Information on EC 4.2.3.18 - abieta-7,13-diene synthase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY
4.2.3.18
-
RECOMMENDED NAME
GeneOntology No.
abieta-7,13-diene synthase
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
(+)-copalyl diphosphate = abieta-7,13-diene + diphosphate
show the reaction diagram
-
-
-
-
(+)-copalyl diphosphate = abieta-7,13-diene + diphosphate
show the reaction diagram
reactions leading from intermediate abieta-8(14)-en-13-yl cation to product are driven by the electrostatic effect of the ionized diphopshate group
Q38710
(+)-copalyl diphosphate = abieta-7,13-diene + diphosphate
show the reaction diagram
gas-phase and enzyme-free post-transition state direct dynamics simulation gas-phase reveal that abietadiene synthase must intervene in order to produce abietadiene selectively, steering this reaction to avoid the generation of a sevenmembered ring containing product which is formed in the enzyme-free reaction
-
(+)-copalyl diphosphate = abieta-7,13-diene + diphosphate
show the reaction diagram
reaction mechanism proceeds via water quenching of the abieta-8(14)-en-13-yl carbocation, resulting in 13-hydroxy-8(14)-abietene
-
REACTION TYPE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
cyclization
-
-
-
-
cyclization
-
bifunctional enzyme that catalyzes two sequential cyclization reactions (class I and clas II synthase reaction) in distinct active sites
elimination of diphosphate
-
-
-
-
PATHWAY
KEGG Link
MetaCyc Link
abietic acid biosynthesis
-
Biosynthesis of secondary metabolites
-
dehydroabietic acid biosynthesis
-
Diterpenoid biosynthesis
-
Metabolic pathways
-
SYSTEMATIC NAME
IUBMB Comments
(+)-copalyl-diphosphate diphosphate-lyase [cyclizing, abieta-7,13-diene-forming]
Part of a bifunctional enzyme involved in the biosynthesis of abietadiene. See also EC 5.5.1.12, copalyl diphosphate synthase. Requires Mg2+.
SYNONYMS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
(-)-abietadiene synthase
-
-
-
-
abietadiene cyclase
-
-
-
-
abietadiene synthase
-
-
abietadiene synthase
B9U4U8
-
abietadiene/levopimaradiene synthase
-
-
cyclase, abietadiene
-
-
-
-
PaLAS
-
-
PdABS
B9U4U8
-
PtTPS-LAS
-
-
TPS-LAS
Q50EK2
-
CAS REGISTRY NUMBER
COMMENTARY
157972-08-2
-
ORGANISM
COMMENTARY
LITERATURE
SEQUENCE CODE
SEQUENCE DB
SOURCE
bifunctional levopimaradiene/abietadiene synthase
SwissProt
Manually annotated by BRENDA team
bifunctional levopimaradiene/abietadiene synthase
-
-
Manually annotated by BRENDA team
bifunctional levopimaradiene/abietadiene synthase
-
-
Manually annotated by BRENDA team
; putative
B9U4U8
UniProt
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
enzyme-free system
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
physiological function
B9U4U8, -
involved in the 2-C-methyl-D-erythritol 4-phosphate pathway
SUBSTRATE
PRODUCT                      
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(+)-copalyl diphosphate
(+-)-13-hydroxy-8(14)-abietene
show the reaction diagram
-
-
initial product of the reaction, thermally unstable. Dehydration of the alcohol products of (+)-copalyl diphosphate, yielding the well established diterpene products levopimaradiene, abietadiene, neoabietadiene, and palustradiene, may occurr due to three conditions of the GC-MS analysis typically used for identification of diterpene synthase products: a hot injector, a hot oven temperature necessary for eluting the compounds, and the high temperature and high energy of the MS and its interface
-
-
(+)-copalyl diphosphate
abieta-7,13-diene + diphosphate
show the reaction diagram
-
-
products are levopimaradiene, abieta-7,13-diene, and sandaracopimaradiene
-
?
(+)-copalyl-diphosphate
(-)-abietadiene + diphosphate
show the reaction diagram
-
-
-
-
?
