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Enzyme Nomenclature
Information on EC 1.14.20.13 - 6beta-hydroxyhyoscyamine epoxidase
for references in articles please use BRENDA:EC1.14.20.13
Word Map on EC 1.14.20.13
- 1.14.20.13
- scopolamine
-
alkaloid
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hyoscyamus
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tropane
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niger
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atropa
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hairy
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belladonna
-
rhizogenes
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anisodus
-
shoot
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datura
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2-oxoglutarate-dependent
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agrobacterium
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1.14.11.11
- pharmacology
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solanaceous
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nicotiana
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bioconversion
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muticus
- synthesis
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anticholinergic
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The enzyme appears in viruses and cellular organisms
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EC NUMBER 
COMMENTARY 
1.14.20.13
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RECOMMENDED NAME
GeneOntology No.
6beta-hydroxyhyoscyamine epoxidase
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
(6S)-6beta-hydroxyhyoscyamine + 2-oxoglutarate + O2 = scopolamine + succinate + CO2 + H2O
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(6S)-6beta-hydroxyhyoscyamine + 2-oxoglutarate + O2 = scopolamine + succinate + CO2 + H2O
proposed mechanism of epoxidation catalyzed by the enzyme, overview
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
oxidation
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redox reaction
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-
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reduction
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-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
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SYSTEMATIC NAME
IUBMB Comments
(6S)-6beta-hydroxyhyoscyamine,2-oxoglutarate oxidoreductase (epoxide-forming)
Requires Fe2+ and ascorbate.
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CAS REGISTRY NUMBER
COMMENTARY
121479-53-6
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
collected from Sari-Garmestan of Mazandaran province in the North of Iran, gene h6h
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
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the enzyme is a member of the 2-oxoglutarate-dependent dioxygenase family
metabolism
physiological function
additional information
metabolism
-
hyoscyamine 6beta-hydroxylase converts hyoscyamine to scopolamine in the last step of scopolamine biosynthetic pathway
metabolism
the enzyme is involved in biosynthesis pathway of tropane alkaloids catalyzing two oxidation steps to form scopolamine from hyoscyamine, overview, cf. EC 1.14.11.11; the enzyme is involved in biosynthesis pathway of tropane alkaloids catalyzing two oxidation steps to form scopolamine from hyoscyamine, overview, cf. EC 1.14.11.11
metabolism
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hyoscyamine 6beta-hydroxylase catalyzes two consecutive oxidation reactions. The first reaction is the hydroxylation of hyoscyamine to 6beta-hydroxyhyoscyamine, Ec 1.14.11.11, and the second is epoxidation of 6beta-hydroxyhyoscyamine yielding scopolamine, the final metabolite in the tropane alkaloid biosynthetic pathway
physiological function
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root cultures overexpressing the enzyme show remarkably elevated levels of scopolamine and anisodamine. Hyoscyamine 6beta-hydroxylases from both Scopolia lurida and Hyoscyamus niger promote anisodamine production at similar levels in Scopolia lurida root cultures. Hyoscyamus niger hyoscyamine 6beta-hydroxylase overexpressing root cultures have more scopolamine in them than Scopolia lurida hyoscyamine 6beta-hydroxylase-overexpressing root cultures
physiological function
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root cultures overexpressing the enzyme show remarkably elevated levels of scopolamine and anisodamine. Hyoscyamine 6beta-hydroxylases from both Scopolia lurida and Hyoscyamus niger promote anisodamine production at similar levels in Scopolia lurida root cultures. Hyoscyamus niger hyoscyamine 6beta-hydroxylase overexpressing root cultures have more scopolamine in them than Scopolia lurida hyoscyamine 6beta-hydroxylase-overexpressing root cultures
physiological function
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overexpression in hairy roots of Atropa baetica leeds to an altered alkaloid profile in which hyoscyamine is entirely converted into scopolamine. Scopolamine accumulation increases up to 9fold amounting to 5.6 mg per g dry weight
physiological function
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Agrobacterium-mediated expression in non-hyoscyamine-producing Nicotiana tabacum and hyoscyamine-producing Hyoscyamus muticus. Transgenic Nicotiana tabacum hairy roots show a more efficient uptake of hyoscyamine from the culture medium and a higher rate of bioconversion of hyoscyamine to scopolamine than those of Hyoscamus muticus. The secretion of scopolamine in Nicotiana tabacum hairy roots is up to 85% of the total scopolamine being released to the culture medium. Exogenous hyoscyamine also causes changes in nicotine alkaloid accumulation in Nicotiana tabacum hairy roots
additional information
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the nitrogen atom in the tropane ring of L-hyoscyamine plays an important role in substrate recognition. Proposed mechanism of epoxidation catalyzed by H6H, overview
additional information
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the transcript level of H6H increased under chromium treatment. This treatment also increases hyoscyamine and scopolamine contents, phenotype with decreased the growth parameters (weights and lengths of the plantlets) and chlorophyll contents and increased proline contents, overview
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
(6S)-hydroxyhyoscyamine + 2-oxoglutarate + O2
scopolamine + succinate + CO2
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-
-
?
