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Literature summary extracted from
- Exploring diterpene metabolism in non-model species transcriptome-enabled discovery and functional characterization of labda-7,13E-dienyl diphosphate synthase from Grindelia robusta (2015), Plant J., 83, 783-793 .
Cloned(Commentary)
| EC Number | Cloned (Comment) | Organism |
|---|---|---|
| 4.2.3.19 | expressed in Escherichia coli BL21DE-C41 cells | Grindelia hirsutula |
| 4.2.3.B67 | GrTPS2, phylogenetic analysis, quantitative real-time PCR expression analysis, recombinant expression of a truncated sequence of GrTPS2 (lacking the first 47 N-terminal amino acids representing the plastidial transit peptide) as His-tagged protein in Escherichia coli strain BL21DE-C41, recombinant expression of enzyme GrTPS2 in Nicotiana benthamiana via Agrobacterium tumefaciens transfection method, coexpression with the class I diTPSs GrTPS4 (ent-kaurene synthase) or GrTPS6 (manoyl oxide synthase) in Nicotiana benthamiana yielding both no detectable products | Grindelia hirsutula |
Natural Substrates/ Products (Substrates)
| EC Number | Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|---|
| 4.2.3.19 | ent-copalyl diphosphate | Grindelia hirsutula | - |
ent-kaurene + diphosphate | - |
? |  |
| 4.2.3.B67 | geranylgeranyl diphosphate | Grindelia hirsutula | via intermediate labda(13E)-labda-13-en-15-yl diphosphate | (13E)-labda-7,13-dien-15-yl diphosphate | - |
? | |
Organism
| EC Number | Organism | UniProt | Comment | Textmining |
|---|---|---|---|---|
| 4.2.3.19 | Grindelia hirsutula | A0A0H4SPM5 | gumweed | - |
| 4.2.3.B67 | Grindelia hirsutula | A0A0H4SRM2 | i.e. Grindelia hirsutula | - |
| 4.2.3.190 | Grindelia hirsutula | - |
- |
- |
Purification (Commentary)
| EC Number | Purification (Comment) | Organism |
|---|---|---|
| 4.2.3.19 | Ni-NTA column chromatography | Grindelia hirsutula |
| 4.2.3.B67 | recombinant His-tagged N-terminally truncated GrTPS2 from Escherichia coli strain BL21DE-C41 | Grindelia hirsutula |
Reaction
| EC Number | Reaction | Comment | Organism | Reaction ID |
|---|---|---|---|---|
| 4.2.3.B67 | geranylgeranyl diphosphate = (13E)-labda-7,13-dien-15-yl diphosphate | via intermediate (13E)-labda-13-en-15-yl diphosphate, the catalytic activity of the Grindelia (13E)-labda-7,13-dien-15-yl diphosphate synthase plausibly involves the protonationinitiated bicyclization of GGPP into the intermediate labda-13-en-15-yl+ cation, followed by deprotonation of the carbocation at the endocyclic methylene at C-7, as opposed to the more common deprotonation at C-8, to yield copalyl diphosphate | Grindelia hirsutula |
Source Tissue
| EC Number | Source Tissue | Comment | Organism | Textmining |
|---|---|---|---|---|
| 4.2.3.B67 | flower | - |
Grindelia hirsutula | - |
| 4.2.3.B67 | leaf | - |
Grindelia hirsutula | - |
| 4.2.3.B67 | additional information | tissue-specific transcript abundance of GrTPS2 in leaves and flowers is accompanied by the presence of an endocyclic 7,13 double bond in (13E)-labda-7,13-dien-15-yl diphosphate. Spatial distribution of terpenoids in Grindelia robusta in extracts of roots, stems, leaves, flowers and resin from flower involucres via gas chromatography/mass spectrometry (GC/MS) analysis, tissue extract terpenoid profile, overview | Grindelia hirsutula | - |
| 4.2.3.B67 | root | despite a 60fold lower transcript abundance, GrTPS2 is also expressed in roots accompanied by the presence of trace amounts of grindelic acid, indicating that diterpenoid biosynthesis may also be active in roots of Grindelia robusta | Grindelia hirsutula | - |
| 4.2.3.190 | additional information | transcription in all organs, consisitient with presence of manoyl oxide in leaves and flowers and to lesser extent also in stem and root tissue | Grindelia hirsutula | - |
Substrates and Products (Substrate)
| EC Number | Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|---|
| 4.2.3.19 | ent-copalyl diphosphate | - |
Grindelia hirsutula | ent-kaurene + diphosphate | - |
? | |
| 4.2.3.B67 | geranylgeranyl diphosphate | via intermediate labda(13E)-labda-13-en-15-yl diphosphate | Grindelia hirsutula | (13E)-labda-7,13-dien-15-yl diphosphate | - |
? | |
| 4.2.3.B67 | geranylgeranyl diphosphate | (E,E,E)-GGPP, via intermediate (13E)-labda-13-en-15-yl diphosphate | Grindelia hirsutula | (13E)-labda-7,13-dien-15-yl diphosphate | - |
? | |
| 4.2.3.B67 | additional information | GrTPS2 is a monofunctional (13E)-labda-7,13-dien-15-yl diphosphate synthase, the reaction product (13E)-labda-7,13-dien-15-yl diphosphate is verified by nuclear magnetic resonance (NMR) analysis | Grindelia hirsutula | ? | - |
? | |
| 4.2.3.190 | (13E)-8alpha-hydroxylabd-13-en-15-yl diphosphate | - |
Grindelia hirsutula | manoyl oxide + diphosphate | - |
? | |
Synonyms
| EC Number | Synonyms | Comment | Organism |
|---|---|---|---|
| 4.2.3.19 | TPS4 | - |
Grindelia hirsutula |
| 4.2.3.B67 | GrTPS2 | - |
Grindelia hirsutula |
| 4.2.3.B67 | labda-7,13E-dienyl diphosphate synthase | - |
Grindelia hirsutula |
| 4.2.3.190 | TPS4 | - |
Grindelia hirsutula |
Temperature Optimum [°C]
| EC Number | Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|---|
| 4.2.3.B67 | 30 | - |
assay at | Grindelia hirsutula |
General Information
| EC Number | General Information | Comment | Organism |
|---|---|---|---|
| 4.2.3.B67 | evolution | the enzyme is a class II diterpene synthase (diTPS, GrTPS2) | Grindelia hirsutula |
| 4.2.3.B67 | metabolism | (13E)-labda-7,13-dien-15-yl diphosphate represents a key intermediate in the formation of grindelanes with GrTPS2 catalyzing the committed reaction step. Diterpenoid biosynthesis in Grindelia robusta, overview | Grindelia hirsutula |
| 4.2.3.B67 | physiological function | enzyme GrTPS2 catalyzes the first committed reaction in the biosynthesis of grindelic acid and related grindelane metabolites. With the formation of (13E)-labda-7,13-dien-15-yl diphosphate, GrTPS2 adds an additional function to the portfolio of monofunctional class II diTPSs, which catalytically most closely resembles the bifunctional labda-7,13E-dien-15-ol synthase of the lycopod Selaginella moellendorffii. Grindelia robusta produces large quantities of these diterpenoids that are secreted through specialized ducts and glandular trichomes as a resinous exudate that covers the surface of the aerial parts of the plant. Among the diterpenoids, grindelic acid is the predominant metabolite and most abundant in resin with 9, 37, and 81fold higher levels as compared with flowers, leaves, and stems, respectively | Grindelia hirsutula |
| 4.2.3.190 | physiological function | no functional interaction with TPS2 | Grindelia hirsutula |
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