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Multi-Substrate Terpene Synthases: Their Occurrence and Physiological Significance.

Pazouki L, Niinemets Ü - Front Plant Sci (2016)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Physiology, Institute of Agricultural and Environmental Sciences, Estonian University of Life Sciences Tartu, Estonia.

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.

No MeSH data available.


Related in: MedlinePlus

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, 2006, 2008; 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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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, 2006, 2008; 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.

Mentions: Phylogenetic analyses indicate that the confirmed multi-substrate enzymes are diffusely spread across different terpene families, indicating a strong convergent nature of this trait (Figure 2), and overall demonstrating a high flexibility for evolution of enzymes with new subcellular compartmentalization and substrate specificity. It has been suggested that the demand for gibberellin production has given rise to the large superfamily of plant terpenoids (Peters, 2010), and thus, all plant terpenoid synthases are believed to originate from an ancient diterpene synthase (Hillwig et al., 2011; Köksal et al., 2011b; Rajabi et al., 2013). These phylogenetically old diterpene synthases are tri-domain, alpha-beta-gamma, proteins that contain a transit peptide (Figure 3; Hillwig et al., 2011; Köksal et al., 2011b; Rajabi et al., 2013). Further evolutionary modifications leading to diversification of product profiles have not only been associated with changes in active center structure, but isoprene and monoterpene synthases have lost the gamma-domain, while sesquiterpene synthases the target peptide and in most cases the gamma-domain (Hillwig et al., 2011; Köksal et al., 2011b; Rajabi et al., 2013). Existence of proteins with mixed substrate specificity allows for developing novel hypotheses about timing of major evolutionary modifications, the loss of γ-domain and transit peptide, in TPSs with different substrate specificity (Figure 3). Analysis of the structure of bi-domain, α-β, kaurene like diterpene synthase from Triticum aestivum (TaKSL5) that can use both ent-copalyl diphosphate to produce ent-kaurene and (E,E)-FDP to produce (E)-nerolidol (Hillwig et al., 2011), suggests that evolution of sesquiterpene synthesis can occur first by loss of γ-domain followed by changes in subcellular localization by loss of transit peptide and further diversification and loss of capacity for use of C20 substrate. Such a possibility is underscored by occurrence of multi-substrate (E)-nerolidol/(E,E)-geranyllinalool synthases in V. vinifera (VvPNLNGl1-VvPNLNGl4 and VvCSENerGl) that have both C15 and C20 substrate use capacity, but lack both the γ-domain and the transit peptide (Martin et al., 2010).


Multi-Substrate Terpene Synthases: Their Occurrence and Physiological Significance.

Pazouki L, Niinemets Ü - Front Plant Sci (2016)

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, 2006, 2008; 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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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, 2006, 2008; 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.
Mentions: Phylogenetic analyses indicate that the confirmed multi-substrate enzymes are diffusely spread across different terpene families, indicating a strong convergent nature of this trait (Figure 2), and overall demonstrating a high flexibility for evolution of enzymes with new subcellular compartmentalization and substrate specificity. It has been suggested that the demand for gibberellin production has given rise to the large superfamily of plant terpenoids (Peters, 2010), and thus, all plant terpenoid synthases are believed to originate from an ancient diterpene synthase (Hillwig et al., 2011; Köksal et al., 2011b; Rajabi et al., 2013). These phylogenetically old diterpene synthases are tri-domain, alpha-beta-gamma, proteins that contain a transit peptide (Figure 3; Hillwig et al., 2011; Köksal et al., 2011b; Rajabi et al., 2013). Further evolutionary modifications leading to diversification of product profiles have not only been associated with changes in active center structure, but isoprene and monoterpene synthases have lost the gamma-domain, while sesquiterpene synthases the target peptide and in most cases the gamma-domain (Hillwig et al., 2011; Köksal et al., 2011b; Rajabi et al., 2013). Existence of proteins with mixed substrate specificity allows for developing novel hypotheses about timing of major evolutionary modifications, the loss of γ-domain and transit peptide, in TPSs with different substrate specificity (Figure 3). Analysis of the structure of bi-domain, α-β, kaurene like diterpene synthase from Triticum aestivum (TaKSL5) that can use both ent-copalyl diphosphate to produce ent-kaurene and (E,E)-FDP to produce (E)-nerolidol (Hillwig et al., 2011), suggests that evolution of sesquiterpene synthesis can occur first by loss of γ-domain followed by changes in subcellular localization by loss of transit peptide and further diversification and loss of capacity for use of C20 substrate. Such a possibility is underscored by occurrence of multi-substrate (E)-nerolidol/(E,E)-geranyllinalool synthases in V. vinifera (VvPNLNGl1-VvPNLNGl4 and VvCSENerGl) that have both C15 and C20 substrate use capacity, but lack both the γ-domain and the transit peptide (Martin et al., 2010).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Physiology, Institute of Agricultural and Environmental Sciences, Estonian University of Life Sciences Tartu, Estonia.

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.

No MeSH data available.


Related in: MedlinePlus