Information on EC 5.5.1.13 - ent-copalyl diphosphate synthase

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The expected taxonomic range for this enzyme is: Eukaryota, Bacteria

EC NUMBER
COMMENTARY hide
5.5.1.13
-
RECOMMENDED NAME
GeneOntology No.
ent-copalyl diphosphate synthase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
geranylgeranyl diphosphate = ent-copalyl diphosphate
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
intramolecular lyase
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Biosynthesis of secondary metabolites
-
-
diterpene phytoalexins precursors biosynthesis
-
-
Diterpenoid biosynthesis
-
-
ent-kaurene biosynthesis I
-
-
kauralexin biosynthesis
-
-
Metabolic pathways
-
-
diterpene phytoalexins precursors biosynthesis
-
-
SYSTEMATIC NAME
IUBMB Comments
ent-copalyl-diphosphate lyase (decyclizing)
Part of a bifunctional enzyme involved in the biosynthesis of kaurene. See also EC 4.2.3.19 (ent-kaurene synthase)
CAS REGISTRY NUMBER
COMMENTARY hide
9055-64-5
in Chemical Abstracts not distinguished from EC 4.2.3.19
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
-
-
Manually annotated by BRENDA team
strain USDA110
-
-
Manually annotated by BRENDA team
strain USDA110
-
-
Manually annotated by BRENDA team
pumpkin
-
-
Manually annotated by BRENDA team
sunflower
-
-
Manually annotated by BRENDA team
bifunctional ent-copalyl diphosphate synthase EC 5.5.1.13, and ent-kaurene synthase, EC 4.2.3.19; gene TPS
-
-
Manually annotated by BRENDA team
cultivar Grand Rapids
UniProt
Manually annotated by BRENDA team
wild cucumber
-
-
Manually annotated by BRENDA team
from mycelia
SwissProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
hybrid white spruce
-
-
Manually annotated by BRENDA team
pea, Torsdag cultivar
-
-
Manually annotated by BRENDA team
68 TPS genes
-
-
Manually annotated by BRENDA team
18 TPS genes
-
-
Manually annotated by BRENDA team
48 TPS genes
-
-
Manually annotated by BRENDA team
strain Lu949
UniProt
Manually annotated by BRENDA team
strain Lu949
UniProt
Manually annotated by BRENDA team
strain KO-3988
-
-
Manually annotated by BRENDA team
strain KO-3988
-
-
Manually annotated by BRENDA team
152 TPS genes
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
malfunction
metabolism
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(E,E,E)-geranylgeranyl diphosphate
ent-copalyl diphosphate
show the reaction diagram
-
-
-
?
copalyl diphosphate
ent-16-alpha-hydroxy-kaurene + ent-kaurene
show the reaction diagram
-
bifunctional copalyl diphosphate synthase/ent-kaurene synthase
86% and 14%, resp.
-
?
copalyl diphosphate
ent-kaurene + diphosphate
show the reaction diagram
geranylgeranyl diphosphate
copalyl diphosphate
show the reaction diagram
geranylgeranyl diphosphate
ent-copalyl diphosphate
show the reaction diagram
geranylgeranyl diphosphate
ent-kaurene
show the reaction diagram
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
(E,E,E)-geranylgeranyl diphosphate
ent-copalyl diphosphate
show the reaction diagram
Q38802
-
-
-
?
copalyl diphosphate
ent-kaurene + diphosphate
show the reaction diagram
Q9UVY5
bifunctional enzyme
-
?
geranylgeranyl diphosphate
copalyl diphosphate
show the reaction diagram
geranylgeranyl diphosphate
ent-copalyl diphosphate
show the reaction diagram
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Co2+
-
or Zn2+, Mg2+, required. Activation in decreasing order: Mg2+, Zn2+, Co2+
Zn2+
-
or Mg2+, Co2+, required. Activation in decreasing order: Mg2+, Zn2+, Co2+
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2'-isopropyl-4'-(trimethylammonium chloride)-5'-methylphenyl-piperidine-1-carboxylate
-
AMO-1618, a quaternary ammonium salt, leads to total enzyme inhibition at 0.001 mM
5-isopropyl-N,N,N,2-tetramethyl-4-[(1-piperidinylcarbonyl)oxy]anilinium chloride
-
CPS1 activity exhibits approximately 70% inhibition by 0.1 mM Amo-1618, whereas the OsCPS2/OsCyc2 activity exhibits approximately 10% inhibition
AMO-1618
-
i.e. 2'-isopropyl-4'-(trimethylammoniumchloride)-5'-methylphenylpiperidine-1-carboxylate
Co2+
-
35-60% relative activity in the presence of 0.1 mM Co2+ compared to Mg2+
EDTA
-
inhibits activity in cell free extract containing the enzyme
Fe2+
-
5-10% relative activity in the presence of 0.1 mM Fe+ compared to Mg2+
geranylgeranyl diphosphate
Mg2+
-
weak inhibition of CPS2/Cyc2 at 5mM Mg2+
Ni2+
-
60-70% relative activity in the presence of 0.1 mM Ni2+ compared to Mg2+
Zn2+
-
CPS1 shows 30% enzymatic activity with 0.1 mM Zn2+ compared to Mg2+
additional information
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-mercaptoethanol
-
5 mM, required for full activity
Tween-80
-
0.1%, required for full activity
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0008 - 0.006
(E,E,E)-geranylgeranyl diphosphate
0.00023
copalyl diphosphate
-
at pH 8
0.00873 - 0.0169
geranylgeranyl diphosphate
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0011 - 0.9
(E,E,E)-geranylgeranyl diphosphate
0.00117
copalyl diphosphate
Phaeosphaeria sp.
