Literature summary for 4.2.3.B67 extracted from

Zerbe, P.; Rodriguez, S.M.; Mafu, S.; Chiang, A.; Sandhu, H.K.; ONeil-Johnson, M.; Starks, C.M.; Bohlmann, J.
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 .

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Cloned(Commentary)

Cloned (Comment)	Organism
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

Cloned (Comment)

Organism

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)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
geranylgeranyl diphosphate	Grindelia hirsutula	via intermediate labda(13E)-labda-13-en-15-yl diphosphate	(13E)-labda-7,13-dien-15-yl diphosphate	-	?

Organism

Organism	UniProt	Comment	Textmining
Grindelia hirsutula	A0A0H4SRM2	i.e. Grindelia hirsutula	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His-tagged N-terminally truncated GrTPS2 from Escherichia coli strain BL21DE-C41	Grindelia hirsutula

Reaction

Reaction	Comment	Organism	Reaction ID
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	a

Source Tissue

Source Tissue	Comment	Organism	Textmining
flower	-	Grindelia hirsutula	-
leaf	-	Grindelia hirsutula	-
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	-
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	-

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
geranylgeranyl diphosphate	via intermediate labda(13E)-labda-13-en-15-yl diphosphate	Grindelia hirsutula	(13E)-labda-7,13-dien-15-yl diphosphate	-	?
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	-	?
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	?	-	?

Synonyms

Synonyms	Comment	Organism
GrTPS2	-	Grindelia hirsutula
labda-7,13E-dienyl diphosphate synthase	-	Grindelia hirsutula

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
30	-	assay at	Grindelia hirsutula

General Information

General Information	Comment	Organism
evolution	the enzyme is a class II diterpene synthase (diTPS, GrTPS2)	Grindelia hirsutula
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
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