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Review
. 2016 Jul 12:7:1019.
doi: 10.3389/fpls.2016.01019. eCollection 2016.

Multi-Substrate Terpene Synthases: Their Occurrence and Physiological Significance

Leila Pazouki  1 , Ülo Niinemets  2
Affiliations
Review

Multi-Substrate Terpene Synthases: Their Occurrence and Physiological Significance

Leila Pazouki et al. Front Plant Sci. .

Abstract

Terpene synthases are responsible for synthesis of a large number of terpenes in plants using substrates provided by two distinct metabolic pathways, the mevalonate-dependent pathway that is located in cytosol and has been suggested to be responsible for synthesis of sesquiterpenes (C15), and 2-C-methyl-D-erythritol-4-phosphate pathway located in plastids and suggested to be responsible for the synthesis of hemi- (C5), mono- (C10), and diterpenes (C20). Recent advances in characterization of genes and enzymes responsible for substrate and end product biosynthesis as well as efforts in metabolic engineering have demonstrated existence of a number of multi-substrate terpene synthases. This review summarizes the progress in the characterization of such multi-substrate terpene synthases and suggests that the presence of multi-substrate use might have been significantly underestimated. Multi-substrate use could lead to important changes in terpene product profiles upon substrate profile changes under perturbation of metabolism in stressed plants as well as under certain developmental stages. We therefore argue that multi-substrate use can be significant under physiological conditions and can result in complicate modifications in terpene profiles.

Keywords: monoterpene synthesis; multi-substrate terpene synthases; prenyltransferases; sesquiterpene synthesis; subcellular compartmentalization; terpene engineering; terpene metabolites.

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Figures

Figure 1
Figure 1
Terpene biosynthetic pathways and their subcellular compartmentalization in plants. Thick arrows denote the classical understanding of terpenoid synthesis compartmentalization among cytosol and plastid (Bohlmann et al., ; Chen et al., ; Tholl and Lee, 2011), reflecting the circumstance that monoterpene and diterpene synthases harboring a chloroplast-targeting peptide are functionally active in plastids and sesquiterpene synthases lacking the target peptide are active in cytosol. However, recent findings of the capacity for multi-substrate use of several mono, sesqui-, and, diterpene synthases suggest that when substrate becomes available, several cytosolic “sesquiterpene” synthases could also operate as monoterpene synthases, and analogously, multi-substrate “monoterpene” and “diterpene” synthases could operate as sesquiterpene synthases in plastids (denoted by thin arrows). In addition, terpenoid synthesis can also potentially occur in mitochondria (Nagegowda, ; Tholl and Lee, ; Dong et al., 2016). For instance, targeting linalool/(E)-nerolidol synthase (FaNES1) from Fragaria ananassa (Table 1 for protein specifics) to the mitochondria led to the production of (E)-nerolidol and homoterpene 4,8-dimethyl nona-1,3,7-triene (DMNT) in transgenic Arabidopsis thaliana plants (Kappers et al., 2005). DMADP, dimethylallyl diphosphate (C5); MEP pathway, 2-C-methyl-D-erythritol 4-phosphate/1-deoxy-D-xylulose 5-phosphate pathway; IDP, isopentenyl diphosphate (C5); FDP, farnesyl diphosphate (C15); GDP, geranyl diphosphate (C10); GGDP, geranylgeranyl diphosphate (C20); NDP, neryl diphosphate (C10).
Figure 2
Figure 2
Phylogenetic tree of terpene synthases (TPS) with confirmed capacity for multi-substrate use (Table 1 for details of product and substrate specificities). The red branch denotes TPS with C5/C10 activity, the black branches with C10/C15 activity, the blue branches with C10/C15/C20 activity, and the green branches with C15/C20 activity. These 40 multi-substrate terpene synthase are from different TPS families including TPS-a, TPS-b, TPS-g, TPS-d, TPS-e, and TPS-f. The tree was constructed by MEGA5 software by UPGMA method (Tamura et al., 2011). The asterisks denote the presence of the conserved arginine-rich RRx8W motif at the N-terminal of the protein that is common in many monoterpene synthases (Chen et al., 2011). The underlined enzymes demonstrate the presence of transit peptide.
Figure 3
Figure 3
Hypothesis of the evolution of multi-substrate enzymes according to two potential routes. Ancient terpenoid synthases underlying the diversity of terpene synthases in plants are tri-domain, α-, β,- and γ-domain proteins with two active sites, one in the α-domain (class I activity) and the other in the β-domain (class II activity) (Christianson, , ; Köksal et al., 2011a,b). The γ-domain without an active site is inserted between the first and second helices of the β-domain (Köksal et al., 2011a,b). These ancient proteins also carry a transit peptide (TP) at the N terminus targeting these proteins to chloroplasts. Through evolution, these complex enzymes have undergone considerable simplification, resulting in changes in catalysis, enzyme subcellular localization, and product and substrate specificities. Class II activity seems to have been lost first (not shown in the figure) and is missing in all confirmed multi-substrate enzymes. A tri-domain terpene synthase functionally active in the cytosol is formed through the loss of the transit peptide from a diterpene synthase. This can be eventually followed by γ-domain loss, resulting in formation of a bi-domain cytosol-active synthase (left). While the transit peptide is maintained, γ-domain loss can first lead to formation of a bi-domain diterpene synthase (e.g., ent-kaurene synthase like synthase in Triticum aestivum, Figure 2, Table 1) and ultimately to a monoterpene synthase. Loss of the transit peptide can further lead to a cytosol-active enzyme (e.g., β-ocimene synthase, AtTPS02, and (E,E)-α-farnesene synthase, AtTPS03, from Arabidopsis thaliana that differ in the subcellular localization due to presence or lack of the transit peptide; Figure 2, Table 1). Changes in substrate specificity are typically also associated with changes in active center size (Köksal et al., 2011b), and thus, the capacity for the use of multiple substrates will critically depend on whether the active center cavity can accommodate substrates of varying size.
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