-
at pH 8
0.0308 - 0.035
geranylgeranyl diphosphate
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.55 - 300
(E,E,E)-geranylgeranyl diphosphate
3768
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.00075
-
at pH 8
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 8
-
more than 90% of maximum activity within this range
6 - 8
-
-
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30 - 40
-
-
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
75000
-
sedimentation velocity
80900
-
CPS2/Cyc2, calculated from amino acid sequence
83000
-
gel filtration
84900
-
CPS1, calculated from amino acid sequence
92000
-
calculated from amino acid sequence
101000
-
x * 101000, calculated, x * 106000, SDs-PAGE of myc-fusion protein
106000
107000
calculated from amino acid sequence
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
-
x * 101000, calculated, x * 106000, SDs-PAGE of myc-fusion protein
monomer
-
gel filtration
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
enzyme complexed with (S)-15-aza-14,15-dihydrogeranylgeranyl thiolodiphosphate, vapor diffusion method, using 100 mM sodium citrate (pH 5.4), 30% (w/v) polyethylene glycol 400, 200 mM KH2PO4
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30
-
activity is fully retained after incubating at 30°C in 0.05 M Tris-HCl at pH 7.0 for 1 h
35
-
pH 5.5, stable for at least 1 h
ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
2-mercaptoethanol
-
5 mM 2-mercaptoethanol is required for full enzymatic activity
Glycerol
-
20% (v/v) glycerol is required for full enzymatic activity
Tween 80
-
0.1% (v/v) Tween 80 is required for full enzymatic activity
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cell free extract; recombinant enzyme, using Sepharose 4B glutathione affinity resin and SDS PAGE electrophoresis
-
HisTrap nickel-affinity column chromatography
Ni-NTA His-Bind resin column chromatography
partial, gradient centrifugation
-
partial, GST recombinant protein
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partial, using ammonium sulfate fractionation
partial, using ammonium sulfate fractionation, column chromatography on Sephadex G25
recombinant enzyme, using Sepharose 4B glutathione affinity resin
recombinant enzyme, using Sepharose 4B glutathione affinity resin and SDS PAGE electrophoresis
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using ammonium sulfate fractionation, Sephadex A-50 chromatography and PAGE electrophoresis
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli
expressed in Escherichia coli and Pichia pastoris
expressed in Escherichia coli BL21(DE3) cells
-
expressed in Escherichia coli C41 cells
expressed in Escherichia coli C41 OverExpress cells
expressed in Escherichia coli Top10 cells
-
expression as myc-fusion protein using Spodoptera frugiperda 21 insect cells. Sequence contains a DVDD motif responsible for copalyl diphosphate synthase activity and a DDYFD motif responsible for ent-kaurene synthase activity
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expression in Escherichia coli of a recombinant GST fused enzyme
gene TPS, genetic organization on the chromosome, genotyping and phylogenetic analysis, detailed overview
OsCPS1ent, expression in Escherichia coli BL21; OsCPS2ent, expression in Escherichia coli BL21
overexpression in Arabidopsis
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D503A
the mutant exhibits a 7fold reduction in kcat
E211A
the mutation results in a nearly 500fold reduction in kcat
N425A
the mutant exhibits a 13fold reduction in kcat
R340A
the mutant exhibits an 850fold reduction in kcat
T421A
the mutant exhibits a 163fold reduction in kcat (KM is increased 2fold)
T421S
the mutant exhibits a 3fold reduction in kcat
D320A
-
the mutation leads to complete enzyme activity
D656A
-
the mutation causes a small reduction in activity
A710C
-
no change in reaction product profile compared to wild-type; site-directed mutagenesis, the mutant shows unaltered activity and reaction product profile compared to the wild-type Jungermannia subulata JsCPS/KS
A710F
-
mutant converts geranylgeranyl diphosphate to ent-kaurene as the sole product; site-directed mutagenesis, the mutant protein shows the same enzymic activity as wild-type Jungermannia subulata JsCPS/KS
A710G
-
no change in reaction product profile compared to wild-type; site-directed mutagenesis, the mutant shows unaltered activity and reaction product profile compared to the wild-type Jungermannia subulata JsCPS/KS
A710L
-
the substitution changes the ratio of ent-kaurene and 16alpha-hydroxy-entkaurane produced. The production of ent-kaurene is increased to the same level as that of 16alpha-hydroxyent-kaurane
A710M
-
mutant converts geranylgeranyl diphosphate to ent-kaurene as the sole product; site-directed mutagenesis, the mutant protein shows the same enzymic activity as wild-type Jungermannia subulata JsCPS/KS
A710N
-
no change in reaction product profile compared to wild-type; site-directed mutagenesis, the mutant shows unaltered activity and reaction product profile compared to the wild-type Jungermannia subulata JsCPS/KS
A710S
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no change in reaction product profile compared to wild-type; site-directed mutagenesis, the mutant shows unaltered activity and reaction product profile compared to the wild-type Jungermannia subulata JsCPS/KS
C717A
-
site-directed mutagenesis, the mutant protein shows the same enzymic activity as wild-type Jungermannia subulata JsCPS/KS
additional information