## 4 Discussion
_Pica abies_ TPS \(E\), \(E\)-alpha-far, _Pinus taeda_ TPS alpha-far, and _Pica engelmannii_\(\times\)_Pica glauca_ TPS (\(E\), \(E\))-alpha-far/(\(E\))-beta-oci in TPS D1 contained an additional motif 15 compared to the other sesqui-TPSs. The absence of these motifs suggests a potential difference in the evolution of mono- and sesqui-TPSs. Furthermore, in the phylogenetic tree, motif 19 was absent in all sesqui-TPS sequences of _Pinus_, including _P. massoniana_. We hypothesize that this may provide evidence for the functional differentiation of TPSs occurring before species differentiation. Previous study has shown that the catalytic function of TPSs is affected by kinetics rather than thermodynamics, meaning that the types of intermediates initially formed in the reaction can affect the formation of subsequent products [62]. The formation of multiple entromeres in the active center can generate a variety of substrates, leading to the formation of by-products in fewer reaction steps [63]. Molecular docking simulations showed that the substrate-binding pockets of sesqui-TPSs were larger than those of mono-TPSs. The substrate of sesqui-TPS is FPP (C15), which contains three double bonds and is more complex than GPP (C10), which contains two double bonds [64]. Because of the increase in the chain length of the substrate, the number of sesquiterpene intermediates is much greater than that of monoteremetries, which enriches the diversity of product structures [65]. Larger pockets may facilitate the entry of substrates and the formation of products. Secondary metabolic enzymes tend to produce mechanistic elasticity during evolution, giving rise to new enzymes with a wider range of substrates and a variety of new products [66]. Some residues are responsible for catalytic elasticity within and around the active sites of TPSs. Changing these residues can regulate the products or catalytic rates [67; 68; 69]. For example, [PERSON] et al. [70] reported that Tyr residues (Y573) in S-limonene synthase may be involved in the earlier steps of the reaction, probably by controlling the conformation of the helical linalyl diphosphate intermediate. The replacement of Ile in _A. thaliana_ entkaurene synthase with Thr can convert it into a pimaradiene synthase, but the catalytic efficiency is reduced by four times [71]. The replacement of nine amino acids in _Nicotiana tabacum_ can accomplish conversion between the catalytic specificities of 5-epi-aristolochene synthase and premnisproidene synthase [68]. Molecular docking between Pm TPSs and the corresponding substrates showed that there were seven kinds of residues in Pm mono-TPSs participating in the catalytic process, Asp, Arg, Tyr, Glu, Trp, Thr, and Cys, which were distributed between helix C and helix K. Asp, Try, Arg, and Lys were found in Pm sesqui-TPSs, distributed between helix D and helix H2, similar to reports from _A. grandis_[31]. Mn\({}^{2+}\) is a common catalyst in biological catalytic reactions, often combining with Asp [72], which may be a reason for the elevated catalytic efficiency of some TPSs in the presence of Mn\({}^{2+}\)[73]. Thus, adding Mn\({}^{2+}\) may accelerate the production of Pm (+)-\(\alpha\)-pinene, Pm seliinene, and Pm longifolene. At a pH \(\geq\) 7, Asp and Glu are deprotonated, facilitating the formation of a carbonium ion intermediate. Therefore, when using Pm mono-TPSsin the catalytic reaction, it is critical to maintain a pH above 7 for stable production. The surfaces of the aromatic groups in Tyr and Trp are positively charged, which can stabilize the carbonium ion intermediate [62]. Because Pm sesqui-TPSs have fewer aromatic groups than Pm mono-TPSs, there may be more binding possibilities for the structures of the intermediates and products in Pm sesqui-TPSs than that in Pm mono-TPSs. During expression vector construction, a purified tag was inserted at the N-terminus to avoid the influence of redundant tag sequences at the C-terminal active site during protein activity tests.