Information on EC 4.2.3.128 - beta-cubebene synthase

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The expected taxonomic range for this enzyme is: Eukaryota

EC NUMBER
COMMENTARY
4.2.3.128
-
RECOMMENDED NAME
GeneOntology No.
beta-cubebene synthase
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
(2E,6E)-farnesyl diphosphate = beta-cubebene + diphosphate
show the reaction diagram
-
-
-
-
(2E,6E)-farnesyl diphosphate = beta-cubebene + diphosphate
show the reaction diagram
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
-
(2E,6E)-farnesyl diphosphate = beta-cubebene + diphosphate
show the reaction diagram
reaction mechanism, overview
B3TPQ6
PATHWAY
KEGG Link
MetaCyc Link
beta-cubebene biosynthesis
-
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.
SYNONYMS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
Cop4
-
-
ambiguous
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Mg25
-
-
-
-
Mg25
B3TPQ6
gene name
sesquiterpene synthase
A8NU13
-
ORGANISM
COMMENTARY
LITERATURE
SEQUENCE CODE
SEQUENCE DB
SOURCE
Coprinopsis cinerea 9/55
-
UniProt
Manually annotated by BRENDA team
gene Mg25
UniProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
evolution
-
the enzyme belongs to the sesquiterpene synthases
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
evolution
B3TPQ6
the terpene synthase Mg25 belongs to the angiosperm sesquiterpene synthase subclass TPS-a. The structural diversity observed in the three Magnolia terpene synthase genes have occurred either by a rapid loss of introns from a common ancestor TPS gene or by a gain of introns into an intron-deficient progenote TPS gene
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
additional information
B3TPQ6
two terpenes dominate the plant sesquiterpene profile, with beta-cubebene accounting for 31.3% of the total and beta-elemene for 25.3%. The enzyme contains the conserved Asp-rich domain, DDXXD, that coordinates substrate binding via the formation of divalent cation salt bridges
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
beta-cubebene + diphosphate
show the reaction diagram
A8NU13
-
-
-
?
(2E,6E)-farnesyl diphosphate
beta-cubebene + diphosphate
show the reaction diagram
-
-
-
-
?
(2E,6E)-farnesyl diphosphate
beta-cubebene + diphosphate
show the reaction diagram
B3TPQ6
-
-
-
?
(2E,6E)-farnesyl diphosphate
beta-cubebene + diphosphate
show the reaction diagram
A8NU13
Cop4 synthesizes delta-cadinene as its major product, cf. EC 4.2.3.13
-
-
?
(2E,6E)-farnesyl diphosphate
beta-cubebene + diphosphate
show the reaction diagram
B3TPQ6
Mg25 converts farnesyl diphosphate predominantly to beta-cubebene
-
-
?
(2E,6E)-farnesyl diphosphate
beta-cubebene + diphosphate
show the reaction diagram
-
conversion of (E,E)-farnesyl diphosphate proceeds via an (E,E)-germacradienyl carbocation in the case of Cop4
-
-
?
(2E,6E)-farnesyl diphosphate
beta-cubebene + diphosphate
show the reaction diagram
Coprinopsis cinerea 9/55
A8NU13
-, Cop4 synthesizes delta-cadinene as its major product, cf. EC 4.2.3.13
-
-
?
(2Z,6E)-farnesyl diphosphate
beta-cubebene + diphosphate
show the reaction diagram
-
conversion of (E,E)-farnesyl diphosphate proceeds via a (6S)-beta-bisabolene carbocation in the case of Cop4
-
-
?
(E)-geraniol diphosphate
?
show the reaction diagram
-
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
-
-
?
additional information
?
-
A8NU13
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
-
-
-
additional information
?
-
B3TPQ6
the dominant reaction products generated by Mg25 are beta-cubebene with 24.5%, alpha-muurolene with 19.3%, cf. EC 4.2.3.125, delta-cadinol with 18.6%, delta-elemene with 16.0%, tau-muurolene with 10.8%, and beta-elemene with 10.8%
-
-
-
additional information
?
