Interrogation of an Enzyme Library Reveals the Catalytic Plasticity of Naturally Evolved [4+2] Cyclases

Chembiochem. 2023 Jul 17;24(14):e202300382. doi: 10.1002/cbic.202300382. Epub 2023 Jun 18.


Stereoselective carbon-carbon bond forming reactions are quintessential transformations in organic synthesis. One example is the Diels-Alder reaction, a [4+2] cycloaddition between a conjugated diene and a dienophile to form cyclohexenes. The development of biocatalysts for this reaction is paramount for unlocking sustainable routes to a plethora of important molecules. To obtain a comprehensive understanding of naturally evolved [4+2] cyclases, and to identify hitherto uncharacterised biocatalysts for this reaction, we constructed a library comprising forty-five enzymes with reported or predicted [4+2] cycloaddition activity. Thirty-one library members were successfully produced in recombinant form. In vitro assays employing a synthetic substrate incorporating a diene and a dienophile revealed broad-ranging cycloaddition activity amongst these polypeptides. The hypothetical protein Cyc15 was found to catalyse an intramolecular cycloaddition to generate a novel spirotetronate. The crystal structure of this enzyme, along with docking studies, establishes the basis for stereoselectivity in Cyc15, as compared to other spirotetronate cyclases.

Keywords: biosynthetic gene cluster; cyclases; enzyme screening; intra-molecular Diels-Alder reaction; polyketides.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Carbon*
  • Catalysis
  • Chemistry Techniques, Synthetic
  • Cycloaddition Reaction
  • Proteins*


  • Proteins
  • Carbon