoracin
  • Oxidoreductases
  • Lyases