# Mechanistic Insights into the Bornyl Diphosphate Synthase from _Lavandula angustifolia_
###### Abstract  
Lavender species hold substantial economic importance due to their widespread cultivation for essential oils (EOs). Lavender EOs contain terpenes essential for industries such as cosmetics, personal care, and pharmaceuticals. In the biosynthetic pathway of EOs, _Lavandula angustifolia_ bornyl diphosphate synthase (LaBPPS) catalyzes the conversion of geranyl diphosphate (GPP) to bornyl diphosphate (BPP). However, the functional mechanisms of LaBPPS remain poorly understood. Here, we conducted mutational experiments based on the molecular docking results, and found that mutations at positions D356A, D360A, R497A, D501A, or E508A led to a 50- to 100-fold reduction in the activity. Deletion of region 1-58 (\(\Delta\)1-58) did not affect activity compared to the wild-type (WT) protein, while deletions of regions 1-74 or 59-74 (\(\Delta\)1-74 or \(\Delta\)59-74) significantly decreased the activity. Conversely, deletion of residues 578-602 (\(\Delta\)578-602) dramatically increased the activity. The _LaBPPS_ gene showed dramatically higher expression levels in flowers compared to other tissues (stems, leaves and roots), peaking at 8:00. Our results provide valuable insights into EO biosynthesis in Lavender and suggest potential strategies for genetic engineering aimed at improving EO quality.  
Lavandula angustifolia; bornyl diphosphate synthase; geranyl diphosphate; enzymatic activity assays; gene expression profiles 20 May 202525 June 20251 July  
Although monoterpenes exhibit significant structural diversity, they all originate from two fundamental five-carbon precursors: isopentenyl pyrophosphate and its isomer, dimethylallylyl pyrophosphate [20; 21]. The enzymatic activity of prenyl transferases facilitates the condensation of these precursors, generating geranyl pyrophosphate and farnesyl pyrophosphate, which serve as key intermediates for the biosynthesis of monoterpenes and sesquiterpen, respectively [4; 22; 23]. Monoterpene synthases, such as bornyl diphosphate synthase, subsequently catalyze the removal of the pyrophosphate group from these linear precursors, leading to the formation of a diverse array of cyclic, linear, hydroxylated, or hydrocarbon monoterpenes [22; 24; 25]. Within the EO biosynthetic pathway, borneol and camphor are synthesized through the formation of bornyl diphosphate (BPP) from geranyl diphosphate (GPP), a reaction catalyzed by bornyl diphosphate synthase (BPPS) [26; 27]. BPP serves as a substrate for the production of borneol, which is then oxidized to camphor by borneol dehydrogenase [26; 27]. Notably, _Lavandula angustifolia_ BPPS (LaBPPS) plays a pivotal role in the biosynthesis of BPP. Therefore, considerable research has focused on understanding the catalytic mechanisms of LaBPPS to gain insights into EO specificity and improve EO quality. However, the structural and mechanistic details of LaBPPS are still not understood.  
Herein, we used AlphaFold2 to predict the three-dimensional structure of LaBPPS. The hydrodynamic radius of LaBPPS was determined to be 5.9 \(\pm\) 0.3 nm. Based on the molecular docking results, mutational experiments were performed. We found that substitutions at D356A, D360A, R497A, D501A, or E508A led to a 50- to 100-fold reduction in enzyme activity. The activity of \(\Delta\)1-58 was indistinguishable from that of the wild-type (WT) protein. In contrast, \(\Delta\)1-74 or \(\Delta\)59-74 caused a significant reduction in the activity compared to the WT protein. Of note, \(\Delta\)578-602 resulted in a dramatic increase in the activity. The expression level of the _LaBPPS_ gene was the highest in the flower among these tissues (flower, leaf, stem, and root), with a peak of expression at 8:00. These findings provide insight into the functional mechanisms of LaBPPS in lavelander, contributing valuable knowledge for enhancing the quality of lavelander essential oils through genetic engineering, which could have implications for the development of cosmetic, personal care, and alternative medicinal products.