(+)-copalyl-diphosphate
(-)-abietadiene + levopimaradiene + neoabietadiene + palustradiene + diphosphate
show the reaction diagram
Q675L4
reaction intermediate is C8-sandaracopimaradienyl cation
bifunctional levopimaradiene/abietadiene synthase produces a mixture of diterpenes
-
?
(+)-copalyl-diphosphate
(-)-abietadiene + levopimaradiene + neoabietadiene + palustradiene + diphosphate
show the reaction diagram
-, Q50EK2
reaction intermediate is C8-sandaracopimaradienyl cation
bifunctional levopimaradiene/abietadiene synthase, about 47% levopimaradiene, 27% abietadiene, 22% neoabietadiene, 3% palustradiene
-
?
(+)-copalyl-diphosphate
abieta-7,13-diene + abieta-8(14),13(15)-diene + abieta-8(14),12-diene + diphosphate
show the reaction diagram
Q38710
reaction intermediate is abieta-8(14)-en-13-yl cation
-
-
?
8alpha-hydroxy-17-nor copalyl diphosphate
17-normanoyl oxide + diphosphate
show the reaction diagram
-
-
-
-
?
copalyl diphosphate
abietadienes + diphosphate
show the reaction diagram
-
class I terpene synthase reaction
-
-
ir
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
(+)-copalyl-diphosphate
(-)-abietadiene + diphosphate
show the reaction diagram
-
-
-
-
?
copalyl diphosphate
abietadienes + diphosphate
show the reaction diagram
-
class I terpene synthase reaction
-
-
ir
METALS and IONS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
K+
-
100 mM included in assay medium
Mg2+
-
10 mM selectively enables the conversion of copalyl diphosphate
Mg2+
-
terpene synthases (TPS) require divalent metal ion co-factors, typically magnesium
INHIBITORS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
14,15-dihydro-15-aza-geranylgeranyl diphosphate
-
-
14,15-dihydro-15-azageranylgeranyl diphosphate
-
-
geranylgeranyl diphosphate
-
substrate inhibition
geranylgeranyl diphosphate
-
-
norpimarenylamine
-
-
-
KM VALUE [mM]
KM VALUE [mM] Maximum
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.0002
-
copalyl diphosphate
-
mutants D404N, R411A
0.0003
-
copalyl diphosphate
-
mutants D402A, D402N, D404E
0.0003
-
copalyl diphosphate
-
mutants E773A, E778A
0.00035
-
copalyl diphosphate
-
-
0.0004
-
copalyl diphosphate
-
D404A
0.0004
-
copalyl diphosphate
-
mutants W358A, D361A, D404A, D405E
0.0004
-
copalyl diphosphate
-
wild-type, mutant D766A
0.0005
-
copalyl diphosphate
-
mutants D402E, D405E, R454A, E499A Y520A
0.0005
-
copalyl diphosphate
-
mutant N765A
0.0005
-
copalyl diphosphate
-
mutants rAS:K86A/R87A
0.0007
-
copalyl diphosphate
-
mutants D405N, R365A
0.0007
-
copalyl diphosphate
-
mutants R762A, E699A, D621A
0.0007
-
copalyl diphosphate
-
mutants ASDELTA:107-868
0.0008
-
copalyl diphosphate
-
mutants T848A, Y841F
0.0009
-
copalyl diphosphate
-
mutant rAS:D96A
0.001
-
copalyl diphosphate
-
mutants E589A, R584A
0.002
-
copalyl diphosphate
-
mutants R586A, T769A
0.003
-
copalyl diphosphate
-
mutant S721A
132
-
copalyl diphosphate
-
-
138
-
copalyl diphosphate
-
mutant D404A
TURNOVER NUMBER [1/s]
TURNOVER NUMBER MAXIMUM[1/s]
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.05
-
copalyl diphosphate
-
mutant E589A
0.2
-
copalyl diphosphate
-
mutant E778A
0.4
-
copalyl diphosphate
-
mutant T617A
0.75
-
copalyl diphosphate
-
-
2
-
copalyl diphosphate
-
mutants D361A, D402A, D402E, D404N, D404A, D404E, D404N, D405A, D405E, D405N, D621A, E499A, R365A, R411A, R454A, W358A, Y520A
pH OPTIMUM
pH MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
7.2
-
-
assay at
8.7
-
-
with copalyl diphosphate as substrate
TEMPERATURE OPTIMUM
TEMPERATURE OPTIMUM MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
30
-
-
assay at
pI VALUE
pI VALUE MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
5.57
-
B9U4U8, -
calculated
SOURCE TISSUE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
SOURCE
-
resin duct epithelial cells are the main site of diterpene biosynthesis in Sitka spruce, diterpenoid biosynthesis is induced in cortical resin duct epithelial cells early upon treatment with methyljasmonate, and immature developing traumatic resin duct epithelial cells produce levopimaradiene/abietadiene synthase
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
MOLECULAR WEIGHT MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
80000
-
-
gel filtration
90000
-
-
gel filtration
SUBUNITS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
?
