Information on EC 4.2.3.46 - alpha-farnesene synthase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
4.2.3.46
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RECOMMENDED NAME
GeneOntology No.
alpha-farnesene synthase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
(2E,6E)-farnesyl diphosphate = (3E,6E)-alpha-farnesene + diphosphate
show the reaction diagram
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Biosynthesis of secondary metabolites
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farnesene biosynthesis
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Sesquiterpenoid and triterpenoid biosynthesis
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SYSTEMATIC NAME
IUBMB Comments
(2E,6E)-farnesyl-diphosphate lyase [(3E,6E)-alpha-farnesene-forming]
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
cultivars ‘Chieftain’ and ‘Hayward’
UniProt
Manually annotated by BRENDA team
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UniProt
Manually annotated by BRENDA team
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UniProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
differences in the relative emissions of (E)-beta-ocimene and (E,E)-alpha-farnesene from accession Wassilewskija, a high-(E)-beta-ocimene emitter, and accession Columbia, a trace-(E)-beta-ocimene emitter, are attributed to allelic variation of closely related, tandem-duplicated terpene synthase genes, TPS02 and TPS03. The Wassilewskija genome contains a functional allele of TPS02 but not of TPS03, while the opposite is the case for Columbia. Recombinant proteins of the functional Wassilewskija TPS02 and Columbia TPS03 genes both show (E)-beta-ocimene and (E,E)-alpha-farnesene synthase activities. Differential subcellular compartmentalization of the two enzymes in plastids and the cytosol is responsible for the ecotype-specific differences in (E)-beta-ocimene/(E,E)-alpha-farnesene emission; differences in the relative emissions of (E)-beta-ocimene and (E,E)-alpha-farnesene from accession Wassilewskija, a high-(E)-beta-ocimene emitter, and accession Columbia, a trace-(E)-beta-ocimene emitter, are attributed to allelic variation of closely related, tandem-duplicated terpene synthase genes, TPS02 and TPS03. The Wassilewskija genome contains a functional allele of TPS02 but not of TPS03, while the opposite is the case for Columbia. Recombinant proteins of the functional Wassilewskija TPS02 and Columbia TPS03 genes both show (E)-beta-ocimene and (E,E)-alpha-farnesene synthase activities. Differential subcellular compartmentalization of the two enzymes in plastids and the cytosol is responsible for the ecotype-specific differences in (E)-beta-ocimene/(E,E)-alpha-farnesene emission
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
(3E,6E)-alpha-farnesene + beta-farnesene + diphosphate
show the reaction diagram
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when farnesyl diphosphate, synthesised from 96% (E,E)-farnesol, is incubated with recombinant protein, (E,E)- and (Z,E)-alpha-farnesene are produced in a ratio of 96:4, respectively
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?
(2E,6E)-farnesyl diphosphate
(3E,6E)-alpha-farnesene + diphosphate
show the reaction diagram
(2E,6E)-farnesyl diphosphate
alpha-farnesene + diphosphate
show the reaction diagram
(2E,6E)-farnesyl diphosphate + H2O
(3E,6E)-alpha-farnesene + diphosphate
show the reaction diagram
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about 95% of total product, plus trace amounts of beta-farnesene
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?
farnesyl diphosphate
(E,E)-alpha-farnesene + diphosphate
show the reaction diagram
sesquiterpene synthase activity
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?
farnesyl diphosphate
(Z,E)-alpha-farnesene + diphosphate
show the reaction diagram
isomers of farnesene produced in apple fruit are (E,E)-alpha and (Z,E)-alpha are in a ratio of 300:1
trace amounts
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?
geranyl diphosphate
(E)-beta-ocimene + beta-myrcene
show the reaction diagram
poor substrate
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?
geranyl diphosphate
(E)-beta-ocimene + diphosphate
show the reaction diagram
geranyl diphosphate
(Z)-beta-ocimene + diphosphate
show the reaction diagram
monoterpene synthase activity
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?
geranyl diphosphate
linalool + (Z)-beta-ocimene + (E)-beta-ocimene + beta-myrcene
show the reaction diagram
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at 18% of the optimised rate for alpha-farnesene synthesis from farnesyl diphosphate
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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
(3E,6E)-alpha-farnesene + diphosphate
show the reaction diagram
farnesyl diphosphate
(E,E)-alpha-farnesene + diphosphate
show the reaction diagram
Q84LB2
sesquiterpene synthase activity
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?
