Information on EC 4.2.3.19 - ent-kaurene synthase

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

EC NUMBER
COMMENTARY hide
4.2.3.19
-
RECOMMENDED NAME
GeneOntology No.
ent-kaurene synthase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ent-copalyl diphosphate = ent-kaurene + diphosphate
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
cyclization
diphosphate lysis
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Biosynthesis of secondary metabolites
-
-
Diterpenoid biosynthesis
-
-
ent-kaurene biosynthesis I
-
-
Metabolic pathways
-
-
SYSTEMATIC NAME
IUBMB Comments
ent-copalyl-diphosphate diphosphate-lyase (cyclizing, ent-kaurene-forming)
Part of a bifunctional enzyme involved in the biosynthesis of ent-kaurene. See also EC 5.5.1.13 (ent-copalyl diphosphate synthase)
CAS REGISTRY NUMBER
COMMENTARY hide
9055-64-5
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
strain USDA110
-
-
Manually annotated by BRENDA team
strain USDA110
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
bifunctional ent-copalyl diphosphate synthase EC 5.5.1.13, and ent-kaurene synthase, EC 4.2.3.19
-
-
Manually annotated by BRENDA team
strain L487
SwissProt
Manually annotated by BRENDA team
strain L487
SwissProt
Manually annotated by BRENDA team
Sitka spruce
UniProt
Manually annotated by BRENDA team
-
UniProt
Manually annotated by BRENDA team
strain Lu949
UniProt
Manually annotated by BRENDA team
strain Lu949
UniProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
physiological function
-
enzyme overexpression results in elevated gibberellic acid production
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
sandaracopimaradiene + diphosphate
show the reaction diagram
-
-
-
?
ent-copalyl diphosphate
16alpha-hydroxy-ent-kaurane + diphosphate
show the reaction diagram
ent-copalyl diphosphate
ent-kaurene + diphosphate
show the reaction diagram
geranylgeranyl diphosphate
ent-kaurene + diphosphate
show the reaction diagram
PpCPS-KS directly synthesizes the ent-kaurene skeleton from GGDP
-
-
?
syn-copalyl diphosphate
?
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
copalyl diphosphate
sandaracopimaradiene + diphosphate
show the reaction diagram
A0A077JGG8
-
-
-
?
ent-copalyl diphosphate
ent-kaurene + diphosphate
show the reaction diagram
syn-copalyl diphosphate
?
show the reaction diagram
A0A077JGG8
-
-
-
?
additional information
?
-
A0A077JGG8
the enzyme prefers normal and syn-copalyl diphosphate over ent-copalyl diphosphate
-
-
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
16-aza-ent-beyerane
-
16-aza-ent-trachylobane
-
2'-isopropyl-4'(trimethylammonium chloride)-5'-methylphenyl piperidine-1-carboxylate
2-(N,N-dimethyl-N-octylammonium bromide)-p-methan-1-ol
AMO-1618
copalyl diphosphate
deoxycholate
-
-
ent-beyeran-16-yl diphosphate
-
iodacetamide
-
-
N,N-dimethylaminoethyl 2,2-diphenylphenylether
N-ethylmaleimide
p-hydroxymercuribenzoate
Phosphone D
Phosphone S
-
sulfhydryl groups
-
-
additional information
-
wide variety of quaternary ammonium compounds with structural similarities to geranylgeranyl diphosphate tested for inhibition, substances shows significant effects on AB activity, but low effects on B activity
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-mercaptoethanol
-
-
dithiothreitol
additional information
-
expression of KS4, KS7, and KS8 is induced by elicitor treatment
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0004 - 0.001
copalyl diphosphate
0.0003
[15-3H]ent-copalyl diphosphate
pseudo-binding constant for [15-3H]ent-copalyl diphosphate with GST-recombinant kaurene synthase
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.000004 - 0.0001
16-aza-ent-beyerane
0.00002 - 0.001
16-aza-ent-trachylobane
0.01
ent-beyeran-16-yl diphosphate
Arabidopsis thaliana
Q9SAK2
in the absence of diphosphate
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.005
-
partially purified enzyme
0.01
-
purified enzyme
0.123
-
purified enzyme
additional information
-
activity in wild-type and trabsgenic KS-overexpressing plants
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.2 - 7.2
-
TES buffer
6.8 - 7.5
-
-
7.2
activity assay
7.2 - 8.2
-
Tris buffer
7.5
-
phosphate buffer
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
23
activity assay at room temperature
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
during embryo development, mRNA levels of KS3L peak at an early stage of zygotic embryogenesis
