Information on EC 1.14.13.119 - 5-epiaristolochene 1,3-dihydroxylase

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The expected taxonomic range for this enzyme is: Nicotiana tabacum

EC NUMBER
COMMENTARY hide
1.14.13.119
-
RECOMMENDED NAME
GeneOntology No.
5-epiaristolochene 1,3-dihydroxylase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
5-epiaristolochene + 2 NADPH + 2 H+ + 2 O2 = capsidiol + 2 NADP+ + 2 H2O
show the reaction diagram
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-
-
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydroxylation
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Sesquiterpenoid and triterpenoid biosynthesis
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SYSTEMATIC NAME
IUBMB Comments
5-epiaristolochene,NADPH:oxygen oxidoreductase (1- and 3-hydroxylating)
A heme-thiolate protein (P-450). Kinetic studies suggest that 1beta-hydroxyepiaristolochene is mainly formed first followed by hydroxylation at C-3. However the reverse order via 3alpha-hydroxyepiaristolochene does occur.
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
1-deoxycapsidiol + NADPH + H+ + O2
capsidiol + NADP+ + H2O
show the reaction diagram
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-
-
-
?
1beta-hydroxy-5-epiaristolochene + NADPH + H+ + O2
capsidiol + NADP+ + H2O
show the reaction diagram
-
the catalytic efficiency for 1beta-hydroxy-5-epiaristolochene is about 10times greater than that for 3alpha-hydroxy-5-epiaristolochene
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-
?
3-deoxycapsidiol + NADPH + H+ + O2
capsidiol + NADP+ + H2O
show the reaction diagram
-
-
-
-
?
3alpha-hydroxy-5-epiaristolochene + NADPH + H+ + O2
capsidiol + NADP+ + H2O
show the reaction diagram
-
the catalytic efficiency for 3alpha-hydroxy-5-epiaristolochene is about 10times lower than that for 1beta-hydroxy-5-epiaristolochene
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-
?
5-epiaristolochene + 2 NADPH + 2 H+ + 2 O2
3-deoxycapsidiol + 2 NADP+ + 2 H2O
show the reaction diagram
5-epiaristolochene + 2 NADPH + 2 H+ + 2 O2
capsidiol + 2 NADP+ + 2 H2O
show the reaction diagram
5-epiaristolochene + NADPH + H+ + O2
1-deoxycapsidiol + NADP+ + H2O
show the reaction diagram
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-
-
-
?
5-epiaristolochene + NADPH + H+ + O2
1beta-hydroxy-5-epiaristolochene + NADP+ + H2O
show the reaction diagram
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the release of an 1beta-hydroxy-5-epiaristolochene intermediate at high 5-epiaristolochene concentrations and a 10fold catalytic preference for 1beta-hydroxy-5-epiaristolochene versus 3alpha-hydroxy-5-epiaristolochene is indicative of a preferred reaction order of hydroxylation at C-1, followed by that at C-3
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-
?
premnaspirodiene + NADPH + H+ + O2
solavetivone + NADP+ + H2O
show the reaction diagram
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-
-
-
?
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
1-deoxycapsidiol + NADPH + H+ + O2
capsidiol + NADP+ + H2O
show the reaction diagram
-
-
-
-
?
3-deoxycapsidiol + NADPH + H+ + O2
capsidiol + NADP+ + H2O
show the reaction diagram
-
-
-
-
?
5-epiaristolochene + 2 NADPH + 2 H+ + 2 O2
3-deoxycapsidiol + 2 NADP+ + 2 H2O
show the reaction diagram
-
3-deoxycapsidiol is a reaction product when EAH is incubated with high concentrations (above 0.4 mM) of the 5-epiaristolochene substrate
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-
?
5-epiaristolochene + 2 NADPH + 2 H+ + 2 O2
capsidiol + 2 NADP+ + 2 H2O
show the reaction diagram
-
-
-
-
?
5-epiaristolochene + NADPH + H+ + O2
1-deoxycapsidiol + NADP+ + H2O
show the reaction diagram
-
-
-
-
?
premnaspirodiene + NADPH + H+ + O2
solavetivone + NADP+ + H2O
show the reaction diagram
-
-
-
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Ancymidol
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dose-dependent inhibition with more than 80% by 0.075 mM ancymidol
dimethyl sulfoxide
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conversion of 5-epiaristolochene to capsidiol activity is inhibited at concentrations above 10% (v/v) dimethyl sulfoxide
ketoconazole
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dose-dependent inhibition with 95% inhibition at 0.1 mM ketoconazole
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
dimethyl sulfoxide
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maximum conversion of 5-epiaristolochene to capsidiol activity is observed at final concentrations of 2-5% (v/v) dimethyl sulfoxide
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00174 - 0.02124
1beta-hydroxy-5-epiaristolochene
0.01518 - 0.0688
5-epiaristolochene
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.132 - 0.582
1beta-hydroxy-5-epiaristolochene
0.037 - 0.553
5-epiaristolochene
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
6.2 - 334.4
1beta-hydroxy-5-epiaristolochene
7909
2.4 - 33.1
5-epiaristolochene
2922
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.025
Ancymidol
Nicotiana tabacum
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pH and temperature not specified in the publication
0.025
ketoconazole
Nicotiana tabacum
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pH and temperature not specified in the publication
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in a WAT11 line of yeast
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expressed in WAT11 yeast strain
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
after an apparent lag phase of 8 h, a rapid induction of hydroxylase activity is observed 10 to 15 h after elicitor addition (cellulase or paraciticein) to the cell cultures, reaching a maximum by 18 h followed by a rather gradual decline of 10 to 20% over the next 8 h
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
I468A
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the mutant produces significant amounts 1beta-hydroxy-5-epiaristolochene, but negligible amounts of capsidiol from 5-epiaristolochene
S368A
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the mutant retains its ability to fully convert 5-epiaristolochene to capsidiol, but the turnover rates for capsidiol formation is 3-13times lower compared with that of the wild type enzyme and is also able to convert 1beta-hydroxy-5-epiaristolochene to capsidiol with kcat values comparable with that of wild type enzyme, but with Km values for 1beta-hydroxy-5-epiaristolochene 4-6times greater compared with that of wild type
S368C
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the mutant exhibits wild type catalytic efficiency for 1beta-hydroxy-5-epiaristolochene biosynthesis, but is devoid of the successive hydroxylation activity for capsidiol biosynthesis
S368F
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devoid of any hydroxylase activity for 5-epiaristolochene or 1beta-hydroxy-5-epiaristolochene
S368I
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devoid of any hydroxylase activity for 5-epiaristolochene or 1beta-hydroxy-5-epiaristolochene
S368T
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the mutant retains its ability to fully convert 5-epiaristolochene to capsidiol, but the turnover rates for capsidiol formation is 3-13times lower compared with that of the wild type enzyme and is also able to convert 1beta-hydroxy-5-epiaristolochene to capsidiol with kcat values comparable with that of wild type enzyme, but with Km values for 1beta-hydroxy-5-epiaristolochene 4-6times greater compared with that of wild type
S368V
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the mutant exhibits wild type catalytic efficiency for 1beta-hydroxy-5-epiaristolochene biosynthesis, but is devoid of the successive hydroxylation activity for capsidiol biosynthesis, the mutant catalyzes the relative equal biosynthesis of 1beta-hydroxy-5-epiaristolochene, 2beta-hydroxy-5-epiaristolochene, and 3beta-hydroxy-5-epiaristolochene from 5-epiaristolochene with wild type efficiency and converts about 1.5% of these monohydroxylated products to their respective ketone forms