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Information on EC 4.2.3.132 - neoabietadiene synthase and Organism(s) Abies grandis and UniProt Accession Q38710
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4.2.3.132 neoabietadiene synthaseIUBMB Comments
Isolated from Abies grandis (grand fir) . This class I enzyme forms about equal proportions of abietadiene, levopimaradiene and neoabietadiene. See also EC 4.2.3.18, abieta-7,13-diene synthase and EC 4.2.3.32, levopimaradiene synthase. An X-ray study of this multifunctional enzyme showed that the class I activity is in the alpha domain, while (+)-copalyl diphosphate synthase activity (EC 5.5.1.12, a class II activity) is in the beta and gamma domains . In Pinus taeda (loblolly pine) the major product is levopimaradiene, with less abietadiene and neoabietadiene .
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The taxonomic range for the selected organisms is: Abies grandis
The enzyme appears in selected viruses and cellular organisms
The enzyme appears in selected viruses and cellular organisms
Synonyms
abietadiene synthase, abietadiene/levopimaradiene synthase, AGAS, NAS, PtTPS-LAS, TPS-LAS, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
abietadiene synthase
AGAS
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PtTPS-LAS
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SYSTEMATIC NAME
IUBMB Comments
(+)-copaly-diphosphate diphosphate-lyase (cyclizing, neoabietadiene-forming)
Isolated from Abies grandis (grand fir) [1]. This class I enzyme forms about equal proportions of abietadiene, levopimaradiene and neoabietadiene. See also EC 4.2.3.18, abieta-7,13-diene synthase and EC 4.2.3.32, levopimaradiene synthase. An X-ray study of this multifunctional enzyme showed that the class I activity is in the alpha domain, while (+)-copalyl diphosphate synthase activity (EC 5.5.1.12, a class II activity) is in the beta and gamma domains [2]. In Pinus taeda (loblolly pine) the major product is levopimaradiene, with less abietadiene and neoabietadiene [3].
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
(+)-copalyl diphosphate
?
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the enzyme converts geranylgeranyl diphosphate and the intermediate (+)-copalyl diphosphate to a nearly equal mixture of abietadiene (31%), levopimaradiene (34%), and neoabietadiene (28%), as well as to three minor products pimara-8(14),15-diene (3%), palustradiene (2%), and sandaracopimaradiene (2%)
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additional information
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abietadiene synthase catalyzes the cyclization and rearrangement of (E,E,E)-geranylgeranyl diphosphate to a mixture of abietadiene double-bond isomers. The enzyme is bifunctional: Protonation across the terminal 14-15 double bond of (E,E,E)-geranylgeranyl diphosphate followed by bicyclization and deprotonation, produces the stable intermediate (+)-copalyl diphosphate (CPP). Then the enzyme ionizes CPP to promote cyclization to the tricyclic perhydrophenanthrene backbone. However, this cyclization is further coupled to a 1,2-methyl migration, by means of intramolecular proton transfer within a pimarenyl intermediate, to generate the C13 isopropyl group characteristic of the abietane skeleton. Finally, deprotonation of the resulting abietenyl carbocation at one of three alternative positions (C7, C12, or C15) leads to the three principal olefin products abieta-7(8),13(14)-diene (abietadiene), abieta-8(14),12(13)-diene (levopimaradiene), and abieta-8(14)-13(15)-diene (neoabietadiene), respectively, as a function of pH
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?
