Information on EC 4.2.3.129 - (+)-sativene synthase

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The expected taxonomic range for this enzyme is: Eukaryota

EC NUMBER
COMMENTARY
4.2.3.129
-
RECOMMENDED NAME
GeneOntology No.
(+)-sativene synthase
-
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
(2E,6E)-farnesyl diphosphate = (+)-sativene + diphosphate
show the reaction diagram
-
-
-
-
SYSTEMATIC NAME
IUBMB Comments
(2E,6E)-farnesyl-diphosphate diphosphate-lyase (cyclizing, (+)-sativene-forming)
Isolated from the fungus Coprinus cinereus. The enzyme also forms (+)-delta-cadinene, beta-copaene, beta-cubebene, and traces of several other sequiterpenoids. See EC 4.2.3.13, (+)-delta-cadinene synthase, EC 4.2.3.127, beta-copaene synthase, and EC 4.2.3.128, beta-cubebene synthase.
SYNONYMS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
Cop4
-
-
ambiguous
-
Cop4
-
gene name
Cop4
Coprinopsis cinerea 9/55
A8NU13
-
-
sesquiterpene synthase
A8NU13
-
ORGANISM
COMMENTARY
LITERATURE
SEQUENCE CODE
SEQUENCE DB
SOURCE
Coprinopsis cinerea 9/55
-
UniProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
evolution
-
the enzyme belongs to the sesquiterpene synthases
malfunction
-
identification of constitutive photomorphogenic mutants, cop2, cop3, and cop4,in which dark-grown seedlings have open and enlarged cotyledons resembling those of light-grown wild-type seedlings. Mutations in each of the three loci alleviate the normal inhibition of cell-type differentiation, cell enlargement, and lateral cell division observed in cotyledons of dark-grown wild-type seedlings, but do not affect plastid differentiation. Cop4 mutation also leads to high-leve1 dark expression of nuclear, but not plastid-encoded, light-inducible genes
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
physiological function
-
the COP4 locus may be involved in both light-signaling and gravity-sensing processes. COP4 modulates cabl promoter activity through a pathway distinct from that of COPl and COP9, nuclear cabl gene encodes a chlorophyll alb binding protein of the photosynthetic light-harvesting complex. Modulation of cabl promoter activity by light and by the cop4 mutation
metabolism
-
epistatic relationships of these three mutations to previously characterized phytochrome-deficient mutations suggest that COP2, COP3, and COP4 may act downstream of phytochrome in the light regulatory pathway
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
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
(+)-sativene + diphosphate
show the reaction diagram
-
-
-
-
?
(2E,6E)-farnesyl diphosphate
(+)-sativene + diphosphate
show the reaction diagram
A8NU13
-
-
-
?
(2E,6E)-farnesyl diphosphate
(+)-sativene + diphosphate
show the reaction diagram
-
-
-
-
?
(2E,6E)-farnesyl diphosphate
(+)-sativene + diphosphate
show the reaction diagram
A8NU13
Cop4 synthesizes delta-cadinene as its major product, cf. EC 4.2.3.13
-
-
?
(2E,6E)-farnesyl diphosphate
(+)-sativene + diphosphate
show the reaction diagram
-
conversion of (E,E)-farnesyl diphosphate proceeds via an (E,E)-germacradienyl carbocation in the case of Cop4
-
-
?
(2Z,6E)-farnesyl diphosphate
(+)-sativene + diphosphate
show the reaction diagram
-
conversion of (E,E)-farnesyl diphosphate proceeds via a (6S)-beta-bisabolene carbocation in the case of Cop4
-
-
?
additional information
?
-
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
(+)-sativene + diphosphate
show the reaction diagram
-
-
-
-
?
(2E,6E)-farnesyl diphosphate
(+)-sativene + diphosphate
show the reaction diagram
-
-
-
-
?
(2E,6E)-farnesyl diphosphate
(+)-sativene + diphosphate
show the reaction diagram
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
-
-
-
METALS and IONS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
Mg2+
-
required
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
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
ACTIVATING COMPOUND
ORGANISM
UNIPROT ACCESSION NO.
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]
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, 30°
0.048
-
(2E,6E)-farnesyl diphosphate
-
Cop6 loop graft mutant Cop6L4, pH 8.0, 30°
0.0707
-
(2E,6E)-farnesyl diphosphate
-
mutant K233I, pH 8.0, 30°
additional information
-
additional information
-
kinetic analysis, overview
-
pH OPTIMUM
pH MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
pH RANGE
pH RANGE MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
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
TEMPERATURE OPTIMUM MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
25
30
-
assay at
SOURCE TISSUE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
SOURCE
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
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
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ENGINEERING
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
H235P
-
site-directed mutagenesis, no production of sativene, 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 only slight reduction in beta-cubebene synthesis, sativene levels are similar to the wild-type. 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 sativene, 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 sativene synthesis. The mutant does no longer show production of cubebol and (-)-germacrene D compared to the wild-type enzyme
additional information
-
generation of a cop4 defective mutant and a cop4/cop1 double mutant
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 and püroduction of sativene is reduced by 50% 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