Information on EC 4.2.3.91 - cubebol synthase

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The expected taxonomic range for this enzyme is: Coprinopsis cinerea

EC NUMBER
COMMENTARY
4.2.3.91
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RECOMMENDED NAME
GeneOntology No.
cubebol synthase
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
(2E,6E)-farnesyl diphosphate + H2O = cubebol + diphosphate
show the reaction diagram
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(2E,6E)-farnesyl diphosphate = cubebol + diphosphate
show the reaction diagram
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SYSTEMATIC NAME
IUBMB Comments
(2E,6E)-farnesyl-diphosphate diphosphate-lyase (cyclizing, cubebol-forming)
Requires Mg2+. The enzyme gives 28% cubebol, 29% (-)-germacrene D, 10% (+)-delta-cadinene and traces of several other sesquiterpenoids. See also EC 4.2.3.75 ()-germacrene D synthase and EC 4.2.3.13 (+)-delta-cadinene synthase.
SYNONYMS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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
cubebol + diphosphate
show the reaction diagram
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conversion of (6E,6E)-farnesyl diphosphate 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
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(2E,6E)-farnesyl diphosphate + H2O
cubebol + diphosphate
show the reaction diagram
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-
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?
(2E,6E)-farnesyl diphosphate + H2O
cubebol + diphosphate
show the reaction diagram
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germacrene D and cubebol are the major products in vitro
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?
additional information
?
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Cop4 is a catalytically promiscuous enzyme that cyclizes (E,E)-farnesyl diphophate into multiple products, including (-)-germacrene D and cubebol (major products). Conversion of (E,E)-farnesyl diphophate by Cop4 proceeds via an (E,E)-germacradienyl carbocation. Cop4 cyclizes (2Z,6E)-farnesyl diphophate via a (6S)-beta-bisabolyl cation intermediate to (6S)-beta-bisabolene. Cop4 converts E-geranyl diphophate into cyclic and acyclic monoterpenes. Cop4 also cyclizes nerolidyl diphosphate. Under alkaline (pH 10) or acidic (pH 5.0) conditions, Cop4 becomes a very selective germacrene D synthase, 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
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additional information
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Cop4 rearranges a secondary cisoid, allylic cation derived from farnesyl diphophate after isomerization (cis-trans pathway of catalysis) into multiple products, with delta-cadinene and beta-copaene as the major products when expressed in Escherichia coli
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
K+
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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
Mn2+
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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
0.011
(2E,6E)-farnesyl diphosphate
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pH 8.0, 30C; pH not specified in the publication, at 37C
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.012
(2E,6E)-farnesyl diphosphate
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pH 8.0, 30C; pH not specified in the publication, at 37C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
5
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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
10
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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
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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
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the catalytic fidelity of Cop4 can be drastically altered by varying reactions conditions
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
4 - 25
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lowering the reaction temperature from 25C to 4C increases the selectivity of Cop4 for (-)-germacrene D and decreases the fraction of structurally unidentified sesquiterpene olefins by half
37
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increasing the reaction temperature to 37C decreases the fidelity of Cop4. At 37C Cop4 generates a relative larger fraction of products beta-cubebene, sativene, delta-cadinene and beta-copaene that are derived from a cadinyl cation intermediate
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Talon resin metal affinity column chromatography
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli BL21 (DE3) cells
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expressed in Escherichia coli strain JM109 or BL21
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
4L6
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the mutant shows a 1000fold-decreased catalytic efficiency compared to the wild type enzyme
H235P
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the mutant makes (-)-germacrene D as the major cyclization product and also synthesizes beta-ylangene not synthesized by wild-type Cop4
K233I
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the mutation has only a moderate effect on product selectivity
N239L
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the mutant makes (-)-germacrene D as the major cyclization product and also synthesizes beta-ylangene not synthesized by wild-type Cop4