Information on EC 4.2.3.24 - amorpha-4,11-diene synthase

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The expected taxonomic range for this enzyme is: Artemisia annua

EC NUMBER
COMMENTARY hide
4.2.3.24
-
RECOMMENDED NAME
GeneOntology No.
amorpha-4,11-diene synthase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
(2E,6E)-farnesyl diphosphate = amorpha-4,11-diene + diphosphate
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
isomerization
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
artemisinin biosynthesis
-
-
Biosynthesis of antibiotics
-
-
Biosynthesis of secondary metabolites
-
-
Sesquiterpenoid and triterpenoid biosynthesis
-
-
SYSTEMATIC NAME
IUBMB Comments
(2E,6E)-farnesyl diphosphate diphosphate-lyase (amorpha-4,11-diene-forming)
Requires Mg2+ and Mn2+ for activity. This is a key enzyme in the biosynthesis of the antimalarial endoperoxide artemisinin [3]. Catalyses the formation of both olefinic [e.g. amorpha-4,11-diene, amorpha-4,7(11)-diene, gamma-humulene and beta-sesquiphellandrene] and oxygenated (e.g. amorpha-4-en-7-ol) sesquiterpenes, with amorpha-4,11-diene being the major product. When geranyl diphosphate is used as a substrate, no monoterpenes are produced [2].
CAS REGISTRY NUMBER
COMMENTARY hide
259213-60-0
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
UniProt
Manually annotated by BRENDA team
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
amorpha-4,11-diene + diphosphate
show the reaction diagram
2-trans,6-trans-farnesyl diphosphate
amorpha-4,11-diene + diphosphate
show the reaction diagram
farnesyl diphosphate
amorpha-4,11-diene + diphosphate
show the reaction diagram
-
-
additionally production of 15 different sesquiterpenoids, detailed reaction scheme
-
?
farnesyl diphosphate
amorpha-4,7-diene + diphosphate
show the reaction diagram
Geranyl diphosphate
?
show the reaction diagram
-
-
-
-
-
geranyl diphosphate
alpha-terpineol + ocimene + myrcene + linalool
show the reaction diagram
-
alpha-terpineol (39%) + ocimene (24%) + myrcene (19%) + linalool (11%)
-
-
?
geranyl diphosphate + H2O
amorpha-4,11-diene + diphosphate
show the reaction diagram
-
substrate in the presence of Mn2+ but not with Mg2+ or Co2+
-
-
?
additional information
?
-
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
2-trans,6-trans-farnesyl diphosphate
amorpha-4,11-diene + diphosphate
show the reaction diagram
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Co2+
-
increase in product selectivity at pH 6.5, inhibitory above 0.5 mM
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Co2+
-
inhibitory above 0.5 mM
Cu2+
-
complete inhibition
Mn2+
-
inhibitory above 0.5 mM
Zn2+
-
complete inhibition
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0006 - 0.008
farnesyl diphosphate
0.0169 - 0.0282
geranyl diphosphate
additional information
additional information
-
the apparent Km value is 3.4 mmol/l
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00078 - 0.0154
(2E,6E)-farnesyl diphosphate
0.00034 - 0.0007
geranyl diphosphate
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.3 - 9.7
(2E,6E)-farnesyl diphosphate
0.012 - 0.042
geranyl diphosphate
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.000002
-
strain BL21DE3 pLysS Tuner, 6 g/l Inducer, sorbitol, betaine; strain BL21DE3 pLysS Tuner, no Inducer; strain BL21DE3 pLysS, without IPTG, sorbitol, betaine
0.000003
-
strain BL21DE3 pLysS Tuner, 1 g/l Inducer, sorbitol, betaine; strain BL21DE3 pLysS Tuner, 3 g/l Inducer, sorbitol, betaine; strain BL21DE3 pLysS Tuner, no Inducer, sorbitol, betaine
0.000008
-
strain BL21DE3 pLysS Tuner, 6 g/l Inducer; strain BL21DE3 pLysS Tuner, without IPTG; strain BL21DE3 pLysS Tuner, without IPTG, sorbitol, betaine
0.000013
-
strain BL21DE3 pLysS Tuner, 1 g/l Inducer; strain BL21DE3 pLysS Tuner, 3 g/l Inducer
0.00002
-