farnesyl diphosphate
(Z,E)-alpha-farnesene + diphosphate
show the reaction diagram
Q84LB2
isomers of farnesene produced in apple fruit are (E,E)-alpha and (Z,E)-alpha are in a ratio of 300:1
trace amounts
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?
geranyl diphosphate
(E)-beta-ocimene + diphosphate
show the reaction diagram
Q84LB2
in monoterpene synthase assays, only (E)-beta-ocimene is produced at much reduced levels
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?
geranyl diphosphate
(Z)-beta-ocimene + diphosphate
show the reaction diagram
Q84LB2
monoterpene synthase activity
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?
additional information
?
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1-methylcyclopropene
after treatment of fruits at harvest with a blocker of ethylene action, AFS1 mRNA declines sharply over the initial 4 weeks of cold storage, and falls to nearly undetectable levels by 8 weeks
Mn2+
above 0.03 mM
Na2MoO4
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10 mM, 96% inhibition
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0026 - 0.0095
(2E,6E)-farnesyl diphosphate
0.0028
geranyl diphosphate
pH 7.5
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0026 - 0.44
(2E,6E)-farnesyl diphosphate
0.0026 - 0.0613
farnesyl diphosphate
0.000005 - 0.03
geranyl diphosphate
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.12 - 51
(2E,6E)-farnesyl diphosphate
81
11
geranyl diphosphate
Actinidia deliciosa
C7SHN9
pH 7.5
175
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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S487K mutant has 35-45% of the wild-type activity with farnesyl diphosphate with no significant alterations in the alpha-farnesene isomer ratios produced and a small decrease in catalytic efficiency (wild-type and S487K respective kcat/Km values of 17.5 and 13.3 mM/s)
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7 - 8.5
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7.3
sesquiterpene synthase assay
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7 - 8
maximal sesquiterpene synthase activity is observed
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.93
calculated from sequence
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
production of (E,E)-alpha-farnesene occurs in the epidermal (peel tissue) or adjacent hypodermal cell layers and the sesquiterpene can accumulate to high levels in the natural epicuticular coating of scald-susceptible apples during the first weeks of storage
Manually annotated by BRENDA team
within floral tissues, expression is highest in petals and stamens
Manually annotated by BRENDA team
within floral tissues, expression is highest in petals and stamens
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
plastidial transit peptide of 25 amino acids, and fluoresence studies using GPF-fusion protein, accession Wassilewskija
Manually annotated by BRENDA team
additional information
AdAFS1 protein sequences lacks predicted N-terminal transit peptide-like sequences for chloroplast targeting
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Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
66000
without myc-epitope tag, determined by PAGE analysis
68000
with myc-epitope tag, determined by PAGE analysis
88100
x * 88100, calculated from sequence
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
x * 88100, calculated from sequence
monomer
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
sesquiterpene synthase activity declined below pH 7.0 and at pH 5.0-5.5, it is reduced more than 50%
694765
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
AFS1 transcript increases about 4fold in peel tissue of apple fruit during the first 4 weeks of storage at 0.5°C
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
recombinant MdAFS1protein is extracted and purified
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
After screening a cDNA library generated from the peel tissue mRNA, a full-length terpene synthase cDNA 1931 nucleotides long is isolated (hot phenol RNA extraction protocol is used). The 1728-bp open reading frame encodes the 576 amino acid protein. Expression of the apple gene in Escherichia coli.
expressed in Escherichia coli
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expression in Escherichia coli
exprsssion in Escherichia coli and Saccharomyces cerevisiae
heterologously expressed in Escherichia coli
mutated enzymes are generated using the QuickChange II site-directed mutagenesis kit. The PCR-based mutagenesis protocol is performed using pET-30a harbouring the MdAFS1 cDNAs as template. The single-site mutant enzymes overexpressed in Escherichia coli.