3-hydroxy-3-phenylpropionyltropine + 2-oxoglutarate + O2
3-hydroxy-3-phenylpropionyl-6-hydroxytropine + succinate + CO2 + H2O
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-
-
-
?
6,7-dehydrohyoscyamine + 2-oxoglutarate + O2
scopolamine + succinate + CO2
-
-
-
-
?
8-methyl-8-azabicyclo[3.2.1]octan-3-yl 2-(4-(dimethylamino)phenyl)acetate + 2-oxoglutarate + O2
?
-
-
-
-
?
8-methyl-8-azabicyclo[3.2.1]octan-3-yl benzoate + 2-oxoglutarate + O2
?
-
-
-
-
?
apoatropine + 2-oxoglutarate + O2
6-hydroxyapoatropine + succinate + CO2 + H2O
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-
-
-
?
l-homatropine + 2-oxoglutarate + O2
6-hydroxyhomatropine + succinate + CO2 + H2O
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-
-
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?
norhyoscyamine + 2-oxoglutarate + O2
6-hydroxynorhyoscyamine + succinate + CO2 + H2O
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-
-
-
?
phenylacetyltropine + 2-oxoglutarate + O2
6-hydroxyphenylacetyltropine + succinate + CO2 + H2O
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-
-
-
?
t-cinnamoyltropine + 2-oxoglutarate + O2
t-cinnamoyl-6-hydroxytropine + succinate + CO2 + H2O
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-
-
-
?
(6S)-6-hydroxyhyoscyamine + 2-oxoglutarate + O2
scopolamine + succinate + CO2
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-
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?
(6S)-6-hydroxyhyoscyamine + 2-oxoglutarate + O2
scopolamine + succinate + CO2
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alkaloid metabolism, scopolamine is formed by oxidative transformation of hyoscyamine in several solanaceous species
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?
(6S)-6beta-hydroxyhyoscyamine + 2-oxoglutarate + O2
scopolamine + succinate + CO2
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-
-
?
(6S)-6beta-hydroxyhyoscyamine + 2-oxoglutarate + O2
scopolamine + succinate + CO2
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-
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-
?
(6S)-6beta-hydroxyhyoscyamine + 2-oxoglutarate + O2
scopolamine + succinate + CO2
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-
-
-
?
(6S)-6beta-hydroxyhyoscyamine + 2-oxoglutarate + O2
scopolamine + succinate + CO2
-
via 6,7-dehydrohyoscyamine or not, two possible pathways for the epoxidation in the biosynthesis of scopolamine, overview
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-
?
(6S)-6beta-hydroxyhyoscyamine + 2-oxoglutarate + O2
scopolamine + succinate + CO2 + H2O
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?
(6S)-6beta-hydroxyhyoscyamine + 2-oxoglutarate + O2
scopolamine + succinate + CO2 + H2O
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?
(6S)-6beta-hydroxyhyoscyamine + 2-oxoglutarate + O2
scopolamine + succinate + CO2 + H2O
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-
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?
additional information
?
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enzyme catalyzes both the hydroxylation of hyoscyamine, reaction of EC 1.14.11.11, and the epoxidation of 6beta-hydroxyhyoscyamine to generate scopolamine
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additional information
?
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hyoscyamine 6beta-hydroxylase, a 2-oxoglutarate-dependent dioxygenase, converts L-hyoscyamine to its 6,7-epoxy derivative, scopolamine, in two sequential steps, the enzyme also catalyzes the reaaction of EC 1.14.11.11. 6,7-Dehydrohyoscyamine, a potential precursor for the last step of epoxidation, is an obligatory intermediate in the biosynthesis of scopolamine
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additional information
?
