## 3 Discussion
mechanism behind these observations, we compared the protein structures between bacterial _trans_-PTs and the one from _Arabidopsis_. Three bacterial structures (PDB IDs: 5H9D from _Staphylococcus aureus_, 1 WKZ from _Thermotoga maritima_, and 1 WY0 from _Pyrococcus horikoshii_) were superposed with the structure of AtGGPPS11 (PDB ID: 5E8L). All these three proteins form homodimers, and the root-mean-square deviation (RMSD) is as low as 1.499 A over 260 residues, displaying very similar overall structures (Figure S7). We further compared the product chain elongation tunnels and found that the three key \(\alpha\) helices (\(\alpha\) helix D, E, and F) are also similar except for some nonessential residues (Figure S8). Thus, the inconsistency with the \"three floors\" model is unlikely related to the monomer structural factors. Some previous work indicated that, in addition to the residues in the \"three floors\", the two aspartates in FARM and SARM were identified as factors influencing the chain length specificity of products in the Gram-positive bacterium _Mycobacterium tuberculosis_[33]. Moreover, the interface of the _trans_-PT homodimer was also proposed to be a new product chain elongation tunnel, in that the blocking residues were rearranged and affected the profile of the final product [14]. These reasons may explain the incorrectly predicted cases for bacterial _trans_-PTs by PTS-Pre; however, further investigations are still needed to verify whether these factors are only specific to bacteria or also cover other species. It also should be noted that the PTS-Pre method was designed on the basis of homodimeric structures and validated only by in vitro assays. However, in some specific species, the _trans_-PTs are in an additional heterodimeric state which contains one large subunit (LSU) and one small subunit (SSU). The kinetic parameters and product profile of the LSU would be changed by the regulatory function of SSU [12]; thus, the predicted results could be different from the genuine products formed in vivo, which require further in vivo investigations case by case. Compared with the most extensively studied mono-, sesqui-, di-, and triterpenes (C10, C15, C20, and C30, respectively), sesterterpenes (C25) constitute the rarest group of terpenids discovered to date, with only about 1300 members being reported [34]. Despite the wide distribution of sesterrepenoids in bacteria, fungi, insects, and marine organisms [19], the corresponding genes and enzymes of plant-originated C25 compounds were only recently reported in limited families including Lamiaceae and Brassica [10; 25; 26]. Our blind prediction and subsequent enzyme assays of randomly selected testing sequences from _Gossypium_ identified a new plant GFPPS (Table 1 and Figure 4), suggesting that more GFPPS-related genes may exist in the plant kingdom. Indeed, further genome mining of the _trans_-PT gene family identified six additional GFPPSs from Malvaccae, Musaceae, and Ranunculaceae families (Table 2 and Figure 4A), which were evolutionarily separated (Figure 4B). Many sesterterpenoids identified in plants possess either highly oxygenated or complex cyclic skeletons and exhibit diverse biological functions such as anti-inflammatory, antimicrobial, and antifeedant bioactivities [19; 34]. The fact that changing only a few key amino acids could alter the enzymatic characteristics of _trans_-PT enzymes suggests that plant GFPPSs independently evolved via gene duplication and neo-functionalization for the adaption of various selection stresses. By using PTS-Pre, the prediction of promising GFPPSs with high credibility in specific species provides the fundamental basis for the discovery of new sesterterpenes and opens up a great opportunity for uncovering the mystery of the distribution and evolutionary origin of sesterterpene biosynthesis in plants.