# Mechanistic Insights into the Bornyl Diphosphate Synthase from _Lavandula angustifolia_
## 4 Discussion  
In this study, AlphaFold2 was employed to predict the three-dimensional structure of LaBPPS. The hydrodynamic radius of LaBPPS was measured to be 5.9 \(\pm\) 0.3 nm. According to the molecular docking results, mutational experiments were performed. Substitutions at D356A, D360A, R497A, D501A, or E508A resulted in a 50- to 100-fold reduction in enzymatic activity. The activity of the \(\Delta\)1-58 mutant was found to be equivalent to that of the wild-type (WT) protein. Deletion of regions 1-74 or 59-74 (\(\Delta\)1-74 or \(\Delta\)59-74) significantly diminished activity compared to the WT protein, while deletion of 578-602 (\(\Delta\)578-602) led to a marked increase in activity. The expression level of the _LaBPPS_ gene was significantly higher in the flower than in the stem and root, and leaf, with a peak at 8:00. These findings provide critical insights into the functional mechanisms of LaBPPS and offer potential strategies for improving the quality of lvender essential oils through genetic engineering.  
Due to the inability to obtain crystals of LaBPPS, we pursued a more detailed investigation into its functional mechanism. To aid in this analysis, we utilized SWISS-MODEL [50; 51; 52; 53] to identify structural homologs of LaBPPS (Table S1). Our search revealed that LaBPPS shared amino acid sequence identities of 53.83%, 53.64%, 53.13%, 50.47%, and 45.81% with terpene synthases from _Mentha spicata_, _Salvia officinalis_, _Salvia fritucosa_, _Thymus vulgaris_, and _Citrus sinensis_, respectively (Table S1). These results provided valuable insights into the structural and functional mechanisms of LaBPPS in lvender.  
Essential oils (EOs) from the genus lvender primarily consist of a limited number of'regular' monoterpenes and their derivatives [16; 54]. For instance, the economically valuable EOs from _Lavandula angustifolia_ and _Lavandula x intermedia_ species are rich in compounds such as linalool, linalool acetate, and 1,8-cineole [55]. In contrast, emerging synthetic biology platforms have shown great promise as an alternative method for producing monoterpencoids [14; 56; 57]. By engineering microbial biosynthetic pathways, sustainable and efficient production systems can be established. Notable successes include the microbial production of geraniol, limonene, linalool, (+)-borneol, citronello, and nerol. (\(-\))-Borneol is primarily derived from geranyl diphosphate (GPP), which is synthesized from the two common C\({}_{5}\) precursors, isopentenyl diphosphate and dimethylallyl diphosphate, through either the cytoplasmic mexulonate (MVA) pathway or the plastid 2-C-methyl-D-erythnitol-4-phosphate (MEP) pathway [10; 17; 55; 56; 58].  
In conclusion, our study presents a novel approach for the thorough investigation of the complex functional mechanisms of LaBPPS in lvender, with the aim of enhancing the quality of lvender EOs.  
The following supporting information can be downloaded at [[https://www.mdpi.com/article/10.3390/cimb47070517/s1](https://www.mdpi.com/article/10.3390/cimb47070517/s1)]([https://www.mdpi.com/article/10.3390/cimb47070517/s1](https://www.mdpi.com/article/10.3390/cimb47070517/s1)).  
Conceptualization, D.L.; data curation, D.L.; formal analysis, D.L.; investigation, D.L., N.L., F.Y., Y.D., H.S., and W.Y.; methodology, D.L.; resources, D.L.; software, D.L.; validation, D.L.; writing--original draft, D.L.; writing--review and editing, D.L. All authors have read and agreed to the published version of the manuscript.  
Our research work is financially supported by grants from the third batch of the 'Tianchi Talent' Young Doctoral Research Grant, Xinjiang Autonomous Region (2025 QNBS001), Xinjiang Key Laboratory of Lavender Conservation and Utilization (LCUZ2405), and Start-up Fund for Doctoral Research Established by Yili Normal University (2024 RCYJ08).  
Not applicable.  
The data presented in this study are available in this article (and Supplementary Materials), and further enquiries can be directed to the corresponding authors.