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.00035
(+)-copalyl diphosphate
-
in 50 mM HEPES, pH 7.2, 100 mM KCl, 7.5 mM MgCl2, 0.02 mM MnCl2, at 30°C
0.0007
(+)-copalyl diphosphate
mutant enzyme R356A, pH and temperature not specified in the publication
0.0014
(+)-copalyl diphosphate
mutant enzyme N451A, pH and temperature not specified in the publication
0.002
(+)-copalyl diphosphate
wild type enzyme, pH and temperature not specified in the publication
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.75
(+)-copalyl diphosphate
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in 50 mM HEPES, pH 7.2, 100 mM KCl, 7.5 mM MgCl2, 0.02 mM MnCl2, at 30°C
1.1
(+)-copalyl diphosphate
mutant enzyme R356A, pH and temperature not specified in the publication
1.9
(+)-copalyl diphosphate
mutant enzyme N451A, pH and temperature not specified in the publication
2.6
(+)-copalyl diphosphate
wild type enzyme, pH and temperature not specified in the publication
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
2000
(+)-copalyl diphosphate
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in 50 mM HEPES, pH 7.2, 100 mM KCl, 7.5 mM MgCl2, 0.02 mM MnCl2, at 30°C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
metabolism
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abietadiene synthase catalyzes the committed step in resin acid biosynthesis by catalyzing the cyclization and rearrangement of (E,E,E)-geranylgeranyl diphosphate to a mixture of abietadiene double-bond isomers. The enzyme is bifunctional: Protonation across the terminal 14-15 double bond of (E,E,E)-geranylgeranyl diphosphate followed by bicyclization and deprotonation, produces the stable intermediate (+)-copalyl diphosphate (CPP). Then the enzyme ionizes the CPP to promote cyclization to the tricyclic perhydrophenanthrene backbone. However, this cyclization is further coupled to a 1,2-methyl migration, by means of intramolecular proton transfer within a pimarenyl intermediate, to generate the C13 isopropyl group characteristic of the abietane skeleton. Finally, deprotonation of the resulting abietenyl carbocation at one of three alternative positions (C7, C12, or C15) leads to the three principal olefin products abieta-7(8),13(14)-diene (abietadiene), abieta-8(14),12(13)-diene (levopimaradiene), and abieta-8(14)-13(15)-diene (neoabietadiene), respectively
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). TPSDV_ABIGR
868
0
99536
Swiss-Prot
Chloroplast (Reliability: 1)
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
hanging drop vapor diffusion method, using 24% (w/v) PEG 8000, 0.1 M sodium citrate, pH 5.1, 0.1 M dibasic ammonium phosphate
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
D621A
the mutation selectively abrogates abietadiene synthase class I activity
N451A
the mutant shows reduced class I abietadiene synthase activity compared to the wild type enzyme
R356A
the mutant shows reduced class I abietadiene synthase activity compared to the wild type enzyme
D621A
-
the mutant shows increased Km and reduced kcat values compared to the wild type enzyme
D625A
-
the mutant shows increased Km and reduced kcat values compared to the wild type enzyme
D766A
-
the mutant shows increased Km and reduced kcat values compared to the wild type enzyme
D845A
-
the mutant shows increased Km and reduced kcat values compared to the wild type enzyme
E589A
-
the mutant shows increased Km and reduced kcat values compared to the wild type enzyme
E699A
-
the mutant shows increased Km and reduced kcat values compared to the wild type enzyme
E773A
-
the mutant shows decreased Km and reduced kcat values compared to the wild type enzyme
E778A
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the mutant shows decreased Km and reduced kcat values compared to the wild type enzyme
N765A
-
the mutant shows increased Km and reduced kcat values compared to the wild type enzyme
R584A
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the mutant shows increased Km and reduced kcat values compared to the wild type enzyme
R586A
-
the mutant shows increased Km and reduced kcat values compared to the wild type enzyme
R762A
-
the mutant shows increased Km and reduced kcat values compared to the wild type enzyme
S721A
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the mutant shows increased Km and reduced kcat values compared to the wild type enzyme
T617A
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the mutant shows increased Km and reduced kcat values compared to the wild type enzyme
T769A
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the mutant shows increased Km and reduced kcat values compared to the wild type enzyme
T848A
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the mutant shows increased Km and reduced kcat values compared to the wild type enzyme
Y841F
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the mutant shows increased Km and reduced kcat values compared to the wild type enzyme
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-80°C, 10 mM Bis-Tris, pH 6.8, 10% (v/v) glycerol, 150 mM KCl, 10 mM MgCl2, and 5 mM dithiothreitol, several months, without significant loss of activity
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
type II ceramic hydroxyapatite column chromatography and Mono Q column chromatography
type II ceramic hydroxyapatite column chromatography and POROS HQ/M column chromatography
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
DELTA84 pseudo-mature form of abietadiene synthase is expressed in Escherichia coli C41 and B834(DE3) cells
truncated abietadiene synthase is expressed in Escherichia coli BL21(DE3) cells
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REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
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
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
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