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EC Tree
IUBMB Comments Part of a bifunctional enzyme involved in the biosynthesis of abietadiene. See also EC 5.5.1.12, copalyl diphosphate synthase. Requires Mg2+.
The taxonomic range for the selected organisms is: Abies grandis The enzyme appears in selected viruses and cellular organisms
Synonyms
pdabs, pttps-las,
more
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(-)-abietadiene synthase
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abietadiene cyclase
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cyclase, abietadiene
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(+)-copalyl diphosphate = abieta-7,13-diene + diphosphate
reactions leading from intermediate abieta-8(14)-en-13-yl cation to product are driven by the electrostatic effect of the ionized diphopshate group
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elimination of diphosphate
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cyclization
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cyclization
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bifunctional enzyme that catalyzes two sequential cyclization reactions (class I and clas II synthase reaction) in distinct active sites
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(+)-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+.
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(+)-copalyl diphosphate
abieta-7,13-diene + diphosphate
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?
(+)-copalyl-diphosphate
abieta-7,13-diene + abieta-8(14),13(15)-diene + abieta-8(14),12-diene + diphosphate
reaction intermediate is abieta-8(14)-en-13-yl cation
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?
geranylgeranyl diphosphate + diphosphate
(+)-copalyl diphosphate
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?
(+)-copalyl-diphosphate
(-)-abietadiene + diphosphate
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?
8alpha-hydroxy-17-nor copalyl diphosphate
17-normanoyl oxide + diphosphate
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?
copalyl diphosphate
abietadienes + diphosphate
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class I terpene synthase reaction
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ir
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(+)-copalyl diphosphate
abieta-7,13-diene + diphosphate
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?
(+)-copalyl-diphosphate
(-)-abietadiene + diphosphate
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?
copalyl diphosphate
abietadienes + diphosphate
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class I terpene synthase reaction
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ir
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K+
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100 mM included in assay medium
Mg2+
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10 mM selectively enables the conversion of copalyl diphosphate
Mg2+
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terpene synthases (TPS) require divalent metal ion co-factors, typically magnesium
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(13alpha)-N,13-dimethylpodocarp-8(14)-en-13-aminium chloride
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14,15-dihydro-15-aza-geranylgeranyl diphosphate
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14,15-dihydro-15-azageranylgeranyl diphosphate
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geranylgeranyl diphosphate
geranylgeranyl diphosphate
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geranylgeranyl diphosphate
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substrate inhibition
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0.0002 - 138
copalyl diphosphate
0.0002
copalyl diphosphate
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mutants D404N, R411A
0.0003
copalyl diphosphate
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mutants E773A, E778A
0.0003
copalyl diphosphate
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mutants D402A, D402N, D404E
0.00035
copalyl diphosphate
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0.0004
copalyl diphosphate
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D404A
0.0004
copalyl diphosphate
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wild-type, mutant D766A
0.0004
copalyl diphosphate
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mutants W358A, D361A, D404A, D405E
0.0005
copalyl diphosphate
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mutant N765A
0.0005
copalyl diphosphate
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mutants D402E, D405E, R454A, E499A Y520A
0.0005
copalyl diphosphate
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mutants rAS:K86A/R87A
0.0007
copalyl diphosphate
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mutants R762A, E699A, D621A
0.0007
copalyl diphosphate
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mutants ASDELTA:107-868
0.0007
copalyl diphosphate
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mutants D405N, R365A
0.0008
copalyl diphosphate
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mutants T848A, Y841F
0.0009
copalyl diphosphate
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mutant rAS:D96A
0.001
copalyl diphosphate
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mutants E589A, R584A
0.002
copalyl diphosphate
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mutants R586A, T769A
0.003
copalyl diphosphate
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mutant S721A
132
copalyl diphosphate
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138
copalyl diphosphate
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mutant D404A
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0.05 - 2
copalyl diphosphate
0.05
copalyl diphosphate
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mutant E589A
0.2
copalyl diphosphate
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mutant E778A
0.4
copalyl diphosphate
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mutant T617A
0.75
copalyl diphosphate
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2
copalyl diphosphate
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mutants D361A, D402A, D402E, D404N, D404A, D404E, D404N, D405A, D405E, D405N, D621A, E499A, R365A, R411A, R454A, W358A, Y520A
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8.7
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with copalyl diphosphate as substrate
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SwissProt
brenda
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brenda
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brenda
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TPSDV_ABIGR
868
0
99536
Swiss-Prot
Chloroplast (Reliability: 1 )
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monomer
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monomer
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1 * 80000, SDS-PAGE
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hanging drop vapor diffusion method, using 24% (v/v) PEG 8000, 0.1 M sodium citrate, pH 5.1, 0.1 M dibasic ammonium phosphate
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A723S
product is switched from abietadienes to 75% isopimara-7,15-diene + 21% sandaracopimaradiene
D621A
this substitution eliminates the subsequently acting class I activity
H348A/D621A
the mutant also produces 8alpha-hydroxy-copalyl diphosphate from geranylgeranyl diphosphate
N451A
this mutation reduces class II (protonation-initiated cyclization) activity about 100fold, with essentially no effect on class I (ionization-initiated cyclization) activity
D361A
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lower turnover with geranylgeranyl diphosphate than wild-type
D402A
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lower turnover with geranylgeranyl diphosphate than wild-type
D402E
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lower turnover with geranylgeranyl diphosphate than wild-type
