posted on 2022-03-16, 17:05authored byXiaoguang Yan, Jiahui Zhou, Jianjun Ge, Weiguo Li, Dongmei Liang, Warispreet Singh, Gary Black, Shengxin Nie, Jian Liu, Meiqing Sun, Jianjun Qiao, Meilan Huang
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.