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Information on EC 4.2.3.19 - ent-kaurene synthase and Organism(s) Arabidopsis thaliana and UniProt Accession Q9SAK2

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EC Tree
     4 Lyases
         4.2 Carbon-oxygen lyases
             4.2.3 Acting on phosphates
                4.2.3.19 ent-kaurene synthase
IUBMB Comments
Part of a bifunctional enzyme involved in the biosynthesis of ent-kaurene. See also EC 5.5.1.13 (ent-copalyl diphosphate synthase)
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This record set is specific for:
Arabidopsis thaliana
UNIPROT: Q9SAK2
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Word Map
The taxonomic range for the selected organisms is: Arabidopsis thaliana
The expected taxonomic range for this enzyme is: Eukaryota, Bacteria
Synonyms
ent-kaurene synthase, ppcps/ks, kaurene synthase, cps/ks, ent-kaurene synthase b, ent-copalyl/ent-kaurene synthase, bifunctional cps/ks, ent-kaur-16-ene synthase, ent-copalyldiphosphate synthase/ent-kaurene synthase, copalyl diphosphate/kaurene synthase, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
ent-kaur-16-ene synthase
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ent-kaurene synthase
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ent-kaurene synthase B
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-
-
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ent-kaurene synthetase B
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-
-
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ent-copalyl diphosphate = ent-kaurene + diphosphate
show the reaction diagram
cyclization mechanism catalyzed by KSs and the Ile mutants, via ent-pimar-15-en-8-yl, ent-beyeran-16-yl, and ent-kauran-16-yl intermediates, detailed overview
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
cyclization
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-
-
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diphosphate lysis
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-
-
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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
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SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
ent-copalyl diphosphate
ent-kaur-16-ene + diphosphate
show the reaction diagram
-
-
-
?
ent-copalyl diphosphate
ent-kaurene + diphosphate
show the reaction diagram
-
-
-
?
8beta-hydroxy-ent-copalyl diphosphate
ent-13-epi-manoyl oxide + ent-kaurene + diphosphate
show the reaction diagram
-
-
-
-
?
ent-copalyl diphosphate
ent-kaurene + diphosphate
show the reaction diagram
additional information
?
-
GC-MS analysis of products, overview
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-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
ent-copalyl diphosphate
ent-kaur-16-ene + diphosphate
show the reaction diagram
-
-
-
?
ent-copalyl diphosphate
ent-kaurene + diphosphate
show the reaction diagram
-
-
-
?
ent-copalyl diphosphate
ent-kaurene + diphosphate
show the reaction diagram
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
16-aza-ent-beyerane
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16-aza-ent-trachylobane
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ent-beyeran-16-yl diphosphate
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
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
in the absence of diphosphate
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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activity in wild-type and trabsgenic KS-overexpressing plants
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.2
activity assay
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
23
activity assay at room temperature
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
production of ent-kaurene as a precursor for important signaling molecules such as the gibberellins seems to have arisen early in plant evolution, with corresponding cyclase(s) present in all land plants (i.e., embryophyta). The relevant enzymes seem to represent fusion of the class II diterpene cyclase that produces the intermediate ent-copalyl diphosphate (ent-CPP) and the subsequently acting class I diterpene synthase that produces ent-kaurene, although the bifunctionality of the ancestral gene is only retained in certain early diverging plants, with gene duplication and sub-functionalization leading to distinct ent-CPP synthases and ent-kaurene synthases (KSs) generally observed
malfunction
substitution of threonine for a conserved isoleucine has been shown to short-circuit the complex bicyclization and rearrangement reaction catalyzed by ent-kaurene synthases (KSs) after initial cyclization, leading to predominant production of ent-pimaradiene, at least in KSs from angiosperms. This effect extends to KSs from earlier diverging plants (i.e., bryophytes), including a bifunctional/KS. A dramatic effect of this single residue switch on product outcome to electrostatic stabilization of the ent-pimarenyl carbocation intermediate formed upon initial cyclization by the hydroxyl introduced by threonine substitution is paralleled by similar effects from substitution of alanine
metabolism
the enzyme produces the diterpene ent-kaur-16-ene, which in vascular plants (i.e., tracheophytes) serves as an intermediate in biosynthesis of the gibberellin phytohormones. Production of ent-kaur-16-ene from the general diterpenoid precursor (E,E,E)-geranylgeranyl diphosphate (GGPP) proceeds via two distinct bicyclization reactions. The first is catalyzed by copalyl diphosphate synthases (CPSs, EC 5.5.1.13) that are representative of class II diterpene cyclases and produce ent-copalyl diphosphate. This is then subsequently further cyclized and rearranged by ent-kaurene synthases (KSs)
physiological function
the enzyme produces the diterpene ent-kaur-16-ene, which in vascular plants (i.e., tracheophytes) serves as an intermediate in biosynthesis of the gibberellin phytohormones
additional information
the hydroxyl group of the Thr638 side chain enables occasional addition of water
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION?
