Information on EC 4.2.3.75 - (-)-germacrene D synthase

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The expected taxonomic range for this enzyme is: Eukaryota, Bacteria

EC NUMBER
COMMENTARY
4.2.3.75
-
RECOMMENDED NAME
GeneOntology No.
(-)-germacrene D synthase
-
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
(2E,6E)-farnesyl diphosphate = (-)-germacrene D + diphosphate
show the reaction diagram
-
-
-
-
(2E,6E)-farnesyl diphosphate = (-)-germacrene D + diphosphate
show the reaction diagram
action of (-)-germacrene D synthase involves a 1,3-hydride shift. Pro-R-H-1 of the substrate (2E,6E)-farnesyl diphosphate becomes H-11 of (-)-germacrene D, while pro-S-H-1 of (2E,6E)-farnesyl diphosphate becomes H-6 of (-)-germacrene D and H-10 of (2E,6E)-farnesyl diphosphate becomes H-7 of (-)-germacrene D
-
(2E,6E)-farnesyl diphosphate = (-)-germacrene D + diphosphate
show the reaction diagram
formation of (-)-germacrene D involves a stereospecific 1,3-hydride shift of H-1si of farnesyl diphosphate
-
(2E,6E)-farnesyl diphosphate = (-)-germacrene D + diphosphate
show the reaction diagram
formation of (-)-germacrene D involves a stereospecific 1,3-hydride shift of H-1si of farnesyl diphosphate
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
germacrene biosynthesis
-
-
Sesquiterpenoid and triterpenoid biosynthesis
-
-
SYSTEMATIC NAME
IUBMB Comments
(2E,6E)-farnesyl-diphosphate diphosphate-lyase [(-)-germacrene-D-forming]
In Solidago canadensis the biosynthesis results in the pro-R hydrogen at C-1 of the farnesy diphosphate ending up at C-11 of the (-)-germacrene D [1]. With Streptomyces coelicolor the pro-S hydrogen at C-1 ends up at C-11 of the (-)-germacrene D [2].
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
bifunctional germacradienol/germacrene D synthase
-
-
Manually annotated by BRENDA team
bifunctional germacradienol/germacrene D synthase
-
-
Manually annotated by BRENDA team
; cv. Chardonnay and cv. Gewürztraminer
SwissProt
Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                      
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(2E,6E)-farnesyl diphosphate
(-)-germacrene D + diphosphate
show the reaction diagram
Q70EZ6
-
-
-
?
(2E,6E)-farnesyl diphosphate
(-)-germacrene D + diphosphate
show the reaction diagram
Q64K29
-
79.4% of olefinic products
-
-
(2E,6E)-farnesyl diphosphate
(-)-germacrene D + diphosphate
show the reaction diagram
-
-
catalyzes the formation of enantiomerically pure (-)-germacrene D
-
?
(2E,6E)-farnesyl diphosphate
(-)-germacrene D + diphosphate
show the reaction diagram
-
-
main product is germacradienol, about 15% formation of (-)-(7S)-germacrene
-
?
(2E,6E)-farnesyl diphosphate
(-)-germacrene D + diphosphate
show the reaction diagram
Q6Q3H3
-
plus about 8% dleta-cadinene
-
-
(2E,6E)-farnesyl diphosphate
(-)-germacrene D + diphosphate
show the reaction diagram
-
conversion of (E,E)-FPP proceeds via an (E,E)-germacradienyl carbocation
main products at pH 8.0 are 29.3% (-)-germacrene D, 28.2% cubebol, 10.4% delta-cadinene, at pH 10.0 is 91% (-)-germacrene D, at pH 5.0 84% (-)-germacrene D plus 12% delta-cadinene
-
-
(2E,6E)-farnesyl diphosphate
(-)-germacrene D + diphosphate
show the reaction diagram
-
-
main product is germacradienol, about 15% formation of (-)-(7S)-germacrene
-
?
(2E,6E)-farnesyl diphosphate
(-)-germacrene D + diphosphate
show the reaction diagram
Vitis vinifera cv. Gewuerztraminer
-
-
plus about 8% dleta-cadinene
-
-
2-trans,6-trans-farnesyl diphosphate
(-)-(7S)-germacrene D + diphosphate
show the reaction diagram
Q6Q3H3
-
-
-
?
2-trans,6-trans-farnesyl diphosphate
(-)-(7S)-germacrene D + diphosphate
show the reaction diagram
Q6Q3H3
strictly enantio-specific reaction of (-)-germacrene D synthase
GC-MS product analysis
-
?
additional information
?
-
Q6Q3H3
cell terpenoid profile determination, no substrate: geranyl diphosphate or geranylgeranyl diphosphate
-
-
-
additional information
?
-
-
no substrate: geranyl diphosphate or geranylgeranyl diphosphate
-
-
-
additional information
?
-
-
Coprinus cinereus Cop4 additionally accepts (2Z,6E)-farnesyl diphosphate to produce 27.7% alpha-acoradiene, 10% amorpha-4,11-diene, and 13.3% cadina-4,11-diene
-
-
-
additional information
?
