Fig 1.
The reaction mechanism of sesquiterpene production starts with farnesyl diphosphate ((E,E)-FPP).
Loss of the diphosphate moiety (OPP) leads to farnesyl cation formation. The farnesyl cation can subsequently be converted to the nerolidyl cation. Acyclic sesquiterpenes (acyclic-F and acyclic-N) are formed from these two cations by proton loss or reaction with water molecules. Possible cyclizations for both cations are indicated in the figure. The subsequently formed cyclic cations undergo modifications and rearrangements to form cyclic sesquiterpenes. Some of these sesquiterpenes (g-A and bcg) themselves act as neutral intermediates which can be re-protonated and undergo further reactions to form more products. Products are also formed from specific charged intermediates such as a 1,2- or 1,3-hydride shift of the 10,1-cyclized farnesyl cation (1,2H, 1,3H) and the cadalane skeleton (cadalanes), which can be formed via either of the two precursor cations, or via acid-induced rearrangement of germacrene D. The 7,1-cyclization of the nerolidyl cation, shown in gray, is not found in plant-derived sesquiterpenes. g-A = germacrene A, g-D = germacrene D, bcg = bicyclogermacrene.
Table 1.
The six structures used for multi-template modelling.
Table 2.
Validation results of three classifiers.
Fig 2.
Characterized STSs visualized using the feature values of the cation-specific residues followed by dimensionality reduction using UMAP [23], which positions STSs with similar feature values closer to each other.
Squares represent farnesyl cation-specific STSs and diamonds represent nerolidyl cation-specific STSs. Each STS is also colored by its cyclization specificity. Enzymes catalyzing products from different precursor cations are marked as triangles.
Fig 3.
A. Thirty cation-specific residues found by the structure-based Clf-str predictor on the tobacco epi-aristolochene synthase (TEAS) structure, colored by region. Terpene synthase motifs are labelled in purple, Mg+2 ions in pink, and a substrate analog in orange and dark pink. The N-terminal domain is shaded with a lower opacity. B. Sequence conservation of Clf-str cation-specific residues across farnesyl and nerolidyl cation-specific STSs, labelled by region and residue position in the TEAS structure. The height of a letter represents its frequency in that position. An insertion/deletion is represented by a black ‘X’. Residue positions which are deleted in the TEAS structure are represented by ‘-’s and correspond to residue 627 and residue 687 respectively in the Abies grandis α-bisabolene synthase structure (PDB ID: 3SDU). Note that if, for a given position, the residues in both logos are similar, this indicates that in spite of similarity in sequence at this position, the farnesyl and nerolidyl cation-specific enzymes are structurally different.
Fig 4.
A. Tobacco epi-aristolochene synthase (TEAS) secondary structure with distal cation-specific co-evolutionary contacts (green arcs), motif residues (purple), and cation-specific residues (colored by region). Helix naming as in Starks et al. [26] B. Sequence-pair conservation of four cation-specific contacts discussed in the text, across farnesyl and nerolidyl cation-specific STSs, and all putative terpene synthases. The height of a pair of letters represents the frequency of the pair appearing in those two positions, with ‘X’ representing gaps. C. Diagrams indicating the proximity of residues labeled B in Fig 3B, as well as the residues that they co-evolve with, to substrate analogs trifluorofarnesyl diphosphate (FFF) co-crystallized with TEAS (left) and farnesyl thiodiphosphate (FPS) co-crystallized with Abies grandis α-bisabolene synthase (AgBIS) (right). Carbon atoms are numbered (white boxes) as in the FFF subtrate analog moeity in PDB ID 5EAU. The closest distance (in Å) between each residue’s β-carbon and a substrate atom is labeled in gray. Two co-evolving contacts (labeled 1 and 2 in A) are colored in green.