Information on EC 4.2.3.17 - taxadiene synthase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
4.2.3.17
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RECOMMENDED NAME
GeneOntology No.
taxadiene synthase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
geranylgeranyl diphosphate = taxa-4,11-diene + diphosphate
show the reaction diagram
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
cyclization
oxidation
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Biosynthesis of secondary metabolites
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Diterpenoid biosynthesis
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Metabolic pathways
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taxadiene biosynthesis (engineered)
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taxol biosynthesis
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SYSTEMATIC NAME
IUBMB Comments
geranylgeranyl-diphosphate diphosphate-lyase (cyclizing; taxa-4,11-diene-forming)
This is the committed step in the biosynthesis of the diterpenoid antineoplastic drug Taxol (paclitaxel). The cyclization involves a 1,5-hydride shift.
CAS REGISTRY NUMBER
COMMENTARY hide
169277-52-5
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
CBS 27992
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Manually annotated by BRENDA team
CBS 27992
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(10,11R)-dihydrogeranylgeranyl diphosphate
(1S,12R)-11,12-dihydroisocembrene + diphosphate
show the reaction diagram
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?
(10,11S)-dihydrogeranylgeranyl diphosphate
(1S,12S)-11,12-dihydroisocembrene + diphosphate
show the reaction diagram
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?
(2E,6E,10Z)-geranylgeranyl diphosphate
(1S,11,12Z)-isocembrene + diphosphate
show the reaction diagram
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?
(E,E,E)-geranylgeranyl diphosphate
taxa-4(5),11(12)-diene + diphosphate
show the reaction diagram
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first committed step of taxol biosynthesis
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?
(E,E,E)-geranylgeranyl diphosphate
taxa-4,11-diene + diphosphate
show the reaction diagram
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mechanism of cyclization. Incubation of (R)-[4-2H1]geranylgeranyl diphosphate gives a 10:10:80 mixture of [5beta-2H1]taxa-3(4),11(12)-diens, [5beta-2H1]taxa-4(20),11(12)-diene and unlabeled taxa-4(5),11(12)-diene
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?
11-desmethylgeranylgeranyl diphosphate
(1S)-12-desmethylisocembrene + diphosphate
show the reaction diagram
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?
7-desmethylgeranylgeranyl diphosphate
(1S,4E,8E,14R,15S)-1,8,14,15-tetramethylbicyclo[9.3.1]pentadeca-4,8,11-triene + diphosphate
show the reaction diagram
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?
7-desmethylgeranylgeranyl diphosphate
(3E,7E,11S,12R)-4,11,12,15-tetramethylbicyclo[9.3.1]pentadeca-1(15),3,7-triene + diphosphate
show the reaction diagram
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?
7-desmethylgeranylgeranyl diphosphate
(6E,10E)-11,15-dimethyl-3-methylidenehexadeca-1,6,10,14-tetraene + diphosphate
show the reaction diagram
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product may be an artefact, but non is formed when enzyme is not present
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?
7-fluorogeranylgeranyl diphosphate
(1R,3E,7Z,11R)-7-fluoro-4,8,12,15,15-pentamethyl-bicyclo[9.3.1]pentadeca-3,7,12-triene
show the reaction diagram
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i.e. endo-7-fluoroverticillene, 25% yield
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?
7-fluorogeranylgeranyl diphosphate
(1S,3E,7Z,11R)-7-fluoro-4,8,15,15-tetramethyl-12-methylene-bicyclo[9.3.1]pentadeca-3,7-diene
show the reaction diagram
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verticille-12-yl carbocation intermediate with an 11R stereocenter, the reaction gives three main products and two further isomers
i.e. exo-7-fluoroverticillene, 37% yield
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?
7-fluorogeranylgeranyl diphosphate
(4Z,11R)-5-fluoro-4,14,15,15-tetramethyl-8-methylene-bicyclo[9.3.1]pentadeca-4,14-diene
show the reaction diagram
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i.e. 4(20)-methylene-7-fluoroverticillene, 26% yield
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?
cyclopropylidene
?
show the reaction diagram
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?
geranylgeranyl diphosphate
taxa-4(20),11(12)-diene + diphosphate
show the reaction diagram
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taxa-4(20),11(12)-diene is formed as a small amount
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?
geranylgeranyl diphosphate
taxa-4(5),11(12)-diene
show the reaction diagram
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product is biochemically synthesized using truncated TS recombinant protein from Escherichia coli, labeling reveals that 10% substrate is converted to the product
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?
