Information on EC 1.14.99.22 - ecdysone 20-monooxygenase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
1.14.99.22
-
RECOMMENDED NAME
GeneOntology No.
ecdysone 20-monooxygenase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ecdysone + AH2 + O2 = 20-hydroxyecdysone + A + H2O
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
redox reaction
-
-
-
-
reduction
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
ecdysone and 20-hydroxyecdysone biosynthesis
-
-
Insect hormone biosynthesis
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-
SYSTEMATIC NAME
IUBMB Comments
ecdysone,hydrogen-donor:oxygen oxidoreductase (20-hydroxylating)
An enzyme from insect fat body or malpighian tubules involving a heme-thiolate protein (P-450). NADPH can act as ultimate hydrogen donor.
CAS REGISTRY NUMBER
COMMENTARY hide
55071-97-1
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
females
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
canton S strain
-
-
Manually annotated by BRENDA team
crayfish
-
-
Manually annotated by BRENDA team
Colorado potato beetle
UniProt
Manually annotated by BRENDA team
flesh-fly
-
-
Manually annotated by BRENDA team
Parasarcophaga argyrostoma
flesh-fly
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
cotton leafworm
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
physiological function
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
alpha-ecdysone + NADPH + O2
20-hydroxyecdysone + NADP+ + H2O
show the reaction diagram
ecdysone + AH2 + O2
20-hydroxyecdysone + A + H2O
show the reaction diagram
ecdysone + NADPH + H+ + O2
20-hydroxyecdysone + NADP+ + H2O
show the reaction diagram
ecdysone + NADPH + O2
20-hydroxyecdysone + NADP+ + H+ + H2O
show the reaction diagram
-
-
-
-
?
ecdysone + NADPH + O2
20-hydroxyecdysone + NADP+ + H2O
show the reaction diagram
ecdysone + reduced acceptor + O2
20-hydroxyecdysone + acceptor + H2O
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
ecdysone + AH2 + O2
20-hydroxyecdysone + A + H2O
show the reaction diagram
ecdysone + NADPH + H+ + O2
20-hydroxyecdysone + NADP+ + H2O
show the reaction diagram
-
-
-
-
?
ecdysone + NADPH + O2
20-hydroxyecdysone + NADP+ + H2O
show the reaction diagram
ecdysone + reduced acceptor + O2
20-hydroxyecdysone + acceptor + H2O
show the reaction diagram
V5K5L7
-
-
-
?
additional information
?
-
-
E20-M preferentially uses NADPH in catalyzing ecdysone conversion to the physiologically active molting hormone, 20-hydroxyecdysone
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-
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
cytochrome P450
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Ferredoxin
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1,4-anthraquinone
-
about 20% residual activity at 10 mM
-
1,9-Dideoxyforskolin
1H-[1,2,4]oxadizolo[4,3-a]-quinoxalin-1-one
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a guanylate cyclase inhibitor, applied with ecdysone agonist RH-5849 results in 63% reduced E20M activity
2-isopropoxy-1,4-naphthoquinone
2-methoxy-1,4-anthraquinone
-
about 18% residual activity at 1 mM
-
2-methoxy-1,4-naphthoquinone
2-propoxy-1,4-naphthoquinone
20-hydroxyecdysone
6-anilinoquinoline-5,6-quinone
-
LY-38583, a guanylate cyclase inhibitor, applied with ecdysone agonist RH-5849 results in 80% reduced E20M activity, addition of guanylate cyclase activator S levels the activity to 78% of control activity
7-O-Hemisuccinyl-7-deacetyl-forskolin
9-hydroxy-1,4-anthraquinone
-
about 40% residual activity at 10 mM
-
9-methoxy-1,4-anthraquinone
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about 45% residual activity at 10 mM
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diquat dibromide
ecdysterone
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competitive inhibition
forskolin
KCN
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25% inhibition
KK-42
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i.e. 1-benzyl-5[(E)-2,6-dimethyl-1,5-heptadienyl]imidazole
methylene blue
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a guanylate cyclase inhibitor, applied with ecdysone agonist RH-5849 results in 86% reduced E20M activity, reversed by addition of dibutyryl-cGMP/theophylline to 87% of control activity
Metyrapone
NADP+
-
competitive inhibition
Non-ionic detergents
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Tween 20, Tween 80 and Triton X-100
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oxidized cytochrome c
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reversed by KCN
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quinizarin
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about 60% residual activity at 10 mM
Tris/HCl-buffer
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-
Tween 80
additional information
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
20-hydroxyecdysone
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injection of 20-hydroxyecdysone stimulates ecdysone 20-hydroxylase activity in the midgut
9-methoxy-1,4-anthraquinone
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about 125% activity at 0.001 mM
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ATP
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increase of activity
dibutyryl-cGMP
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activates E20M activity by 50fold, diminished by injections of guanylate cyclase inhibitors alone or combined with ecdysone agonist RH-5849
Juvenile hormone
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L-dihydroxyphenylalanine
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-
octopamine
additional information
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00125
alpha-ecdysone
-
-
0.000000367 - 0.025
ecdysone
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.3
1,4-anthraquinone
Manduca sexta
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at pH 7.5 and 35C
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0.04
2-methoxy-1,4-anthraquinone
Manduca sexta
-
at pH 7.5 and 35C
-
1
9-hydroxy-1,4-anthraquinone
Manduca sexta
-
at pH 7.5 and 35C
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2
9-methoxy-1,4-anthraquinone
Manduca sexta
-
at pH 7.5 and 35C
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60
quinizarin
Manduca sexta
-
at pH 7.5 and 35C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.00000000617
Parasarcophaga argyrostoma
-
fat body
0.0000000103
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fat body
0.00000041
-
mid- and hindgut
0.00000075
-
microsomes, fat body
0.00000115
-
malphigian tubules
0.000003
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microsomes, malphigian tubules
0.00000562
-
malphigian tubules, 10000 x g pellet
0.0000197
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fat body
0.0000466
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mitochondrial enzyme system
0.0001721
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midgut
0.0021
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microsomes
27
-
recombinant GST-fusion protein, purified
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.8 - 8
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potassium phosphate buffer
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 8.5
-
-
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
additional information
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
60000
-
? * 60000, SDS-PAGE
77700
-
? * 77700, sequence determination
103000
-
? * 103000, SDS-PAGE, recombinant GST-fusion protein
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
bovine serum albumin stabilizes
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instable, linear activity under standard assay conditions for 10 min
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-30C, microsomal preparations with 30% v/v glycerol
-
-80C, quick frozen membranes, 15% glycerol v/v, at least 1 month
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4C to -20C storage for prolonged periods not possible due to unstability of the enzyme, appreciable loss of activity
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
not: purification of the enzyme from microsomes failed due to unstability of the enzyme
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recombinant GST-fusion protein
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solubilization of the enzyme from microsomes with detergent Synperonic NP10
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cDNA isolated from non-diapause eggs, expression in Escherichia coli BL21 as GST-fusion protein
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cloned CYP6H1 cDNA is probably identical with purified enzyme
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expressed in Sf9 insect cells
expression in S2 cells
gene SgShd, DNA and amino acid sequence determination and analysis
into a pSTBlue-1 Acceptor vector for sequencing, recombinant baculovirus harboring the BmE20OHase cDNA is constructed using the BAC-TO-BAC baculovirus expression system
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
ecdysone 20-hydroxylase expression is not induced by 20-hydroxyecdysone
-
L-dihydroxyphenylalanine and octopamine feeding increases ecdysone 20-monooxygenase activity by a factor of 1.6 and 1.7, respectively
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information