D402N
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lower turnover with geranylgeranyl diphosphate than wild-type
D404E
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lower turnover with geranylgeranyl diphosphate than wild-type
D404N
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lower turnover with geranylgeranyl diphosphate than wild-type
D405A
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lower turnover with geranylgeranyl diphosphate than wild-type
D405E
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lower turnover with geranylgeranyl diphosphate than wild-type
D405N
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lower turnover with geranylgeranyl diphosphate than wild-type
D625A
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no effect on geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type
D766A
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no effect on geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type
D845A
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no effect on geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type
DELTA:107-868
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lower turnover with copalyl diphosphate than wild-type
DELTA:85-849
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no turnover with copalyl diphosphate
E499A
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lower turnover with geranylgeranyl diphosphate than wild-type
E589A
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no effect on geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type
E699A
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no effect on geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type
E773A
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no effect on geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type
E778A
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no effect on geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type
N765A
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no effect on geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type
R365A
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lower turnover with geranylgeranyl diphosphate than wild-type
R411A
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lower turnover with geranylgeranyl diphosphate than wild-type
R454A
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lower turnover with geranylgeranyl diphosphate than wild-type
R584A
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no effect on geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type
R586A
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no effect on geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type
R762A
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no effect on geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type
rAS:D96A
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nearly the same turnover with copalyl diphosphate like wild-type
rAS:K86A/R87A
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lower turnover with copalyl diphosphate than wild-type
S721A
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no effect on geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type
T617A
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no effect on geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type
T848A
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no effect on geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type
W358A
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lower turnover with geranylgeranyl diphosphate than wild-type
Y520A
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lower turnover with geranylgeranyl diphosphate than wild-type
Y841F
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no effect on geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type
D404A
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D404A
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unreactive with geranylgeranyl diphosphate
D621A
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D621A
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unreactive with (+)-copalyl diphosphate
D621A
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eliminates class I activity
T769A
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no effect on geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type
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-80°C, purified enzyme in 10 mM Bis-Tris, pH 6.8, 10% (v/v) glycerol,150 mM KCl, 10 mM MgCl2, and 5 mM dithiohtreitol, several months, without significant loss of activity
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Mono Q column chromatography and type II ceramic hydroxyapatite column chromatography
purification using Ni-NTA His-bind resin in batch mode
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expressed in Escherichia coli C41 OverExpress and B834(DE3) cells
expression in Escherichia coli
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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.
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Ravn, M.M.; Coates, R.M.; Flory, J.E.; Peters, R.J.; Croteau, R.
Stereochemistry of the cyclization-rearrangement of (+)-copalyl diphosphate to (-)-abietadiene catalyzed by recombinant abietadiene synthase from Abies grandis
Org. Lett.
2
573-576
2000
Abies grandis
brenda
Peters, R.J.; Flory, J.E.; Jetter, R.; Ravn, M.M.; Lee, H.J.; Coates, R.M.; Croteau, R.B.
Abietadiene synthase from grand fir (Abies grandis): characterization and mechanism of action of the "pseudomature" recombinant enzyme
Biochemistry
39
15592-15602
2000
Abies grandis
brenda
Peters, R.J.; Ravn, M.M.; Coates, R.M.; Croteau, R.B.
Bifunctional abietadiene synthase: Free diffusive transfer of the (+)-copalyl diphosphate intermediate between two distinct active sites
J. Am. Chem. Soc.
123
8974-8978
2001
Abies grandis
brenda
Peters, R.J.; Croteau, R.B.
Abietadiene synthase catalysis: conserved residues involved in protonation-initiated cyclization of geranylgeranyl diphosphate to (+)-copalyl diphosphate
Biochemistry
41
1836-1842
2002
Abies grandis
brenda
Peters, R.J.; Croteau, R.B.
Abietadiene synthase catalysis: mutational analysis of a prenyl diphosphate ionization-initiated cyclization and rearrangement
Proc. Natl. Acad. Sci. USA
99
580-584
2002
Abies grandis
brenda
Ravn, M.M.; Peters, R.J.; Coates, R.M.; Croteau, R.
Mechanism of abietadiene synthase catalysis: stereochemistry and stabilization of the cryptic pimarenyl carbocation intermediates
J. Am. Chem. Soc.
124
6998-7006
2002
Abies grandis
brenda
Peters, R.J.; Carter, O.A.; Zhang, Y.; Matthews, B.W.; Croteau, R.B.
Bifunctional abietadiene synthase: mutual structural dependence of the active sites for protonation-initiated and ionization-initiated cyclizations
Biochemistry
42
2700-2707
2003
Abies grandis
brenda
Vogel, B.S.; Wildung, M.R.; Vogel, G.; Croteau, R.
Abietadiene synthase from grand fir (Abies grandis). cDNA isolation, characterization, and bacterial expression of a bifunctional diterpene cyclase involved in resin acid biosynthesis
J. Biol. Chem.
271
23262-23268
1996
Abies grandis
brenda
Wilderman, P.R.; Peters, R.J.
A single residue switch converts abietadiene synthase into a pimaradiene specific cyclase
J. Am. Chem. Soc.
129
15736-15737
2007
Abies grandis (Q38710)
brenda
Zhou, K.; Peters, R.J.
Investigating the conservation pattern of a putative second terpene synthase divalent metal binding motif in plants
Phytochemistry
70
366-369
2009
Abies grandis
brenda
Zhou, K.; Gao, Y.; Hoy, J.A.; Mann, F.M.; Honzatko, R.B.; Peters, R.J.
Insights into diterpene cyclization from structure of bifunctional abietadiene synthase from Abies grandis
J. Biol. Chem.
287
6840-6850
2012
Abies grandis (Q38710)
brenda
Criswell, J.; Potter, K.; Shephard, F.; Beale, M.H.; Peters, R.J.
A single residue change leads to a hydroxylated product from the class II diterpene cyclization catalyzed by abietadiene synthase
Org. Lett.
14
5828-5831
2012
Abies grandis (Q38710)
brenda