Isolation and Characterization of Two Monoterpene Synthases and a Sesquiterpene Synthase from Asarum heterotropoides

<b>Background</b>: <i>Asarum heterotropoides</i>, a prominent medicinal plant in China, is well known for producing an abundance of monoterpenes and sesquiterpenes, which constitute the primary components of its essential oil and serve as the principal active compounds of the species. However, the biosynthetic pathways for these terpenoids remain largely unelucidated. <b>Methods</b>: Gas chromatography-mass spectrometry analysis, in vitro enzyme assay, subcellular localization experiment and molecular docking were used to characterize the function of terpene synthase from <i>A. heterotropoides</i>. <b>Results</b>: In this study, we isolated and characterized two monoterpene synthases and one sesquiterpene synthase from <i>A. heterotropoides</i>. These enzymes exhibit promiscuous activities, accepting geranyl pyrophosphate and farnesyl pyrophosphate as substrates to yield a variety of monoterpene and sesquiterpene products in in vitro enzymatic assays. All three enzymes possess a conserved RRx8W motif, a hallmark typically associated with TPS-b and TPS-d monoterpene synthases involved in cyclic monoterpene formation. However, these two monoterpene synthases yield linear instead of cyclic products. The sesquiterpene synthase (AhTPS3) is a second example of TPS-a terpene synthase harboring such motif. <b>Conclusions</b>: Our findings significantly expand our understanding of terpene biosynthesis, especially the role of RRx8W motif.