Structural Insights into Monoterpene Cyclisation of Limonene Synthase from Cannabis sativa

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Terpenes represent the largest and most structurally diverse class of natural products, playing crucial roles in plant defence, ecological interactions, and environmental adaptation. Cannabis sativa is distinguished by its rich terpene profile, which contributes to its aroma, flavour, and pharmacological properties. Among these, limonene is a major monoterpene with commercial relevance in the fragrance, flavouring, and solvent industries, making it a prime target for metabolic engineering. Terpene biosynthesis is mediated by terpene synthase enzymes that catalyse the conversion of isoprenoid diphosphates into structurally diverse terpene scaffolds. Despite significant advances in terpene biochemistry, structural characterisation of C. sativa TPSs remains untouched. In this study, we present the first crystal structure of (-)-limonene synthase from C. sativa, providing mechanistic insights into monoterpene biosynthesis. The structure, solved at 3.2 Å resolution, reveals an "open" conformation, characterised by a solvent-accessible active site and disordered loop regions near the catalytic pocket, representing a pre-catalytic state, facilitating substrate access and positioning. Biochemical characterisation confirmed limonene synthase as a highly specific monoterpene synthase, producing (–)-limonene as the predominant product from geranyl diphosphate, with minor amounts of eight other monoterpenes. Kinetic analysis yielded a K_m of 7.809 ± 0.678 μM and a k_cat of 0.0204 s⁻¹, indicating moderate catalytic efficiency consistent with other plant-derived monoterpene synthases. These findings enhance our understanding of TPS function and lay the groundwork for enzyme engineering to optimise terpene biosynthesis for industrial and biotechnological applications.