B9U4U8, -
x * 97900, calculated
monomer
-
1 * 80000, SDS-PAGE
monomer
-
-
Crystallization/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
homology model of the second active site of enzyme based on the structure of 5-epiaristolochene synthase from Nicotiana tabacum, Protein Data Bank ID code 5EAT
Q675L4
Purification/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
purification using Ni-NTA His-bind resin in batch mode
-
Cloned/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
expression in Escherichia coli
-
Site-directed mutagenesis is carried out via PCR amplification with overlapping mutagenic primers, and the mutant genes verify by complete sequencing. The resulting wild type and mutant genes are then transferred via directional recombination to the T7-promoter and N-terminal 6his fusion expression vector pDEST17. Use of the pDEST17 vector results in a 25 amino acid residue linker between the 6 His-tag and the cloned protein.
-
transfection to Arabidopsis thaliana, expression in Escherichia coli mutants
B9U4U8, -
expression of green fluorescent fusion protein in tobacco cells; expression of PtTPS-LAS in pET100 vector
-, Q50EK2
EXPRESSION
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
after methyl jasmonate treatment initially suppressed; decrease of transcript levels until third day after treatment of trees with 1 mM methyl jasmonate
B9U4U8, -
induced by mechanical wounding; transcript levels 2- or 3fold higher in wood than in other tissues and organs, increase of transcript levels one day after mechanical wounding
B9U4U8, -
ENGINEERING
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
A723S
Q38710
product is switched from abietadienes to 75% isopimara-7,15-diene + 21% sandaracopimaradiene
D361A
-
lower turnover with geranylgeranyl diphosphate than wild-type
D402A
-
lower turnover with geranylgeranyl diphosphate than wild-type
D402E
-
lower turnover with geranylgeranyl diphosphate than wild-type
D402N
-
lower turnover with geranylgeranyl diphosphate than wild-type
D404A
-
unreactive with geranylgeranyl diphosphate
D404A
-
-
D404E
-
lower turnover with geranylgeranyl diphosphate than wild-type
D404N
-
lower turnover with geranylgeranyl diphosphate than wild-type
D405A
-
lower turnover with geranylgeranyl diphosphate than wild-type
D405E
-
lower turnover with geranylgeranyl diphosphate than wild-type
D405N
-
lower turnover with geranylgeranyl diphosphate than wild-type
D621A
-
unreactive with (+)-copalyl diphosphate
D621A
-
-
D621A
-
eliminates class I activity
D625A
-
no effect of geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type
D766A
-
no effect of geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type
D845A
-
no effect of geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type
DELTA:107-868
-
lower turnover with copalyl diphosphate than wild-type
DELTA:85-849
-
no turnover with copalyl diphosphate
E499A
-
lower turnover with geranylgeranyl diphosphate than wild-type
E589A
-
no effect of geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type
E699A
-
no effect of geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type
E773A
-
no effect of geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type
E778A
-
no effect of geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type
N765A
-
no effect of geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type
R365A
-
lower turnover with geranylgeranyl diphosphate than wild-type
R411A
-
lower turnover with geranylgeranyl diphosphate than wild-type
R454A
-
lower turnover with geranylgeranyl diphosphate than wild-type
R584A
-
no effect of geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type
R586A
-
no effect of geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type
R762A
-
no effect of geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type
rAS:D96A
-
nearly the same turnover with copalyl diphosphate like wild-type
