The formation of rings in carbon backbones is essential for the biological activity of many natural products. The polyether tetronate antibiotics tetronasin, tetronomycin, and tetromadurin (SF2487/A80577) are notable for their ring diversity, each possessing a tetronate, cyclohexane, tetrahydropyran, and at least one tetrahydrofuran ring. The antibiotic activity and complexity of these polyether tetronates has led to research on their biosynthesis from actinomycete bacteria. Despite this, the mechanism of stereospecific cyclohexane and tetrahydropyran formation has remained mysterious. Although no formal [4+2] cycloaddition (Diels-Alder reaction) is predicted in the biosynthesis of these compounds, the biosynthetic gene clusters of all three were found to contain a pair of genes that encode homologues of two different [4+2] cyclases previously identified in complex spirotetronate pathways. Specific gene deletions demonstrated that both classes of [4+2] cyclase homologue are essential for polyether tetronate biosynthesis. In the tetronasin producer, Streptomyces longisporoflavus, deletion of the [4+2] cyclase enzyme homologue gene tsn11 resulted in production of a new metabolite that was characterised as an open form of tetronasin lacking both cyclohexane and tetrahydropyran rings. Incubating this metabolite with purified Tsn11 resulted in the production of an unknown intermediate labelled T-22. The structure of T-22 was determined using NMR to contain an unexpected oxadecalin moiety but still lack the tetrahydropyran ring, implicating Tsn11 as catalysing an apparent inverse-electron-demand hetero-Diels-Alder reaction. Remarkably, incubating T-22 with purified Tsn15, the other [4+2] cyclase homologue, formed the tetrahydropyran ring and fragmented the oxadecalin moiety to a cyclohexane ring, producing tetronasin. To gain structural insight into the novel activity of Tsn15 it was successfully crystallised. The structure of Tsn15 was then solved at 1.8 Å using SAD phasing; and Brazilian collaborators solved a Tsn15-ligand structure at 1.7 Å. Tsn15 shares the same eight-stranded β-barrel fold as its [4+2] cyclase homologues. The two main mechanisms considered here for the Tsn15-catalysed conversion of T-22 into tetronasin were a general acid/ base or a pericyclic mechanism. Site-directed mutagenesis of the Tsn15 active site indicated that none of the acid/base amino acid side chains were essential for activity, favouring instead the pericyclic mechanism.