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Information on EC 4.2.3.128 - beta-cubebene synthase and Organism(s) Coprinopsis cinerea and UniProt Accession A8NU13
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4.2.3.128 beta-cubebene synthaseIUBMB Comments
Isolated from the fungus Coprinus cinereus. The enzyme also forms (+)-delta-cadinene, beta-copaene, (+)-sativene and traces of several other sequiterpenoids [2-4]. It is found in many higher plants such as Magnolia grandiflora (Southern Magnolia) together with germacrene A . See EC 4.2.3.13, (+)-delta-cadinene synthase, EC 4.2.3.127, beta-copaene synthase, EC 4.2.3.129, (+)-sativene synthase, and EC 4.2.3.23, germacrene A 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
vvtps, caryophyllene/cubebene synthase, vvshiraztps-y2, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
Mg25
-
-
-
-
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
(2E,6E)-farnesyl diphosphate = beta-cubebene + diphosphate
reaction mechanism, structure-function relationship, overview. The reaction mechanism for (E,E)-FPP cyclization by Cop4 involves the 1,10-cyclization of a cisoid neryl cation to form a (Z,E)-germacradienyl cation, which undergoes a 1,3-hydride shift to form an allylic carbocation that is either deprotonated to yield (-)-germacrene D, the major product of Cop4, or 1,6 cyclized to the bicyclic cadinyl cation
PATHWAY SOURCE
PATHWAYS
-
-
SYSTEMATIC NAME
IUBMB Comments
(2E,6E)-farnesyl-diphosphate diphosphate-lyase (cyclizing, beta-cubebene-forming)
Isolated from the fungus Coprinus cinereus. The enzyme also forms (+)-delta-cadinene, beta-copaene, (+)-sativene and traces of several other sequiterpenoids [2-4]. It is found in many higher plants such as Magnolia grandiflora (Southern Magnolia) together with germacrene A [1]. See EC 4.2.3.13, (+)-delta-cadinene synthase, EC 4.2.3.127, beta-copaene synthase, EC 4.2.3.129, (+)-sativene synthase, and EC 4.2.3.23, germacrene A 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
(2Z,6E)-farnesyl diphosphate
beta-cubebene + diphosphate
conversion of (E,E)-farnesyl diphosphate proceeds via a (6S)-beta-bisabolene carbocation in the case of Cop4
-
-
?
(E)-geraniol diphosphate
?
Cop4 converts 30% of (E)-geraniol diphosphate into cyclic monoterpene products. Limonene is synthesized as the major cyclic monoterpene product. Limonene can be derived from either a cisoid, exo- or cisoid, endo-conformation of the initial geranyl cation of the initial geranyl cation. Exo-conformation yields (Z)-beta-ocimene and linalool, while the endoconformation would give (E)-beta-ocimene
-
-
?
(2E,6E)-farnesyl diphosphate
beta-cubebene + diphosphate
-
-
-
?
(2E,6E)-farnesyl diphosphate
beta-cubebene + diphosphate
Cop4 synthesizes delta-cadinene as its major product, cf. EC 4.2.3.13
-
-
?
(2E,6E)-farnesyl diphosphate
beta-cubebene + diphosphate
conversion of (E,E)-farnesyl diphosphate proceeds via an (E,E)-germacradienyl carbocation in the case of Cop4
-
-
?
additional information
?
-
Cop4 cultures produce several sesquiterpene compounds, e.g. beta-cubebene, sativene, beta-copaene, and cubebol
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-
?
additional information
?
-
Cop4 is a catalytically promiscuous enzyme that cyclizes (2E,6E)-farnesyl diphosphate into multiple products, including (-)-germacrene D and cubebol. Cop 4 produces several volatile sesquiterpene products, including delta-cadinene as the major product, when expressed in Escherichia coli
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-
?
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
(2E,6E)-farnesyl diphosphate
beta-cubebene + diphosphate
-
-
-
?
(2E,6E)-farnesyl diphosphate
beta-cubebene + diphosphate
Cop4 synthesizes delta-cadinene as its major product, cf. EC 4.2.3.13
-
-
?
additional information
?
