Enzymatic synthesis of natural (+)-aristolochene from a non-natural substrateElectronic supplementary information (ESI) available: Synthesis of FDP and 7-methylene-FDP, gas chromatograms, MS spectra, 1H-NMR spectra and molecular calculations. See DOI: 10.1039/c6cc08164a

The sesquiterpene cyclase aristolochene synthase from Penicillium roquefortii (PR-AS) has evolved to catalyse with high specificity (92%) the conversion of farnesyl diphosphate (FDP) to the bicyclic hydrocarbon (+)-aristolochene, the natural precursor of several fungal toxins. Here we report that PR-AS converts the unnatural FDP isomer 7-methylene farnesyl diphosphate to (+)-aristolochene via the intermediate 7-methylene germacrene A. Within the confined space of the enzyme's active site, PR-AS stabilises the reactive conformers of germacrene A and 7-methylene germacrene A, respectively, which are protonated by the same active site acid (most likely HOPP i ) to yield the shared natural bicyclic intermediate eudesmane cation, from which (+)-aristolochene is then generated. Aristolochene synthase from Penicillium roqueforti converts 7-methylene-FDP, a substrate the enzyme never encounters in nature, to the natural product (+)-aristolochene.