Information on EC 4.2.3.132 - neoabietadiene synthase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
4.2.3.132
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RECOMMENDED NAME
GeneOntology No.
neoabietadiene synthase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
(+)-copalyl diphosphate = neoabietadiene + diphosphate
show the reaction diagram
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Biosynthesis of secondary metabolites
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Diterpenoid biosynthesis
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neoabietic acid biosynthesis
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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].
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
loblolly pine
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
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
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(+)-copalyl diphosphate
?
show the reaction diagram
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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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?
(+)-copalyl diphosphate
neoabietadiene + diphosphate
show the reaction diagram
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?
additional information
?
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Mg2+
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required for activity
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00035 - 0.002
(+)-copalyl diphosphate
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.75 - 2.6
(+)-copalyl diphosphate
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2000
(+)-copalyl diphosphate
Abies grandis
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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
2440
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.5
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calculated from amino acid sequence
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
97500
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x * 97500, calculated from amino acid sequence
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
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x * 97500, calculated from amino acid sequence
homotetramer
x-ray crystallography
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
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
Sephadex G-50 gel filtration
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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
expressed in Escherichia coli Codon plus BL21 RIL cells
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truncated abietadiene synthase is expressed in Escherichia coli BL21(DE3) cells
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D625A
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the mutant shows increased Km and reduced kcat values compared to the wild type enzyme
D766A
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the mutant shows increased Km and reduced kcat values compared to the wild type enzyme
D845A
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the mutant shows increased Km and reduced kcat values compared to the wild type enzyme
E589A
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the mutant shows increased Km and reduced kcat values compared to the wild type enzyme
E699A
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the mutant shows increased Km and reduced kcat values compared to the wild type enzyme
E773A
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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
N451A
the mutant shows reduced class I abietadiene synthase activity compared to the wild type enzyme
N765A
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the mutant shows increased Km and reduced kcat values compared to the wild type enzyme
R356A
the mutant shows reduced class I abietadiene synthase activity 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
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the mutant shows increased Km and reduced kcat values compared to the wild type enzyme
R762A
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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