KSB_ARATH
785
0
89622
Swiss-Prot
Chloroplast (Reliability: 3)
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
I368A
the mutant produces ent-pimara-8(14),15-diene, ent-kaur-16-ene, 8alpha-hydroxy-ent-pimara-15-ene, and ent-pimara-7,15-diene from ent-copalyl diphosphate
I368S
the mutant produces ent-pimara-8(14),15-diene and 8alpha-hydroxy-ent-pimara-15-ene from ent-copalyl diphosphate
I368T
the mutant produces ent-pimara-8(14),15-diene from ent-copalyl diphosphate
I368V
the mutant produces ent-kaur-16-ene from ent-copalyl diphosphate
I638A
site-directed mutagenesis, the mutant shows altered product spectrum compared to the wild-type enzyme, the mutant produces a mixture of four products, pedominantly ent-pimara-8(14),15-diene, and a small amount of the double bond isomer ent-pimara-7,15-diene, and variable amounts of ent-kaur-16-ene, as well as substantial amounts of 8a-hydroxy-ent-pimar-15-ene
I638S
site-directed mutagenesis, the mutant shows altered product spectrum compared to the wild-type enzyme, the mutant produces a mixture of four products, pedominantly ent-pimara-8(14),15-diene, and a small amount of the double bond isomer ent-pimara-7,15-diene, and variable amounts of ent-kaur-16-ene, as well as substantial amounts of 8a-hydroxy-ent-pimar-15-ene
I638T
site-directed mutagenesis, the mutant shows altered product spectrum compared to the wild-type enzyme, the mutant produces a mixture of four products, pedominantly ent-pimara-8(14),15-diene, and small amounts of the double bond isomer ent-pimara-7,15-diene and 8a-hydroxy-ent-pimar-15-ene, and variable amounts of ent-kaur-16-ene
I638V
site-directed mutagenesis, the mutant shows unaltered product spectrum compared to the wild-type enzyme and produces just ent-kaur-16-ene
additional information
-
construction of transgenic Arabidopsis thaliana plants, using the Agrobacterium tumefaciens infection system, overexpressing KS leads to increased ent-kaurene production but not to an increase in bioactive gibberellins, no altered morphology or phenotype compared to wild-type plants
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
purified by affinity chromatography
recombinant wild-type and mutant enzymes from Escherichia coli strain C41(DE3)
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
as a GST-fusion protein for expression in Escherichia coli
expressed in Escherichia coli OverExpress C41 cells
sequence comparisons, recombinant expression of wild-type and mutant enzymes in Escherichia coli strain C41(DE3)
overexpression of CPS and KS in transgenic Arabidopsis thaliana plants, expression in Escherichia coli
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REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Fleet, C.M.; Yamaguchi, S.; Hanada, A.; Kawaide, H.; David, C.J.; Kamiya, Y.; Sun, T.P.
Overexpression of AtCPS and AtKS in Arabidopsis confers increased ent-kaurene production but no increase in bioactive gibberellins
Plant Physiol.
132
830-839
2003
Arabidopsis thaliana
Manually annotated by BRENDA team
Roy, A.; Roberts, F.G.; Wilderman, P.R.; Zhou, K.; Peters, R.J.; Coates, R.M.
16-Aza-ent-beyerane and 16-Aza-ent-trachylobane: potent mechanism-based inhibitors of recombinant ent-kaurene synthase from Arabidopsis thaliana
J. Am. Chem. Soc.
129
12453-12460
2007
Arabidopsis thaliana (Q9SAK2), Arabidopsis thaliana
Manually annotated by BRENDA team
Mafu, S.; Potter, K.C.; Hillwig, M.L.; Schulte, S.; Criswell, J.; Peters, R.J.
Efficient heterocyclisation by (di)terpene synthases
Chem. Commun. (Camb.)
51
13485-13487
2015
Arabidopsis thaliana, Fusarium fujikuroi (S0EA85), Fusarium fujikuroi CBS 195.34 / IMI 58289 / NRRL A-6831 (S0EA85), Oryza sativa, Physcomitrium patens, Physcomitrium patens (A5A8G0), Sinorhizobium fredii
Manually annotated by BRENDA team
Jia, M.; Peters, R.J.
Extending a single residue switch for abbreviating catalysis in plant ent-kaurene synthases
Front. Plant Sci.
7
1765
2016
Arabidopsis thaliana (Q9SAK2), Marchantia polymorpha subsp. ruderalis (A0A176VPW0), Oryza sativa Japonica Group (Q0JA82), Physcomitrium patens (A5A8G0), Picea glauca (D2XEB3), Selaginella moellendorffii (A0A077JGG8)
Manually annotated by BRENDA team