Terpene Synthases as Metabolic Gatekeepers in the Evolution of Plant Terpenoid Chemical Diversity

Abstract

Terpenoids comprise tens of thousands of small molecule natural products that are widely distributed across all domains of life. Plants produce by far the largest array of terpenoids with various roles in development and chemical ecology. Driven by selective pressure to adapt to their specific ecological niche, individual species form only a fraction of the myriad plant terpenoids, typically representing unique metabolite blends. Terpene synthase (TPS) enzymes are the gatekeepers in generating terpenoid diversity by catalyzing complex carbocation-driven cyclization, rearrangement, and elimination reactions that enable the transformation of a few acyclic prenyl diphosphate substrates into a vast chemical library of hydrocarbon and, for a few enzymes, oxygenated terpene scaffolds. The seven currently defined clades (a-h) forming the plant TPS family evolved from ancestral triterpene synthase- and prenyl transferase-type enzymes through repeated events of gene duplication and subsequent loss, gain, or fusion of protein domains and further functional diversification. Lineage-specific expansion of these TPS clades led to variable family sizes that may range from a single TPS gene to families of more than 100 members that may further function as part of modular metabolic networks to maximize the number of possible products. Accompanying gene family expansion, the TPS family shows a profound functional plasticity, where minor active site alterations can dramatically impact product outcome, thus enabling the emergence of new functions with minimal investment in evolving new enzymes. This article reviews current knowledge on the functional diversity and molecular evolution of the plant TPS family that underlies the chemical diversity of bioactive terpenoids across the plant kingdom.

Keywords: natural products; plant chemical diversity; plant specialized metabolism; terpene synthases; terpenoid biosynthesis; terpenoids.

Figure 1

Schematic overview of major terpenoid biosynthetic pathways. All terpenoids are derived from two isomeric 5-carbon precursors, isopentenyl diphosphate (IPP), and dimethylallyl diphosphate (DMAPP). In turn, IPP and DMAPP are formed via two pathways, the cytosolic mevalonate (MVA) pathway originating from acetyl-CoA and the pyruvate and glyceraldehyde-3-phosphate (G3P)–derived 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway located in the plastids. However, active transfers of IPP, DMAPP, GPP, and FPP across the plastidial membrane enable some degree of pathway cross-talk. In addition, interconversion of IPP and DMAPP with their respective monophosphate forms IP and DMAP by IP kinase (IPK) and Nudix hydrolase enzymes can impact pathway flux in terpenoid metabolism. Except for isoprene and hemiterpene (C₅) biosynthesis, condensation of IPP and DMAPP units generates prenyl diphosphate intermediates of different chain length. Condensation of IPP and DMAPP yields geranyl diphosphate (GPP) as the precursor to monoterpenoids (C₁₀), fusing GPP with an additional IPP affords the sesquiterpenoid (C₁₅) precursor farnesyl diphosphate (FPP), and fusing FPP with IPP generates geranylgeranyl diphosphate (GGPP) en route to diterpenoids (C₂₀). Furthermore, condensation of two FPP or two GGPP molecules forms the central substrates of triterpenoid (C₃₀) and carotenoids (C₄₀), respectively. Terpene synthases (TPS) are key gatekeepers in the biosynthesis of C₁₀–C₂₀ terpenoids, catalyzing the committed scaffold-forming conversion of the respective prenyl diphosphate substrates into a range of hydrocarbon or oxygenated structures. These TPS products can then undergo various oxygenations through the activity of cytochrome P450 monooxygenases (P450), followed by further possible functional decorations, ultimately giving rise to more than 80,000 distinct natural products. AACT, acetoacetyl-CoA thiolase; CMK, 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase; DXR, 1-deoxy-D-xylulose 5-phosphate reductase; DXS, 1-deoxy-D-xylulose 5-phosphate synthase; HDR, (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate reductase; HDS, (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate synthase; HMGR, 3-hydroxy-3-methylglutaryl-CoA reductase; HMGS, 3-hydroxy-3-methylglutaryl-CoA synthase; IDI, isopentenyl diphosphate isomerase; MCT, MEP cytidyltransferase; MDD, mevalonate-5-diphosphate decarboxylase; MDS, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase; MK, mevalonate kinase; P450, cytochrome P450-dependent monooxygenase; PHY, phytoene synthase; PMK, phosphomevalonate kinase; PT, prenyl transferase; SCS, squalene synthase; SQE, squalene epoxidase; TPS, terpene synthase; TTS, triterpene synthase.

Figure 2

Schematic overview of representative carbocation-driven reactions catalyzed by prenyl transferases (PTs) and diterpene synthases (TPSs). (A) PT catalyzed head-to-tail condensation of isopentenyl diphosphate (IPP) with its positional isomer dimethylallyl diphosphate (DMAPP) via ionization of the allylic diphosphate ester bond (OPP) and subsequent coupling between the resulting carbocation and the C3–C4 double bond of IPP. Deprotonation of the carbocation intermediate yields geranyl diphosphate (GPP). (B–C) Conversion of geranylgeranyl GGPP by class II diTPSs using protonation-initiated cyclization of GGPP to facilitate scaffold rearrangement into bicyclic prenyl diphosphates of ent-copalyl diphosphate (ent-CPP) (B) and related scaffolds of distinct stereochemistry and hydroxylation (C). (D–E) Class I diTPS-catalyzed conversion of bicyclic prenyl diphosphate intermediates via ionization of the diphosphate moiety and subsequent cyclization and rearrangement through various 1,2-hydride and methyl migrations to form, for example, ent-kaurene (D) and a range of other labdane diterpene scaffolds (E).

