# Mechanistic Insights into the Bornyl Diphosphate Synthase from _Lavandula angustifolia_
## 3 Results
### The N- and C-Terminus Regulated the Catalytic Activity of LaBPPS  
The N-terminal and C-terminal regions are located at the periphery of the catalytic pocket (Figure 6a). Deletion of amino acids 578-602 from the C-terminus (\(\Delta\)578-602) resulted in a significant increase in enzyme activity (Figure 6b). This suggested that the 578-602 region of the C-terminus undergoes structural changes that may hinder substrate (GPP) binding to the catalytic cavity, thereby inhibiting the catalytic reaction. These findings indicated that the flexibility of region 578-602 plays a crucial role in modulating the activity of LaBPPS.  
For the N-terminal deletion, the activity of the \(\Delta\)1-58 mutant was comparable to that of the full-length protein (Figure 6b), indicating that the region from residues 1 to 58 is not critical for the activity of LaBPPS. To further investigate the functional mechanisms, additional truncations of LaBPPS were performed. Deletion of 1-74 (\(\Delta\)1-74) resulted in a significant decrease in activity (Figure 6b). In an effort to further explore this, the activity of the \(\Delta\)59-74 mutant was assessed, indicating a dramatic reduction in activity (Figure 6b). These findings suggested that the 59-74 region, rather than the 1-58 region, plays a key role in the catalytic activity of LaBPPS.  
Figure 5: **Enzymatic characterization of LaBPPS to assess the bornyl diphosphate synthase activity.****(a)** Sequence alignment of conserved residues from various species. A0A185 NNC6, _Lavandula angustifolia_ subsp. _engustifolia_; A0A2H4 RG16, _Phyla dulcis_ (_Aztec sweet herb, Lippiia dulcis_); O81192, _Salvia officinalis (Sage_); A0A6M6 CCF6, _Salvia officinalis (Sage_); A0A9 ESIEG7, _Artemisia annua_ (_Sweet wormwood_); A0A2Z4K4Q8, _Wurfbainia villosa_; A0A7L7 TA7, _Wurfbainia villosa_; A0A7L7T473, _Wurfbainia longilularis_. (**b)** The activities of wild-type (WT) LaBPPS and specific mutants were quantified. Mutants D356A, D360A, R497A, D501A, or E508A exhibited a reduction in the activity ranging from 50- to 100-fold. The activity of WT LaBPPS was set to 100%.  
On the other hand, the Michaelis constant (\(K_{m}\)) values for wild-type (WT) LaBPPS and \(\Delta\)1-58 were 29.37 uM and 28.12 uM, respectively, both of which were significantly lower than those of \(\Delta\)1-74 (41.72 uM) and \(\Delta\)59-74 (38.49 uM), but considerably higher than the \(K_{m}\) value of \(\Delta\)578-602 (14.83 uM) (Table 4). In contrast, the catalytic constant (\(K_{cat}\)) values for WT LaBPPS and \(\Delta\)1-58 (6.23 min\({}^{-1}\) for WT and 6.78 min\({}^{-1}\) for \(\Delta\)1-58) were notably higher than those of \(\Delta\)1-74 (3.19 min\({}^{-1}\)) and \(\Delta\)59-74 (3.37 min\({}^{-1}\)) (Table 4), yet lower than the value for \(\Delta\)578-602 (11.71 min\({}^{-1}\)) (Table 4). These results suggest that the \(\Delta\)578-602 mutant exhibits a higher substrate affinity and a faster reaction rate than the WT protein. This implies that the 578-602 region may partially obstruct substrate GPP binding to the catalytic cavity, thereby inhibiting the catalytic reaction.