strain BL21DE3 pLysS, 6 g/l Inducer; strain BL21DE3 pLysS, no Inducer; strain BL21DE3 pLysS, no Inducer, sorbitol, betaine; strain BL21DE3 pLysS Tuner, 0.1 mM IPTG; strain BL21DE3 pLysS Tuner, 0.2 mM IPTG; strain BL21DE3 pLysS Tuner, 0.5 mM IPTG; strain BL21DE3 pLysS, without IPTG
0.00003
-
strain BL21DE3, 0.1 mM IPTG; strain BL21DE3, 0.2 mM IPTG; strain BL21DE3, 0.5 mM IPTG; strain BL21DE3, 1 g/l Inducer; strain BL21DE3, 3 g/l Inducer; strain BL21DE3, 6 g/l Inducer; strain BL21DE3 pLysS, 0.1 mM IPTG; strain BL21DE3 pLysS, 0.2 mM IPTG; strain BL21DE3 pLysS, 0.5 mM IPTG; strain BL21DE3 pLysS, 1 g/l Inducer; strain BL21DE3 pLysS, 1 g/l Inducer, sorbitol, betaine; strain BL21DE3 pLysS, 3 g/l Inducer; strain BL21DE3 pLysS, 3 g/l Inducer, sorbitol, betaine; strain BL21DE3 pLysS, 6 g/l Inducer, sorbitol, betaine; strain BL21DE3 Tuner, 0.1 mM IPTG; strain BL21DE3 Tuner, 0.2 mM IPTG; strain BL21DE3 Tuner, 0.5 mM IPTG; strain BL21DE3 Tuner, no Inducer; strain BL21DE3 Tuner, without IPTG
0.00005
-
strain BL21DE3, no Inducer; strain BL21DE3 Tuner, 1 g/l Inducer; strain BL21DE3 Tuner, 3 g/l Inducer; strain BL21DE3 Tuner, 6 g/l Inducer; strain BL21DE3, without IPTG
0.00006
-
strain BL21DE3, without IPTG, sorbitol, betaine
0.00008
-
strain BL21DE3 Tuner, without IPTG, sorbitol, betaine
0.0001
-
strain BL21DE3 Tuner, 1 g/l Inducer, sorbitol, betaine
0.00012
-
strain BL21DE3, 1 g/l Inducer, sorbitol, betaine; strain BL21DE3, no Inducer, sorbitol, betaine; strain BL21DE3 Tuner, 3 g/l Inducer, sorbitol, betaine
0.00013
-
strain BL21DE3, 3 g/l Inducer, sorbitol, betaine; strain BL21DE3 Tuner, 6 g/l Inducer, sorbitol, betaine; strain BL21DE3 Tuner, no Inducer, sorbitol, betaine
0.00015
-
strain BL21DE3, 0.2 mM IPTG, sorbitol, betaine; strain BL21DE3, 6 g/l Inducer, sorbitol, betaine
0.0002
-
strain BL21DE3, 0.1 mM IPTG, sorbitol, betaine; strain BL21DE3, 0.5 mM IPTG, sorbitol, betaine; strain BL21DE3 pLysS, 0.1 mM IPTG, sorbitol, betaine; strain BL21DE3 pLysS, 0.2 mM IPTG, sorbitol, betaine; strain BL21DE3 pLysS, 0.5 mM IPTG, sorbitol, betaine; strain BL21DE3 pLysS Tuner, 0.1 mM IPTG, sorbitol, betaine; strain BL21DE3 Tuner, 0.1 mM IPTG, sorbitol, betaine; strain BL21DE3 Tuner, 0.2 mM IPTG, sorbitol, betaine; strain BL21DE3 Tuner, 0.5 mM IPTG, sorbitol, betaine
0.00025
-
strain BL21DE3 pLysS Tuner, 0.2 mM IPTG, sorbitol, betaine; strain BL21DE3 pLysS Tuner, 0.5 mM IPTG, sorbitol, betaine
3.53
pH 7.5, 35C, recombinant enzyme
additional information
Strains transformed with the yeast-conform allele (ADSm) are more efficient in terms of production of amorpha-4,11-diene than those transformed with the plant gene. Production of amorpha-4,11-diene in the engineered yeasts containing ADSm gene is 10-20fold higher than the control
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.5 - 7
-
broad
7
-
activity assay
7.5 - 9
-
broad
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.5 - 7
-
-
7.5
-
pH-minimum, increasing enzyme activity with increasing pH above 7.5
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30
-
activity assay
37
-
activity assay
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.3
calculated
5.59
calculated
5.66
calculated
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
-
expressed in 10-40% of the glandular trichomes of mature leaf
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
56000
-
gel filtration
69300
-
detemined by SDS-PAGE, recombinant protein extended with 17 amino acids after fusion to the His-tag
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
sequence contains no plastidial targeting sequence
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
3D-modeling of structure. Predicted model shows that the putative ADS3963 protein contains 72% of alpha-helices, 23% beta-turns and 26% of random coils. Based on docking studies with substrate farnesyl diphosphate, the stretches Ala321-Ala324-Lys-398, Ala234-Val237-Phe283-Thr286-Tyr-287, Ser94-Met95-Trp141-Trp430-Asn434, Ser94-Arg96-Glu104-Leu107-Lys142-Lys431, Lys137-Arg143-Ile147-Ala150-Gln151-Leu478 and Ser218-Gly219-Tyr224-Arg228-Cys352-Met356-Aln450 may constitute to substrate binding