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hyoscyamine 6beta-hydroxylase, a 2-oxoglutarate-dependent dioxygenase, converts L-hyoscyamine to its 6,7-epoxy derivative, scopolamine, in two sequential steps. The catalytic efficiency of AbH6H, especially for the second oxidation, is low. The epoxidation step is much slower than the hydroxylation step. Substrate analogues 1-methylpiperidin-4-yl 2-phenylacetate, 8-methyl-8-azabicyclo[3.2.1]octan-3-yl 3-phenylpropanoate, 8-oxabicyclo[3.2.1]octan-3-yl 2-phenylacetate, and 4 are no substrates for the enzyme
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additional information
?
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the enzyme also catalyzes the reaaction of EC 1.14.11.11
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additional information
?
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enzyme catalyzes both the hydroxylation of hyoscyamine, reaction of EC 1.14.11.11, and the epoxidation of 6beta-hydroxyhyoscyamine to generate scopolamine. Epoxidase activity is low compared to the hydroxylase activity. The binding of the substrates, hyoscyamine and 2-oxoglutarate, to the enzyme induces significant conformational changes
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additional information
?
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enzyme catalyzes both the hydroxylation of hyoscyamine, reaction of EC 1.14.11.11, and the epoxidation of 6beta-hydroxyhyoscyamine to generate scopolamine
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additional information
?
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enzyme is able to catalyze hydroxylation, dehydrogenation and in vitro epoxidation reactions all giving scopolamine. Hydroxyl rebound is unlikely to take place during the cyclization reaction and the hydroxylase versus oxidative cyclase activity is correlated with the presence of an exo-hydroxy group having syn-periplanar geometry with respect to the adjacent H atom to be abstracted
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
(6S)-6-hydroxyhyoscyamine + 2-oxoglutarate + O2
scopolamine + succinate + CO2
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alkaloid metabolism, scopolamine is formed by oxidative transformation of hyoscyamine in several solanaceous species
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?
(6S)-hydroxyhyoscyamine + 2-oxoglutarate + O2
scopolamine + succinate + CO2
A2IBI0
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-
-
?
6,7-dehydrohyoscyamine + 2-oxoglutarate + O2
scopolamine + succinate + CO2
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-
-
-
?
additional information
?
-
-
hyoscyamine 6beta-hydroxylase, a 2-oxoglutarate-dependent dioxygenase, converts L-hyoscyamine to its 6,7-epoxy derivative, scopolamine, in two sequential steps, the enzyme also catalyzes the reaaction of EC 1.14.11.11. 6,7-Dehydrohyoscyamine, a potential precursor for the last step of epoxidation, is an obligatory intermediate in the biosynthesis of scopolamine
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(6S)-6beta-hydroxyhyoscyamine + 2-oxoglutarate + O2
scopolamine + succinate + CO2
A2IBI0
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-
?
(6S)-6beta-hydroxyhyoscyamine + 2-oxoglutarate + O2
scopolamine + succinate + CO2
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-
-
-
?
(6S)-6beta-hydroxyhyoscyamine + 2-oxoglutarate + O2
scopolamine + succinate + CO2
-
-
-
-
?
(6S)-6beta-hydroxyhyoscyamine + 2-oxoglutarate + O2
scopolamine + succinate + CO2
-
via 6,7-dehydrohyoscyamine or not, two possible pathways for the epoxidation in the biosynthesis of scopolamine, overview
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?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Fe2+
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INHIBITORS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
(1R,2R,4S,5S,7s)-7-{[(2S)-3-hydroxy-2-phenylpropanoyl]oxy}-9,9-dimethyl-3-oxa-9-azoniatricyclo[3.3.1.02,4]nonane
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; slight inhibition by the substrate analogue
3,4-dihydroxybenzoate
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competitive inhibitor with respect to 2-oxoglutarate
8-methyl-8-azabicyclo[3.2.1]octan-3-yl 2-(4-(dimethylamino)phenyl)acetate
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Pyridine 2,4-dicarboxylate
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competitive inhibitor with respect to 2-oxoglutarate
additional information
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synthesis and inhibitory potencies of substrate analogues, overview. No inhibition by substrate analogues 1-methylpiperidin-4-yl 2-phenylacetate, 8-methyl-8-azabicyclo[3.2.1]octan-3-yl 3-phenylpropanoate, and 8-oxabicyclo[3.2.1]octan-3-yl 2-phenylacetate
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additional information
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no marked effect on enzyme activity by addition of NAD+, NADH, NADP+, NADPH, ATP + MgSO4, FAD, FMN, pyrroloquinoline quinone, acetyl-CoA, 6,7-dimethyl-5,6,7,8-tetrahydrofolate, phenazine methosulfate, 2,6-dichlorophenolindophenol, cytochrome c and H2O2
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
ascorbate
in absence of ascorbate, 14% of the activity in presence of 4 mM
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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pI VALUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
additional information
additional information
no expression in stem, leaf, pistil, petal, and sepal tissues
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LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
39238
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1 * 39238, recombinant enzyme, mass spectrometry, 1 * 39369, sequence calculation
39369
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1 * 39238, recombinant enzyme, mass spectrometry, 1 * 39369, sequence calculation
43000
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x * 64000, recombinant GST-tagged enzyme, SDS-PAGE, x * 43000, recombinant His-tagged enzyme, SDS-PAGE
64000
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x * 64000, recombinant GST-tagged enzyme, SDS-PAGE, x * 43000, recombinant His-tagged enzyme, SDS-PAGE
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SUBUNITS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
monomer
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1 * 39238, recombinant enzyme, mass spectrometry, 1 * 39369, sequence calculation
?
