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

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. Diels‐Alderases perform an essential step in the biosynthesis of bioactive spirotetronates. To expand the understanding of such enzymes a cyclase library was created, which made it possible to identify a novel spirotetronate cyclase from a metagenome mining approach. Structural elucidation of both the enzyme by X‐ray crystallography and the product by NMR helped us to gain further insights into the essential features of how these enzymes perform complex cyclisations.