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purified by immobilized metal affinity chromatography on a column loaded with Co2+
-
purified by immobilized metal affinity chromatography on a column loaded with Ni2+
-
recombinant enzyme with his-tag
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
; expression in Escherichia coli with His-tag
-
Escherichia coli DH10B is used for cloning and strain DH1 is used for expression, developing and optimizing of a simple cloning system for expression of the amorphadiene biosynthetic pathway. The pathway genes are originally constructed by assembly of the operons onto three different plasmids, each with a different copy number and Escherichia coli promoter. Escherichia coli DH1 harboring pAM45MevT and pADS exhibits a significant increase in amorphadiene production (21 mg/l at 75 h), which is 3fold higher than the original base-strain DH1 pMevTpMBISpADS. To increase ADS expression, transforming of DH1pAM92 with pADS is done, resulting in a combined ADS gene dosage of 30-45 copies/cell. The strain exhibits an increase in amorphadiene production over DH1pAM92, but still does not approach the peak titers obtained with DH1pAM45pADS
-
expression in Escherichia coli
-
expression of a synthetic Artemesia annua amorphadiene synthase gene under control of a strong constitutive promoter ((p)gpdA) in Aspergillus nidulans yields altered product distribution. The transformants produces only small amounts of amorphadiene, but much larger amounts of similar sesquiterpenes normally produced as minor by-products in plants. Expression of ADS in Escherichia coli produces almost exclusively amorpha-4,11-diene. The host environment can greatly impact the terpenes produced from terpene synthases.
-
into the pET28 vector for expression in Escherichia coli BL21DE3, BL21DE3 Tuner, BL21DE3 pLysS and BL21DE3 pLysS Tuner cells using different inducing agents
-
into the pET30a+ vector for expression in Escherichia coli
-
into the pYeDP60 vector for expression in Saccharomyces cerevisiae, and, in addition, introduced into the yeast genome by homologous recombination
-
The ADS gene is mutated to the yeast-conform variant ADSm (based on the original DNA sequence of the wild-type gene, the plant gene codons are modified to Saccharomyces cerevisiae preferred with the intact protein primary structure, production in a transformed yeast.) The 8 low-usage codons, AGG (Arg), CGA (Arg), CGG (Arg), CGC (Arg), CCG (Pro), CTC (Leu), GCG (Ala) and ACG (Thr) in ADSm gene are changed with bias codons in Saccharomyces cerevisiae. The synonymous codons of 5 amino acids are changed into one preferred codon in ADSm gene, such as Gln (CAA), Glu (GAA), Cys (TGT), Arg (AGA) and Gly (GGT), which can improve the abundance of cognate tRNA when translated. Three genes, encoding the FPP synthase, HMG-CoA reductase and ADS, are plasmid-transformed together.
the open reading frame of amorpha-4,11-diene synthase is fused to the green fluorescent proten reporter gene for introducing into the Arabidopsis protoplasts
-
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
strongly induced by methyl jasmonate and chitosan
-
temporary peak in expression after salicylic acid treatment
-
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
protein is recovered from the inclusion body fraction
-
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
agriculture
drug development
medicine
-
amorpha-4,11-diene is a presursor of artemisinin, artemisinin and derivates are used for the treatment of malaria
pharmacology
-
amorpha-4,11-diene is a precursor of artemisinin, an important agent in the treatment of malaria, produced via oxidation
synthesis