Figure 3

Schematic overview of the proposed structural and evolutionary relationships among terpene synthases (TPSs) as based on known protein structures. Proposed progenitors of TPSs include ancestral bacterial class II diTPSs with a signature α-barrel βγ-domain harboring a catalytic DxDD motif, here exemplified by the ent-copalyl diphosphate synthase from Streptomyces platensis (PDB 5BP8; Rudolf et al., 2016). These, in turn, are related to ancestral triterpene synthases (TTS) and bacterial class I diterpene synthases (diTPSs) that adopt the signature α-domain structure with a conserved DDx₂D motif, here exemplified by ent-kaurene synthase from Bradyrhizobium japonicum (PDB 4W4R; Liu et al., 2014) closely related to ancestral prenyltransferases (PT). Fusion of the ancestral monofunctional genes will have given rise to diTPSs with three helical domains (αβγ) represented here by Abies grandis abietadiene synthase (PDB 3S9V; Zhou et al., 2012a). Duplication and subsequent loss of activity in the βγ- and α-domains, respectively, lead to the emergence of monofunctional plant class II, here represented by Arabidopsis thaliana ent-copalyl diphosphate synthase, (PDB 4LIX; Köksal et al., 2014) and class I diTPSs, here represented by Taxus brevifolia taxadiene synthase (PDB 3P5P; Köksal et al., 2011). Through further loss of the γ-domain and various neo-functionalization and specialization events, the large classes of βα-domain class mono- and sesqui-TPSs will have arisen. Domain colors illustrate the γ-domain (orange), the β-domain (blue), the α-domain (red), as well as the conserved DxDD (green) and DDx₂D (cyan) motifs.

Figure 4

Distribution of terpene-biosynthetic enzyme families across major orders across the plant kingdom. PT, prenyltransferases; MTPSL microbial-type terpene synthases (TPSs); TPS-h, Selaginella-specific bifunctional class II/I diterpene synthases (diTPSs); TPS-d, gymnosperm-specific class I mono-, sesqui-, di-TPSs, and bifunctional class II/I diTPSs; TPS-c, monofunctional class II diTPSs; TPS-e/f, monofunctional class I diTPSs; TPS-g, monofunctional class I mono-, sesqui-, and di-TPSs; TPS-a, monofunctional class I sesqui- and di-TPSs; TPS-b, monofunctional class I mono-TPSs.

Figure 5

Schematic overview of prominent linear, polycyclic and macrocyclic diterpene scaffolds that are formed through the activity of monofunctional class I diTPSs that are capable of directly converting geranylgeranyl diphosphate (GGPP) as a substrate.

Figure 6

Examples of active site residues with impact on class II or class I TPS product specificity. (Center) Structure of Abies grandis abietadiene synthase (PDB 3S9V; Zhou et al., 2012a) that adopts the prototypical α-helical TPS fold with variations in three domains γ (orange), β (blue), and α (red). Relative locations of the highlighted active site residues are indicated A–F. (A) A widely conserved His-Asn dyad is critical for stereo-specificity of Arabidopsis ent-CPP synthase (PDB 4LIX; Köksal et al., 2014) and other ent-CPP synthases (Mann et al., 2010; Potter et al., 2014; Mafu et al., 2015). Substitution of this dyad can result in the formation of the alternate clerodienyl diphosphate product (Potter et al., 2014; Pelot et al., 2016). (B) His501 in the rice syn-CPP synthase OsCPS4 is critical for the stereo-specific formation of syn-CPP and is conserved in known syn-CPP synthases but not class II diTPS producing alternate CPP stereoisomers (Potter et al., 2016a; Pelot et al., 2018). (C) A conserved Met residue in ent-kaurene synthases from Picea glauca and other species was shown to control ent-kaurene formation (Xu et al., 2007b; Zerbe et al., 2012a). (D) Mutational studies of corresponding Ser-Ile-Ala-Leu and Ser-Ile-Ser-Leu motifs located at the hinge region of helix G1/2 of P. abies levopimaradiene/abietadiene and isopimaradiene synthase showed their critical role in producing abietane or pimarane scaffolds (Keeling et al., 2008). (E) Three residues were identified in the active site of Artemisia annua β-farnesene synthase, reciprocal exchange of which to corresponding residues in A. annua amorphadiene synthase that control activation (Tyr402), reversion (Tyr430), and restoration (Val476) of cyclization capacity (Salmon et al., 2015). (F) Two residues, Trp324 and His579, were shown in limonene synthase of Mentha spicata to control the reactions cascade toward the natural product 4(S)-limonene (Srividya et al., 2015). The signature catalytic motifs of the class II (DxDD, green) and class I (DDx₂D, cyan; NSE/DTE, magenta) active sites are highlighted. Protein abbreviations: AtECPS, Arabidopsis thaliana ent-copalyl diphosphate (CPP) synthase; OsCPS4, Oryza sativa syn-CPP synthase; PvCPS3, Panicum virgatum 8,13-CPP synthase; SmCPS, Salvia miltiorrhiza (+)-CPP synthase; MvCPS1, Marrubium vulgare peregrinol diphosphate synthase; SdCPS2, Salvia divinorum clerodienyl diphosphate (KPP) synthase; PpCPS/KS, Physcomitrella patens CPP/ent-kaurene synthase; AgAS, Abies grandis abietadiene synthase; PgEKS, Picea glauca ent-kaurene synthase; PtTPS19, Populus trichocarpa ent-kaurene synthase; PaLAS, Picea abies levopimaradiene/abietadiene synthase; PaISO, P. abies isopimaradiene synthase; AaBFS, Artemisia annua β-farnesene synthase; AaADS, A. annua amorphadiene synthase.