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
expression is induced in leaves by elicitor and insect treatment; expression is induced in leaves by elicitor and insect treatment
expression of AdAFS1 is significantly higher in flowers than in leaf tissue. Within floral tissues, expression is highest in petals and stamens. AdAFS1 expression is low in sepals in both sexes and also in ‘Hayward’ pistils. AdAFS1 expression in ‘Hayward’ is co-ordinated with anthesis, with low levels of expression in unopened flower buds and high levels detected in open flowers. The temporal accumulation patterns of AdAFS1 mRNA is constitutive. No difference in expression between male and female flowers. AdAFS1 expression is similar in leaves of both cultivars ‘Chieftain’ and ‘Hayward’
expression of pMdAFS1 is repressed by 1-methylcyclopropene treatment, pMdAFS1 expression is controlled by ethylene
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induced by Lymantria dispar feeding, up to 136fold increase in transcript abundance
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MdAFS1 transcript abundance increases rapidly in untreated Cortland and Law Rome fruit, reaching close to maximal values after 2 weeks of storage, whereas in Idared controls it increases through 4 weeks, declines slightly, and then increases to a maximum by 10 weeks. 1-Methylcyclopropene treatment initially suppresses MdAFS1 expression in all three cultivars. However, Cortland and Law Rome escape from this suppression after 10-15 weeks, with MdAFS1 transcript abundance increasing to maximal control levels by 15-20 weeks. In Cortland, but not in Law Rome, this delayed increase in expression occurrs sooner in green than in red tissue. MdAFS1 transcript levels increase gradually in 1-methylcyclopropene-treated Idared fruit, but throughout storage remains much lower than the maximum levels in untreated fruit
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D326A
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alpha-farnesene synthase, monoterpene synthase and prenyltransferase activities are lost in the mutant
D326A/D330A
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alpha-farnesene synthase, monoterpene synthase and prenyltransferase activities are lost in the mutant
D484A
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85% loss of sesquiterpene synthase activity compared with wild-type enzyme. Little change in K+-independent activity; 85% loss of sesquiterpene synthase activity compared with wild-type enzyme when K+ is present
S485A
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exhibits marginally increased sesquiterpene synthase activities, and an approximate 2fold increase in monoterpene synthase activity compared with the wild-type enzyme; mutant exhibits marginally increased sesquiterpene synthase activities, and an approximate 2fold increase in monoterpene synthase activity compared with the wild-type enzyme
S487A
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95% decrease in sesquiterpene synthase activity compared with wild-type enzyme. Little change in K+-independent activity; 95% decrease in sesquiterpene synthase activity compared with wild-type enzyme when K+ is present
S487K
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mutant has 35–45% of the wild-type activity with farnesyl diphosphate, with no significant alterations in the alpha-farnesene isomer ratios produced and a small decrease in catalytic efficiency; The S487K mutant has 35-45% of the wild-type activity with farnesyl diphosphate, with no significant alterations in the alpha-farnesene isomer ratios produced and a small decrease in catalytic efficiency
S488A
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mutant shows decreases in both sesqui- and monoterpene synthase activities compared with the WT enzyme, with mono-TPS activity being reduced more than sesquiterpene synthase activity. Sesquiterpene synthase (alpha-farnesene) products produced by the mutated and wild-type enzymes are identical, there are no significant alterations in the ratios of the alpha-farnesene isomers produced.; mutant shows decreases in both sesqui- and mono-terpene synthases activities, compared with the wild-type enzyme, with mono-terpene synthases activity being reduced more than sesqui-terpene synthases activity
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
myc-tagged and untagged AFS1 expressed protein in bacterial inclusion-body fractions is urea-denatured, purified and renatured prior to assay of enzymatic activity.
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
agriculture
Because diphenylamine treatment leaves unwanted chemical residues on the fruit, restricts export markets, and creates environmental concerns, a long-range molecular genetic strategy for control of scald by reduction of (E,E)-alpha-farnesene synthesis in scald-susceptible apples is searched. The success of this strategy will rely on our ability to identify, clone, and characterize key genes involved in alpha-farnesene biosynthesis and its regulation by ethylene.
synthesis
expression in Sacchaomyces cerevisiae leads to a titer of (-)-alpha-copaene of 9 mg/l at 48 h and around 7 mg/l in Eschrichia coli, and the titer of (+)-delta-cadinene is 6 mg/l in Saccharomyces cerevisiae and 3.5 mg/l in Escherichia coli