Characterization and Crystal Structures of a Cubebol-Producing Sesquiterpene Synthase from Antrodia cinnamomea

Antrodia cinnamomea is an endemicspecies found in Taiwan, known for its medicinal properties in treatingvarious discomforts, including inflammation, diarrhea, abdominal pain,and other diseases. A. cinnamomea containsterpenoids that exhibit numerous bioactivities, making them potentialfood additives. This discovery piqued our interest in uncovering theirbiosynthetic pathway. Herein, we conducted functional and structuralcharacterization of a sesquiterpene synthase Cop4 from A. cinnamomea (AcCop4). Through gas chromatography-massspectrometry analysis, we observed that AcCop4 catalyzes the cyclizationof farnesyl pyrophosphate (FPP), primarily producing cubebol. Cubebolis widely used as a long-lasting cooling and refreshing agent in thefood industry. The structure of AcCop4, complexed with pyrophosphateand magnesium ions, revealed the closure of the active site facilitatedby R311. Interestingly, binding of pyrophosphate and magnesium ionsdid not cause any significant conformational change in the G1/2 helixof AcCop4, indicating that the apo form is not fully open. This high-resolutionstructure serves as a solid basis for understanding the biosyntheticmechanism of AcCop4 and supports further production and modificationof cubebol for its applications in the food industry.