Manually annotated by BRENDA team
young leaf blade; young leaf blade
Manually annotated by BRENDA team
inflorescence meristem; inflorescence meristem
Manually annotated by BRENDA team
-
highest activity 3 days after imbibition
Manually annotated by BRENDA team
-
highest activity 4 days after imbibition
Manually annotated by BRENDA team
internodal stem; internodal stem
Manually annotated by BRENDA team
additional information
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
45000
-
gel filtration, molecular weight less than 45000 Da
83000
-
gel filtration
87993
x * 87993, calculated
88015
x * 88015, calculated
89000
-
molecular weight of native enzyme predicted from DNA sequence
90000
-
gel filtration
106000
copalyl diphosphate synthase-kaurene synthase fusion protein, determined by SDS-PAGE and Western blot analysis
107000
predicted from cDNA sequence
130000
-
SDS-PAGE, cloned fusion protein
131000
-
molecular weight of cloned fusion protein predicted from DNA sequence
132000
-
SDS-PAGE, cloned fusion protein
430000
-
sucrose density gradient centrifugation, crude enzyme
450000
-
sucrose density gradient centrifugation, purified enzyme
490000
-
gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
x * 87993, calculated; x * 88015, calculated
tetramer
4 * 107000
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
no activity in fresh crude extracts from seedlings for ent-kaurene formation from geranyl-geranyl diphosphate, activity detected after some days of storage in liquid nitrogen, little effect of liquid nitrogen storage on EC 4.2.3.19 activity
-
sensitive on freezing and thawing
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-180°C, no activity in fresh crude extracts from seedlings for ent-kaurene formation from geranyl-geranyl diphosphate, activity detected after some days of storage in liquid nitrogen, little effect of liquid nitrogen storage on EC 4.2.3.19 activity
-
-20°C, 25% glycerol, several months, no loss of activity for purified enzyme
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cloned enzyme from E. coli and native enzyme from endosperm
-
from E. coli
-
HisSpinTrap column chromatography
-
HisTrap nickel-affinity column chromatography
Ni-NTA column chromatography
partial, copurified with ent-kaurene synthase A (EC 5.5.1.13) activity
-
purified by affinity chromatography
separation of activities A and B achieved by hydrophobic interaction chromatography
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
as a GST-fusion protein for expression in Escherichia coli
DNA and amino acid sequence determination of genes KS1-9, chromosomal localizations, gene KS9 is a pseudogene, phylogenetic analysis, overview
-
expressed in Escherichia coli as fusion protein with maltose-binding protein, enzyme exclusively shows EC 4.2.3.19 activity
-
expressed in Escherichia coli BL21(DE3) cells
-
expressed in Escherichia coli BL21DE3-C41 cells
-
expressed in Escherichia coli C41 cells
expressed in Escherichia coli JM109 as glutathione-S-transferase fusion protein, enzyme shows EC 4.2.3.19 and EC 5.5.1.13 activity
-
expressed in Escherichia coli JM109, enzyme shows EC 4.2.3.19 and EC 5.5.1.13 activity
expressed in Escherichia coli M15(DE3) cells
expressed in Escherichia coli strain C41
-
expressed in Escherichia coli strain Top10F-
expressed in Escherichia coli Top10 cells
in Escherichia coli as glutathione-S-transferase fusion protein
-
into the pTD1 vector as a fusion of copalyl diphosphate synthase and ent-kaurene synthase for in vitro expression
overexpression of CPS and KS in transgenic Arabidopsis thaliana plants, expression in Escherichia coli
-
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
expression of isoform ks2L is lower than isoform ks3L; expression of isoform ks2L is lower than isoform ks3L
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D132A
-
low activity
D320A
-
low activity
D656A
-
no activity
A710C
no change in reaction product profile compared to wild-type
A710F
mutant converts geranylgeranyl diphosphate to ent-kaurene as the sole product
A710G
no change in reaction product profile compared to wild-type
A710M
mutant converted geranylgeranyl diphosphate to ent-kaurene as the sole product
A710N
no change in reaction product profile compared to wild-type
A710S
no change in reaction product profile compared to wild-type
D635A/D636A
mutation of the DDYFD motif in the kaurene synthase domain
additional information