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 ACCESSION NO.
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 ACCESSION NO.
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
SEQUENCE CODE
SEQUENCE DB
SOURCE
SUBSTRATE
PRODUCT                      
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
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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?
(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 ACCESSION NO.
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]
KM VALUE [mM] Maximum
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.011
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(2E,6E)-farnesyl diphosphate
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pH 8.0, 30C; pH not specified in the publication, at 37C
TURNOVER NUMBER [1/s]
TURNOVER NUMBER MAXIMUM[1/s]
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.012
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(2E,6E)-farnesyl diphosphate
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pH 8.0, 30C; pH not specified in the publication, at 37C
pH OPTIMUM
pH MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
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
pH RANGE MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
additional information
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the catalytic fidelity of Cop4 can be drastically altered by varying reactions conditions
TEMPERATURE RANGE
TEMPERATURE MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
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 ACCESSION NO.
LITERATURE
Talon resin metal affinity column chromatography
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Cloned/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
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 ACCESSION NO.
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