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Information on EC 4.2.3.127 - beta-copaene synthase and Organism(s) Coprinopsis cinerea and UniProt Accession A8NU13
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4.2.3.127 beta-copaene synthaseIUBMB Comments
Isolated from the fungus Coprinus cinereus. The enzyme also forms (+)-delta-cadinene, beta-cubebene, (+)-sativene and traces of several other sequiterpenoids [1-3]. beta-Copaene is formed in the presence of Mg2+ but not Mn2+ . See EC 4.2.3.13, (+)-delta-cadinene synthase, EC 4.2.3.128, beta-cubebene synthase, and EC 4.2.3.129, (+)-sativene synthase.
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The taxonomic range for the selected organisms is: Coprinopsis cinerea
The enzyme appears in selected viruses and cellular organisms
The enzyme appears in selected viruses and cellular organisms
Synonyms
Cop4, CoTPS2, sesquiterpene synthase, TPS2, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
(2E,6E)-farnesyl diphosphate = beta-copaene + diphosphate
cyclization reaction mechanism, overview
(2E,6E)-farnesyl diphosphate = beta-copaene + diphosphate
the reaction mechanism requires first the isomerization of all-trans (E,E)-FPP at the 2,3 double bond to generate a cisoid-farnesyl cation. Cop4 then catalyzes a 1,10 ring closure of this farnesyl cation isomer to form the secondary cis-germacrene-dienyl cation. Cop4 generates a tertiary cadinyl cation which upon deprotonation yields its major cyclization product delta-cadinene. The cadinyl cation reacts further to yield other cyclization products beta-cubebene, cubebol, beta-copaene, and sativene
SYSTEMATIC NAME
IUBMB Comments
(2E,6E)-farnesyl-diphosphate diphosphate-lyase (cyclizing, bet6a-copaene-forming)
Isolated from the fungus Coprinus cinereus. The enzyme also forms (+)-delta-cadinene, beta-cubebene, (+)-sativene and traces of several other sequiterpenoids [1-3]. beta-Copaene is formed in the presence of Mg2+ but not Mn2+ [2]. See EC 4.2.3.13, (+)-delta-cadinene synthase, EC 4.2.3.128, beta-cubebene synthase, and EC 4.2.3.129, (+)-sativene synthase.
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
(E)-geranyl diphosphate
?
the enzyme accepts (E)-geranyl diphosphate as a substrate, but the catalytic efficiency with the shorter prenyl-diphosphate substrate is lower compared to their longer farnesyl diphosphate substrate
-
-
?
(2E,6E)-farnesyl diphosphate
beta-copaene + diphosphate
-
-
-
?
(2E,6E)-farnesyl diphosphate
beta-copaene + diphosphate
Cop4 produces 10 different sesquiterpene products with delta-cadinene and beta-copaene as the major products in vivo
-
-
?
(2E,6E)-farnesyl diphosphate
beta-copaene + diphosphate
conversion of (E,E)-farnesyl diphosphate proceeds via an (E,E)-germacradienyl carbocation in the case of Cop4. Cyclization of all-trans-farnesyl diphosphate compared to the cyclization of the cis-trans isomer of farnesyl diphosphate serving as a surrogate for the secondary cisoid neryl cation intermediate generated by sesquiterpene synthases capable of isomerizing the C2-C3 bond of all-trans-farnesyl diphosphate. (Z,E)-FPP is cyclized via a (6S)-beta-bisabolene carbocation by Cop4
-
-
?
additional information
?
-
Cop4 synthesizes delta-cadinene as its major product, cf. EC 4.2.3.13. Cop4 cultures produce several sesquiterpene compounds betacubebene, sativene, beta-copaene, and cubebol
-
-
?
additional information
?
-
Cop4 is a catalytically promiscuous enzyme that cyclizes (E,E)-farnesyl diphosphate into multiple products, including (-)-germacrene D and cubebol. But changing the pH of the reaction drastically alters the fidelity of Cop4 and makes it a highly selective enzyme
-
-
?
additional information
?
-
Escherichia coli strains expressing Cop4 accumulate delta-cadinene as the major terpenoid, accounting for about 40% of the total sesquiterpenes detected
-
-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
additional information
?
-
Cop4 synthesizes delta-cadinene as its major product, cf. EC 4.2.3.13. Cop4 cultures produce several sesquiterpene compounds betacubebene, sativene, beta-copaene, and cubebol
-
-
?