geranylgeranyl diphosphate
taxa-4(5),11(12)-diene + diphosphate
show the reaction diagram
geranylgeranyl diphosphate
taxa-4,11-diene + diphosphate
show the reaction diagram
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
(E,E,E)-geranylgeranyl diphosphate
taxa-4(5),11(12)-diene + diphosphate
show the reaction diagram
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first committed step of taxol biosynthesis
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?
geranylgeranyl diphosphate
taxa-4(5),11(12)-diene + diphosphate
show the reaction diagram
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taxadiene biosynthetic pathway
precursor of the chemotherapeutic agent taxol
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ir
geranylgeranyl diphosphate
taxa-4,11-diene + diphosphate
show the reaction diagram
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-hydroxyethyl jasmonate
the transcription level of the TS gene is up-regulated in response to exogenous 2-hydroxyethyl jasmonate, HEJ
methyl jasmonate
the transcription level of the TS gene is up-regulated in response to exogenous methyl jasmonate, MJ
taxol
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exogenous Taxol (paclitaxel) causes an considerable increase in taxadiene synthase activity, the expression level peaks at day 4 with a rapid decrease at day 8, reaching a second, lower, peak at day 12. By day 16, ts mRNA is only detectable in the untreated control at a very low level. The concentration of taxol that most effectively induces ts mRNA expression is 50 mg/l compared to the control, the transcript levels are 2.5-, 3.1- and 3.3fold higher at 4, 8 and 12 days of culture.
additional information
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coexpression of geranylgeranyl diphosphate synthase gene from Sulfolobus acidocaldarius and codon optimzing of the Taxus chinensis taxadiene synthase gene increases the production of taxadiene in Saccharomyces cerevisiae
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0026 - 0.0159
geranylgeranyl diphosphate
0.025
Mg2+
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00004 - 0.000177
geranylgeranyl diphosphate
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
32
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assay at
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
79000
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79000, SDS-PAGE
98300
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calculation from amino acid sequence
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
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79000, SDS-PAGE
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
X-ray crystal structure of a truncation variant lacking the transit sequence and an additional 27 residues at the N-terminus, complexed with 13-aza-13,14-dihydrocopalyl diphosphate, at 1.82 A resolution, and 2-fluorogeranylgeranyl diphosphate, at 2.25 A resolution. The structure reveals a modular assembly of three alpha-helical domains. The C-terminal catalytic domain is a class I terpenoid cyclase, which binds and activates substrate GGPP with a three-metal ion cluster. The N-terminal domain and a third insertion domain together adopt the fold of a vestigial class II terpenoid cyclase
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Purification of the recombinant truncated TS protein as histag is performed using nickel-nitrilotriacetic acid affinity chromatography
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cDNA encoding taxa-4(5),11(12)-diene synthase is introduced in Nicotiana sylvestris for specific expression in trichome cells.
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cDNA is directly cloned into the pGemT-Easy vector, sequenced and cloned into the plant binary transformation vector pBC35. Furthermore it is subcloned into pBluescript, released and ligated into pRD12. The pBC35 and pRD12 constructs are transferred to Agrobacterium tumefaciens and used to transform tomato cotyledons
expressed in Physcomitrella patens
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expression in Escherichia coli
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Expression of Taxus chinensis taxadiene synthase (expression of a transgene encoding the pseudomature form which lacks the N-terminal 74-amino acid residue plastid-targeting sequence) in Saccharomyces cerevisiae alone does not increase taxadiene levels because of insufficient levels of the universal diterpenoid precursor geranylgeranyl diphosphate. Coexpression of Taxus chinensis taxadiene synthase and geranylgeranyl diphosphate synthase fails to increase levels. Replacement the Taxus chinensis geranylgeranyl diphosphate synthase with its counterpart from Sulfolobus acidocaldarius, which does not compete with steroid synthesis, and codon optimized the Taxus chinensis taxadiene synthase gene to ensure high-level expression, resulting in a increase in taxadiene as well as significant amounts of geranylgeraniol, suggesting taxadiene levels can be increased even further.
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for expression in Escherichia coli BL21DE3 cells
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overexpression in Escherichia coli
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
H579R
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accepts farnesyl diphosphate as substrate
additional information
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construction of truncation variant lacking the transit sequence and an additional 27 residues at the N-terminus, crystallization data
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
drug development
medicine
synthesis