-
Vitis vinifera cv. Gewuerztraminer
-
no substrate: geranyl diphosphate or geranylgeranyl diphosphate
-
-
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
2-trans,6-trans-farnesyl diphosphate
(-)-(7S)-germacrene D + diphosphate
show the reaction diagram
Q6Q3H3
-
-
-
?
additional information
?
-
Q6Q3H3
cell terpenoid profile determination
-
-
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
K+
-
replacement of Mg2+ by Mn2+ results in loss of product beta-copaene. Presence of K+ decreases the (?)-germacrene D yield and instead increases the overall yield of tricyclic sesquiterpene olefins
Mg2+
Q70EZ6
required. Bi-dentate Mg2+-binding by residue 303 is not obligatory
Mg2+
Q6Q3H3
-
Mn2+
-
replacement of Mg2+ by Mn2+ results in loss of product beta-copaene. In the presence of the Mn2+, Cop4 favors a reaction path that ends after one cyclization in (?)-germacrene D
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.0029
(2E,6E)-farnesyl diphosphate
Q70EZ6
25°C, pH not specified in the publication
0.011
(2E,6E)-farnesyl diphosphate
-
pH 8.0, 30°C
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.012
(2E,6E)-farnesyl diphosphate
-
pH 8.0, 30°C
0.03
(2E,6E)-farnesyl diphosphate
Q70EZ6
25°C, pH not specified in the publication
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
1
(2E,6E)-farnesyl diphosphate
-
pH 8.0, 30°C
81
10.3
(2E,6E)-farnesyl diphosphate
Q70EZ6
25°C, pH not specified in the publication
81
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
5
-
at pH 5.0 cyclization can proceed via a 1,6 ring closure to also yield a small amount of delta-cadinene, 12% of total sesquiterpene products, in addition to (?)-germacrene D
7
Q6Q3H3
assay at
10
-
at pH 10, the cyclization reaction ends with the hydride shift and deprotonation of the germacradienyl cation to yield (?)-germacrene D, 91% of total sesquiterpene products
additional information
-
under both alkaline and acidic conditions Cop4 becomes a very selective enzyme with only one major product (?)-germacrene D compared to three major compounds produced under neutral reaction conditions. At pH 10, the cyclization reaction ends with the hydride shift and deprotonation of the germacradienyl cation to yield (?)-germacrene D, 91% of total sesquiterpene products, while at pH 5.0 cyclization can proceed via a 1,6 ring closure to also yield a small amount of delta-cadinene, 12% of total sesquiterpene products, in addition to (?)-germacrene D
pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
-
the catalytic fidelity of Cop4 can be drastically altered by varying reactions conditions
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
30
Q6Q3H3
assay at
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
4 - 25
-
lowering the reaction temperature from 25°C to 4°C increases the selectivity of Cop4 for (?)-germacrene D and decreases the fraction of structurally unidentified sesquiterpene olefins by half
37
-
increasing the reaction temperature to 37°C decreases the fidelity of Cop4. At 37°C Cop4 generates a relative larger fraction of products beta-cubebene, sativene, delta-cadinene and beta-copaene that are derived from a cadinyl cation intermediate
pI VALUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
4.5
Q6Q3H3
calculated
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
Q6Q3H3
strongest hybridization signals with RNA from flower budsand strongly reduced transcript levels in pre- and postanthesis open flowers and in set flowers at early fruit onset
Manually annotated by BRENDA team
Q6Q3H3
young
Manually annotated by BRENDA team
Q64K29
local and systemic emission of (-)-germacrene D ist induced by feeding forest tent caterpillars. Transcript level of Tps1 is strongly induced in responce to herbivory
Manually annotated by BRENDA team
Q6Q3H3
vegetative tissues of young leaves
Manually annotated by BRENDA team
Q6Q3H3
green stem
Manually annotated by BRENDA team
Vitis vinifera cv. Gewuerztraminer
-
green stem
-
Manually annotated by BRENDA team
Vitis vinifera cv. Gewuerztraminer
-
vegetative tissues of young leaves
-
Manually annotated by BRENDA team
additional information
Q6Q3H3
no transcripts in fruit
Manually annotated by BRENDA team
additional information
Vitis vinifera cv. Gewuerztraminer
-
no transcripts in fruit
-
Manually annotated by BRENDA team
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
?
Q6Q3H3
x * 64300, calculated
?
Q64K29
x * 65000, calculated
?
Q70EZ6
x * 65163, calculated
?
Vitis vinifera cv. Gewuerztraminer
-
x * 64300, calculated
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
Q64K29
expression in Escherichia coli
Q70EZ6
DNA and amino acid sequence determination and analysis of Vitis vinifera cv. Gewürztraminer, phylogenetic tree, expression in Escherichia coli strain BL21(DE3)-RIL; expression in Escherichia coli
Q6Q3H3
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
transcript level of Tps1 in leaf is strongly induced by feeding forest tent caterpillars and follows diurnal rhythm
Q64K29
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
D303E
Q70EZ6
less than 0.25% of wild-type activity
D303N
Q70EZ6
mimicking of corresponding amino acid in (+)-germacrene D synthase Sc11. 10% of the wild-type, activity, reduction in catalytic constant by a factor of 10, with no change in Km of farnesyl diphosphate, product composition or chirality