Terpene synthases (TPS) catalyze the cyclization of
the acyclic prenyl diphosphate precursor and are responsible for
the abundance of the natural product terpene in nature. The
biosynthesis of terpenoid by nonseed plant TPS is not well
understood due to the highly dynamic feature of the cyclization
reaction and the unavailability of the enzyme structure. Here we
discovered a class I TPS JeST4 from Jungermannia exsertifolia and
elucidated its catalytically active structure in complex with the
substrate and the key carbocation intermediates during catalysis.
We found that D106 is critical for the enzyme’s activity by
mediating the gate formed with R294 or R225. Further, we
identified two hotspot regi... More
Terpene synthases (TPS) catalyze the cyclization of
the acyclic prenyl diphosphate precursor and are responsible for
the abundance of the natural product terpene in nature. The
biosynthesis of terpenoid by nonseed plant TPS is not well
understood due to the highly dynamic feature of the cyclization
reaction and the unavailability of the enzyme structure. Here we
discovered a class I TPS JeST4 from Jungermannia exsertifolia and
elucidated its catalytically active structure in complex with the
substrate and the key carbocation intermediates during catalysis.
We found that D106 is critical for the enzyme’s activity by
mediating the gate formed with R294 or R225. Further, we
identified two hotspot regions from the coevolution study and
computational simulations, and the G91S and R242K mutations improved the conversion rate by 39- and 11-fold, respectively.
Remarkably, in both variants, R294 is able to stabilize the substrate pyrophosphate group, analogous to the dominating interaction
network observed in the distantly related bacterial TPSs. Further, NMR and molecular dynamics simulations indicated the formation
of an unusual C10(S)-bicyclogermacrene. Our research demonstrates the capacity of computing-informed engineering of nonseed
plant TPS. The discovery of the new TPS enzyme from nonseed land plant and computing-guided engineering would potentiate the
exploitation of the ultracheap enzyme as a potential biocatalyst for the production of the valuable terpenoid products.