Unexpected enzyme-catalysed [4+2] cycloaddition and rearrangement in polyether antibiotic biosynthesis.

Enzymes catalysing remarkable Diels-Alder-like [4+2] cyclisations have been previously implicated in the biosynthesis of spirotetronate and spirotetramate antibiotics. Biosynthesis of the polyether antibiotic tetronasin is not anticipated to require such steps, yet the tetronasin gene cluster encodes enzymes Tsn11 and Tsn15, homologous to authentic [4+2] cyclases. Here we show that deletion of Tsn11 led to accumulation of a late-stage intermediate, in which the two central rings of tetronasin, and four of its 12 asymmetric centres, remain unformed. reconstitution showed that Tsn11 catalyses an apparent inverse-electron-demand hetero Diels-Alder-like [4+2] cyclisation of this species to an unexpected oxadecalin compound, which is then rearranged by Tsn15 to form tetronasin. To gain structural and mechanistic insight into the activity of Tsn15, a 1.7 Å crystal structure of a Tsn15-substrate complex has been solved.