The One Ring: A Monocycle Producing Class II Diterpene Cyclase from Isodon leucophyllus

Class II diterpene cyclases (DTCs) have long been recognized to mediate distal cyclization of geranylgeranyl pyrophosphate (GGPP) into bicyclic labdane-related products through an acid-base catalytic mechanism, leaving the pyrophosphate 'head' group intact. Although distal monocyclic diterpenoids naturally occur, these are historically attributed to tetraterpenoid carotenoid degradation rather than direct enzymatic cyclization. Here, the discovery of a novel DTC from Isodon leucophyllus is reported, which produces the monocyclic (+)-trixagyl pyrophosphate and is, hence, termed IleuTPS, marking the first reported instance of such DTC activity. Notably, IleuTPS harbors an unusual E³⁶⁶XDD instead of the prototypical DXDD motif, where the "middle" aspartate functions as the catalytic acid and can also act as the base. Mutational analysis demonstrated that the E366D variant produces substantial amounts of the rearranged monocycle leucophyllusyl pyrophosphate, and small amounts of bicyclic products, suggesting that the native glutamate typically serves as the catalytic base. Isotope labeling experiments, quantum chemical calculations, and TerDockin simulations were further employed to delineate the enzymatic mechanism, highlighting an unusual fragmentation process underlying monocycle formation and underscoring the critical role of catalytic bases in dictating product outcome. Preliminary evolutionary analyses indicate that IleuTPS arose via early gene duplication and neofunctionalization, with such activity broadly distributed across plants and potentially other taxa. This study not only unlocks new avenues for exploring the functions of long-overlooked DTCs but also reshapes our understanding of distal monocyclic diterpenoid biosynthesis.