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x * 64000, recombinant GST-tagged enzyme, SDS-PAGE, x * 43000, recombinant His-tagged enzyme, SDS-PAGE
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GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
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OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20°C, purified recombinant enzyme, 20 mM phosphate, 2 months, loss of approximately 20% of activity
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4°C, purified recombinant enzyme, 20 mM phosphate, 4 days, loss of over 50% activity
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
recombinant N- or C-terminally His-tagged from Escherichia coli by nickel affinity chromatography
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recombinant N-terminally His- or GST-tagged enzyme from Escherichia coli strain BL21 (DE3) by nickel or glutathione affinity chromatography, respectively
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
DNA and amino acid sequence determination and analysis, overexpression of N- or C-terminally His-tagged enzyme in Escherichia coli strain BL21 (DE3), AbH6H loses of the first amino acid methionine during transcription
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DNA and amino acid sequence determination and analysis, overexpression of N-terminally His- or GST-tagged enzyme in Escherichia coli strain BL21 (DE3)
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expression in Escherichia coli
overexpression of the enzyme in hairy roots of transgenic Anisodus acutangulus using Agrobacterium tumefaciens strain C58C1 transfection method, coexpression with tropinone reductase I; overexpression of the enzyme in hairy roots of transgenic Anisodus acutangulus using Agrobacterium tumefaciens strain C58C1 transfection method, coexpression with tropinone reductase I
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
S14P/K97A
-
mutant displays 2.3fold improved epoxidation activity compared with the wild-type enzyme
additional information
enhancied production of tropane alkaloids in transgenic Anisodus acutangulus hairy root cultures by overexpressing tropinone reductase I and hyoscyamine-6beta-hydroxylase Agrobacterium-mediated gene transfer technology, quantification of tropanalkaloid contents in transgenic lines, overview; enhancied production of tropane alkaloids in transgenic Anisodus acutangulus hairy root cultures by overexpressing tropinone reductase I and hyoscyamine-6beta-hydroxylase, quantification of tropan alkaloid contents in transgenic lines, overview
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pharmacology
tropane alkaloids including hyoscyamine, anisodamine, scopolamine and anisodine, are used medicinally as anticholinergic agents with increasing market demand, improvement of production by metabolic engineering introduction of genes encoding the branch-controlling enzyme tropinone reductase I and the downstream rate-limiting enzyme hyoscyamine-6beta-hydroxylase; tropane alkaloids including hyoscyamine, anisodamine, scopolamine and anisodine, are used medicinally as anticholinergic agents with increasing market demand, improvement of production by metabolic engineering introduction of genes encoding the branch-controlling enzyme tropinone reductase I and the downstream rate-limiting enzyme hyoscyamine-6beta-hydroxylase
synthesis
synthesis
-
Escherichia coli cells harboring mutant S14P/K97A in a 5-l-bioreactor produce scopolamine via a single enzyme-mediated two-step transformation from 500 mg/l hyoscyamine in 97% conversion
synthesis
-
overexpression in hairy roots of Atropa baetica leeds to an altered alkaloid profile in which hyoscyamine is entirely converted into scopolamine. Scopolamine accumulation increases up to 9fold amounting to 5.6 mg per g dry weight
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LINKS TO OTHER DATABASES (specific for EC-Number 1.14.20.13)
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