# Biochemistry of Terpenes and Recent Advances in Plant Protection
## 2 Biosynthesis of Terpenes
### Terpene Synthases (TPSs), Classes, and Terpenes Encoded  
The structural diversity of terpenes is due to the broad terpene synthases superfamily (TPSs). Over a hundred TPS genes and prenyltransferases are known [46; 47]. Some of the enzymes involved in the post-cyclization alteration of terpen include methyltransferases, P450s, and NAD+-dependent dehydrogenases [48].  
TPS of gymnosperm origin is different from angiosperms. The entire TPS length, according to the Pfam database, includes N-terminal and C-terminal domains. While the N-terminal has a single motif, the C-terminal has two aspartate-rich motifs [49]. DDxxD and NSE/DTE motifs on the C-terminal domain are responsible for divalent ion(s) coordination and stabilization of water molecules on the active site [49]. The length of TPS varies depending on the terpene class. Generally, monoterpenes synthases are 600-650 amino acid residues long. Sesquiterpenes are 550-580 amino acid residues long, and about 380-440 amino acid lengths are found in diterpenes. TPSs were initially grouped into six subfamilies based on their amino acids full-length or whether they are angiosperms- or gymnosperms-based. TPS sub-families arising from gymnosperm and angiosperm clads are both species-specific and non-species-specific. For example, in Arabidopsis, 10 genes encoding the synthesis of sesquiterpenes and the putative diterpene synthases are close relatives compared to those in other angiosperms. Among the two TPSs families (Class I and II), the subfamilies including TPSa, TPSb, TPSc, TPSd, TPSe, and TPSf are known. However, TPSf and TPSe were recently merged as TPSe/f because they originate from the same ancestral clad, and also, TPSf is a derivative of TPSe. Moreover, TPSg and TPSh from Angiosperms and _Selaginella moellendorffii_ have recently been discovered [50; 51; 52].  
The advent of the next-generation sequencing tool paved the way for the transcriptional studies of many plant genomes, including rice, maize, Arabidopsis, and tomato, resulting in the revelation of enormous functionally elucidated terpene synthase families. The Arabidopsis genome encodes 32 full-length TPSs. Twenty-two of which belong to the TPSa subfamily, and 6 are members of the TPSb subfamily. One each belongs to TPSc and TPSg subfamilies, respectively. The remaining two are from the TPSg subfamily. A total of 34 and 24 TPS subfamilies have been identified in _Oryza sativa_ and _Sorghum bicolor_, respectively. Furthermore, _Solanum Lycopersicum_ (tomato), _Populus trichocarpa_ (black cottonwood), and _S. moellendorffii_ genomes contain 44, 32, and 14 TPSs, respectively [52; 53; 54].  
Figure 3: Biosynthesis of isoprenoid precursors (IPP and DMAPP) [40; 41; 42; 43; 44; 45] (structures were drawn and analyzed with ChemDraw software, version 20.0.0.41).  
TPSa members have no conserved arginine/tryptophan motif RRX8W. They encode only sesquiterpene synthases in dicot and monocot plants. Contrary to TPSa, TPSb and TPSg are angiosperm-specific subfamilies. TPSb has a conserved R(R)X8W motif and is responsible for launching isomeric cyclization reactions. The TPSg subfamily lacks the R(R)X8W motif in its coding protein and plays a role in the biosynthesis of acyclic monoterpenes that comprise volatile organic compounds (VOCs) [53; 55; 56]. Some members of the TPSg subfamily produce acyclic sesqui- and diterpene products aside from monoterpenes [52]. The subfamily TPSc, identified with the DXDD motif instead of DDX, is primarily present in terrestrial plants. TPSd is gymnosperm-specific and encodes mono-, sesqui-, and di-terpene synthases. Furthermore, TPSe/f subfamily members are mainly found in vascular plants, encoding kaurene synthases, and copalyl diphosphate synthases. They function in the biosynthesis of gibberellin. The TPSh subfamily is the sole precursor to _S. moellendorffii_, capable of encoding DXDD and DDX motifs [52].