(2E,6E)-farnesyl diphosphate
beta-copaene + diphosphate
Cop4 produces 10 different sesquiterpene products with delta-cadinene and beta-copaene as the major products in vivo
-
-
?
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Mg2+
additional information
Mg2+
dependent on, two consensus sequences - an aspartate rich DDXXD/E and a NSE/DTE motif - located at the entrance of the active site coordinate a trinuclear Mg2+ cluster. In reactions where Mg2+ is replaced with either Mn2+ or K+ is the disappearance of beta-copaene
additional information
in presence of the divalent cation Mn2+, Cop4 favors a reaction path that ends after one cyclization in (-)-germacrene D
additional information
the enzyme contains the metal-binding DDXXD motif
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.011
(2E,6E)-farnesyl diphosphate
wild-type enzyme, and mutants H235P and N239L, pH 8.0, 30°C
0.048
(2E,6E)-farnesyl diphosphate
Cop6 loop graft mutant Cop6L4, pH 8.0, 30°C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
additional information
changing the pH of the reaction drastically alters the fidelity of Cop4 and makes it a highly selective enzyme
additional information
the product profile of the wild-type and mutant enzymes is highly dependent on the pH value, overview
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
lowering the reaction temperature from 25°C to 4°C increases the selectivity of Cop4 for (-)-germacrene D. Increasing the reaction temperature to 37°C has the opposite effect and decreases the fidelity of Cop4. At this temperature Cop4 generates a relative larger fraction of products beta-cubebene, sativene delta-cadinene, and beta-copaene that are derived from a cadinyl cation intermediate
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
additional information
the product profile of the wild-type and mutant enzymes is highly dependent on the pH value, the loop mutants show a much larger pH activity range, overview
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
additional information
structural homology modeling of Cop4 using the crystal structure of aristolochene synthase from Aspergillus terreus. Several polar side chains in the H-alpha1 loops of Cop4 and Cop3 move closer to side chains in the metal-binding DDXXD motif
additional information
structural homology modeling, hydrogen bond interactions and metal ion coordination in the diphosphate bound closed conformation of the Cop models, overview
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
H235P
site-directed mutagenesis, the mutation converts Cop4 into a much more selective enzyme that produces (-)-germacrene D as the major cyclization product with 50% of total sesquiterpenes products. The mutant makes beta-ylangene, which is a diastereomer of beta-copaene and not synthesized by wild-type Cop4
K233I
site-directed mutagenesis, the mutant shows a highly altered product profile compared to the wild-type enzyme with decrease in beta-copaene amounts. Cop4 loop mutant K2331 also becomes more selective for ()-germacrene D under acidic or basic reaction conditions, although less so than the wild-type enzyme
N238L
site-directed mutagenesis, the mutant shows a slightly altered product profile compared to the wild-type enzyme with increase in beta-copaene amounts
N239L
site-directed mutagenesis, the mutation converts Cop4 into a much more selective enzyme that produces (-)-germacrene D as the major cyclization product with 50% of total sesquiterpenes products. The mutant makes beta-ylangene, which is a diastereomer of beta-copaene and not synthesized by wild-type Cop4
T236L
site-directed mutagenesis, the mutant shows a slightly altered product profile compared to the wild-type enzyme with increase in beta-copaene amounts
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expression of wild-type and mutant Cop4 in Escherichia coli strain JM109
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Agger, S.; Lopez-Gallego, F.; Schmidt-Dannert, C.
Diversity of sesquiterpene synthases in the basidiomycete Coprinus cinereus
Mol. Microbiol.
72
1181-1195
2009
Coprinopsis cinerea (A8NU13)
Lopez-Gallego, F.; Wawrzyn, G.; Schmidt-Dannert, C.
Selectivity of fungal sesquiterpene synthases: Role of the active sites H-1alpha loop in catalysis
Appl. Environ. Microbiol.
76
7723-7733
2010
Coprinopsis cinerea (A8NU13)
Lopez-Gallego, F.; Agger, S.A.; Abate-Pella, D.; Distefano, M.D.; Schmidt-Dannert, C.
Sesquiterpene synthases Cop4 and Cop6 from Coprinus cinereus: catalytic promiscuity and cyclization of farnesyl pyrophosphate geometric isomers
ChemBioChem
11
1093-1106
2010
Coprinopsis cinerea (A8NU13)
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