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 hide
4.2.3.128
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RECOMMENDED NAME
GeneOntology No.
beta-cubebene synthase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
(2E,6E)-farnesyl diphosphate = beta-cubebene + diphosphate
show the reaction diagram
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
beta-cubebene biosynthesis
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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.
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
UniProt
Manually annotated by BRENDA team
gene Mg25
UniProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
physiological function
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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
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(2E,6E)-farnesyl diphosphate
beta-cubebene + diphosphate
show the reaction diagram
(2Z,6E)-farnesyl diphosphate
beta-cubebene + diphosphate
show the reaction diagram
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conversion of (E,E)-farnesyl diphosphate proceeds via a (6S)-beta-bisabolene carbocation in the case of Cop4
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?
(E)-geraniol diphosphate
?
show the reaction diagram
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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
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?
additional information
?
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
(2E,6E)-farnesyl diphosphate
beta-cubebene + diphosphate
show the reaction diagram
additional information
?
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
NaCl
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does not affect the product specificity of Cop4 significantly at 1 M
additional information
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the enzyme contains the metal-binding DDXXD motif
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
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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
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.011 - 0.0707
(2E,6E)-farnesyl diphosphate
additional information
additional information
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kinetic analysis, overview
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.5
maximal activity at pH 7.5, dominated by four products: beta-cubebene, alpha-muurolene, delta-cadinol, and tau-muurolene
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 10
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mutant enzyme K233I
6.5 - 8.5
no activity below or above
8
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wild-type enzyme and mutant H235P
additional information
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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
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25 - 30
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assay at
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
Mg25 is much more evident in young developing leaf tissues than in older, more mature leaves
Manually annotated by BRENDA team
high expression level
Manually annotated by BRENDA team
additional information
Mg25 transcript levels are low or below detection limits in tepal and carpel tissues
Manually annotated by BRENDA team
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression of wild-type and mutant enzymes in Escherichia coli strain JM109
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gene cop4, DNA and amino acid sequence determination and analysis, phylogenetic analysis, recombinant expression in Escherichia coli strain JM109
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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
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
H235P
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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
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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
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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
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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
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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
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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