rAS:K86A/R87A
-
lower turnover with copalyl diphosphate than wild-type
S721A
-
no effect of geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type
T617A
-
no effect of geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type
T769A
-
no effect of geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type
T769A
-
is active
T769G
-
is active
T769S
-
is active
T848A
-
no effect of geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type
W358A
-
lower turnover with geranylgeranyl diphosphate than wild-type
Y520A
-
lower turnover with geranylgeranyl diphosphate than wild-type
Y841F
-
no effect of geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type
A620T
-
130% of wild-type productivity. Products are 87% levopimaradiene, 11% abieta-7,13-diene, and 2% sandaracopimaradiene
A727S
-
inactive
A729G
-
2% of wild-type productivity. Product is 100% sandaracopimaradiene
C618N
-
20% of wild-type productivity. Products are 91% levopimaradiene, 9% abieta-7,13-diene, and traces of sandaracopimaradiene
E777A
-
inactive
G854T
-
140% of wild-type productivity. Products are 79% levopimaradiene, 14% abieta-7,13-diene, and 7% sandaracopimaradiene
I855L
-
70% of wild-type productivity. Products are 90% levopimaradiene, 7% abieta-7,13-diene, and 3% sandaracopimaradiene
K723S
-
180% of wild-type productivity. Products are 90% levopimaradiene, 7% abieta-7,13-diene, and 3% sandaracopimaradiene
L619F
-
150% of wild-type productivity. Products are 94% levopimaradiene, 6% abieta-7,13-diene, and traces of sandaracopimaradiene
L696Q
-
160% of wild-type productivity. Products are 86% levopimaradiene, 7% abieta-7,13-diene, and 7% sandaracopimaradiene
M593I
-
370% of wild-type productivity. Products are 83% levopimaradiene, 12% abieta-7,13-diene, and 5% sandaracopimaradiene
N769A
-
inactive
N838E
-
220% of wild-type productivity. Products are 92% levopimaradiene, 6% abieta-7,13-diene, and 2% sandaracopimaradiene
V731L
-
8% of wild-type productivity. Product is 100% levopimaradiene
Y700F
-
500% of wild-type productivity. Products are 80% levopimaradiene, 15% abieta-7,13-diene, and 5% sandaracopimaradiene
Y700H
-
40% of wild-type productivity. Products are 71% levopimaradiene, traces of abieta-7,13-diene, and 29% sandaracopimaradiene
Y700M
-
500% of wild-type productivity. Products are 80% levopimaradiene, 13% abieta-7,13-diene, and 7% sandaracopimaradiene
Y700W
-
400% of wild-type productivity. Products are 85% levopimaradiene, 7% abieta-7,13-diene, and 8% sandaracopimaradiene
A713S
Q675L4
products are isopimaradiene and sandaracopimaradiene
V717L
Q675L4
no change in product
W679L/Y686H/A713S/V717L
Q675L4
products are isopimaradiene and sandaracopimaradiene
Y686H
Q675L4
no change in product
Y686H/A713S
Q675L4
main products are isopimaradiene and sandaracopimaradiene
Y686H/A713S A713S
Q675L4
main products are isopimaradiene and sandaracopimaradiene
Y686H/A713S/V717L
Q675L4
products are isopimaradiene and sandaracopimaradiene
G651V
Q675L4
no change in product
additional information
Q675L4
swapping of residues 568–640 of isopimaradiene synhase to corresponding residues 560-632 of levopimaradiene/abietadiene synthase results in complete reversion of the product profiles of the two enzymes
APPLICATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
synthesis
-
increase of levopimaradiene synthesis in Escherichia coli by amplification of the flux toward isopentenyl diphosphate and dimethylallyl diphosphate precursors and reprogramming the rate-limiting downstream pathway by generating combinatorial mutations in geranylgeranyl diphosphate synthase and levopimaradiene synthase. The most productive pathway, combining precursor flux amplification and mutant synthases, confers approximately 2600fold increase in levopimaradiene levels. A maximum titer of approximately 700 mg/l is obtained by cultivation in a benchscale bioreactor