-
Coprinopsis cinerea 9/55
A8NU13
Cop4 cultures produce several sesquiterpene compounds, e.g. beta-cubebene, sativene, beta-copaene, and cubebol
-
-
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
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
beta-cubebene + diphosphate
show the reaction diagram
-
-
-
-
?
(2E,6E)-farnesyl diphosphate
beta-cubebene + diphosphate
show the reaction diagram
A8NU13
Cop4 synthesizes delta-cadinene as its major product, cf. EC 4.2.3.13
-
-
?
(2E,6E)-farnesyl diphosphate
beta-cubebene + diphosphate
show the reaction diagram
B3TPQ6
Mg25 converts farnesyl diphosphate predominantly to beta-cubebene
-
-
?
(2E,6E)-farnesyl diphosphate
beta-cubebene + diphosphate
show the reaction diagram
Coprinopsis cinerea 9/55
A8NU13
Cop4 synthesizes delta-cadinene as its major product, cf. EC 4.2.3.13
-
-
?
additional information
?
-
A8NU13
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
-
-
-
additional information
?
-
B3TPQ6
the dominant reaction products generated by Mg25 are beta-cubebene with 24.5%, alpha-muurolene with 19.3%, cf. EC 4.2.3.125, delta-cadinol with 18.6%, delta-elemene with 16.0%, tau-muurolene with 10.8%, and beta-elemene with 10.8%
-
-
-
additional information
?
-
Coprinopsis cinerea 9/55
A8NU13
Cop4 cultures produce several sesquiterpene compounds, e.g. beta-cubebene, sativene, beta-copaene, and cubebol
-
-
-
METALS and IONS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
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
Mg2+
B3TPQ6
required, maximal activities at Mg2+ concentrations of 1 to 10 mM
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
Mn2+
B3TPQ6
activates, naximally at concentrations of 1 mM or less
additional information
-
the enzyme contains the metal-binding DDXXD motif
ACTIVATING COMPOUND
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
additional information
-
increasing the reaction temperature to 37C 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
-
KM VALUE [mM]
KM VALUE [mM] Maximum
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.011
-
(2E,6E)-farnesyl diphosphate
-
wild-type enzyme, and mutants H235P and N239L, pH 8.0, 30C
0.048
-
(2E,6E)-farnesyl diphosphate
-
Cop6 loop graft mutant Cop6L4, pH 8.0, 30C
0.0707
-
(2E,6E)-farnesyl diphosphate
-
mutant K233I, pH 8.0, 30C
additional information
-
additional information
-
kinetic analysis, overview
-
pH OPTIMUM
pH MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
7.5
-
B3TPQ6
maximal activity at pH 7.5, dominated by four products: beta-cubebene, alpha-muurolene, delta-cadinol, and tau-muurolene
8
-
-
assay at
pH RANGE
pH RANGE MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
5
10
-
mutant enzyme K233I
6.5
8.5
B3TPQ6
no activity below or above
8
-
-
wild-type enzyme and mutant H235P
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 37C 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
TEMPERATURE OPTIMUM MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
24
26
B3TPQ6
-
25
30
-
assay at
SOURCE TISSUE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
SOURCE
B3TPQ6
high expression level
Manually annotated by BRENDA team
B3TPQ6
Mg25 is much more evident in young developing leaf tissues than in older, more mature leaves
Manually annotated by BRENDA team
additional information
B3TPQ6
Mg25 transcript levels are low or below detection limits in tepal and carpel tissues
Manually annotated by BRENDA team
SUBUNITS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
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
additional information
-
structural modeling, structure-function relationship, overview. Cop4 has a large binding pocket in the open conformation
additional information
B3TPQ6
the enzyme contains the conserved Asp-rich domain, DDXXD, that coordinates substrate binding via the formation of divalent cation salt bridges
Cloned/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
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
-
gene Mg25, DNA and amino acid sequence determination and analysis, Mg25 has one single intron located near the 5' terminus of the gene, expression analysis and phylogenetic analysis
B3TPQ6
ENGINEERING
ORGANISM
UNIPROT ACCESSION NO.
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
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
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
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