-
Cop4 cultures produce several sesquiterpene compounds, e.g. beta-cubebene, sativene, beta-copaene, and cubebol
-
-
?
additional information
?
-
Cop4 is a catalytically promiscuous enzyme that cyclizes (2E,6E)-farnesyl diphosphate into multiple products, including (-)-germacrene D and cubebol. Cop 4 produces several volatile sesquiterpene products, including delta-cadinene as the major product, when expressed in Escherichia coli
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-
?
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Mg2+
Mn2+
substitution of Mg2+ with Mn2+ as the divalent metal ion shifts the product profile of Cop4 to germacrene D, disfavoring subsequent ring closures that produce the cadinyl cation and its tricyclic descendents
NaCl
does not affect the product specificity of Cop4 significantly at 1 M
additional information
the enzyme contains the metal-binding DDXXD motif
Mg2+
required, substitution of Mg2+ with Mn2+ as the divalent metal ion shifts the product profile of Cop4 to germacrene D, disfavoring subsequent ring closures that produce the cadinyl cation and its tricyclic descendents. 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 that ligands the diphosphate moiety of the isoprenoid substrate, positions the isoprenyl chain in the binding pocket and triggers closure of the active site along with diphosphate cleavage to generate an initial transoid, allylic carbocation
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
increasing the reaction temperature to 37°C 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
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
additional information
kinetic analysis, overview
-
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
changing the pH of the reaction drastically alters the fidelity of Cop4 and makes it a highly selective enzyme. Increasing the reaction temperature to 37°C 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. The histidine side chain in the Cop4 loop, in particular, has a strong impact on the net charge of the loop at different pH values
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
physiological function
the enzyme belongs to the sesquiterpene synthases that are responsible for the cyclization of farnesyl diphosphate into a myriad of structurally diverse compounds with various biological activities
additional information
additional information
directed mutations of the H-alpha1 loop have a marked effect on the product profile Cop4, loop mutations in Cop4 also implicate specific residues responsible for the pH sensitivity of the enzyme. In vivo analysis of sesquiterpene product profiles of H-alpha1 loop mutants, overview. Mutation of K233, presumed to interact with the second Asp92 in the DDXXD motif of Cop4, does not significantly change the overall product promiscuity of Cop4, though beta-cubebene, with 27% of total sesquiterpene products, does become the major product
additional information
structural modeling, structure-function relationship, overview. Changing the pH of the reaction drastically alters the fidelity of Cop4 and makes it a highly selective enzyme
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
additional information
structural modeling, structure-function relationship, overview. Cop4 has a large binding pocket in the open conformation
additional information
structure comparison, homology structural modeling of Cop enzymes, overview. Cop4 has a large active site cavity that undergoes substantial conformational change in the model upon ligand binding
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, mutation of K233, interacting with the second Asp92 in the DDXXD motif of Cop4, does not significantly change the overall product promiscuity of Cop4, though beta-cubebene 4 does become the major product
N238L
site-directed mutagenesis, the mutant shows a altered product profile compared to the wild-type enzyme with a slight reduction in beta-cubebene synthesis. The mutant does no longer show production of cubebol and has reduced (-)-germacrene D synthesis activity compared to the wild-type enzyme, synthesis of beta-cubebene, beta-copaene, delta-cadinene, and alpha-cubebene
N239L
site-directed mutagenesis, no production of beta-cubebene, 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 altered product profile compared to the wild-type enzyme with an increase in beta-cubebene synthesis. The mutant does no longer show production of cubebol and (-)-germacrene D compared to the wild-type enzyme
additional information
directed mutations of the H-alpha1 loop have a marked effect on the product profile Cop4, loop mutations in Cop4 also implicate specific residues responsible for the pH sensitivity of the enzyme. H-alpha1 loop swap between Cop4 and Cop6 shifts Cop4 to a germacrene D synthase
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expression of wild-type and mutant enzymes in Escherichia coli strain JM109
gene cop4, DNA and amino acid sequence determination and analysis, phylogenetic analysis, recombinant expression 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), Coprinopsis cinerea ATCC MYA-4618 (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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