BRENDA - Enzyme Database
show all sequences of 1.14.11.11

Efficient biosynthesis of rare natural product scopolamine using E. coli cells expressing a S14P/K97A mutant of hyoscyamine 6beta-hydroxylase AaH6H

Cao, Y.D.; He, Y.C.; Li, H.; Kai, G.Y.; Xu, J.H.; Yu, H.L.; J. Biotechnol. 211, 123-129 (2015)

Data extracted from this reference:

Cloned(Commentary)
Cloned (Commentary)
Organism
expression of wild-type and mutant enzymes in Escherichia coli
Anisodus acutangulus
Engineering
Protein Variants
Commentary
Organism
K97A
the shows a 3.3fold improved hydroxylation activity compared with the wild-type enzyme
Anisodus acutangulus
K97E
the shows a 2.7fold improved hydroxylation activity compared with the wild-type enzyme
Anisodus acutangulus
K97G
the shows a 1.7fold improved hydroxylation activity compared with the wild-type enzyme
Anisodus acutangulus
S14H
the shows a 2.4fold improved hydroxylation activity compared with the wild-type enzyme
Anisodus acutangulus
S14P
the shows a 3.7fold improved hydroxylation activity compared with the wild-type enzyme
Anisodus acutangulus
S14P/K97A
double mutant shows a 4.4fold improved hydroxylation activity compared with the wild-type enzyme, and the in vivo epoxidation activity is also improved by 2.3times. With this mutant, in a 5 l bioreactor with a working volume of 3 l, scopolamine is produced via a single-enzyme-mediated two-step transformation from hyoscyamine of 500 mg/l in 97% yield (1.068 g)
Anisodus acutangulus
KM Value [mM]
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
0.0348
-
L-hyoscyamine
pH 8.0, 35°C, mutant enzyme S14P/K97A
Anisodus acutangulus
0.0416
-
L-hyoscyamine
pH 8.0, 35°C
Anisodus acutangulus
Natural Substrates/ Products (Substrates)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
ID
L-hyoscyamine + 2-oxoglutarate + O2
Anisodus acutangulus
the enzyme catalyzes the hydroxylation of (-)-hyoscyamine and the subsequent epoxidation of 6beta-hydroxyhyoscyamine to form scopolamine, a natural alkaloid
(6S)-hydroxyhyoscyamine + succinate + CO2
-
-
?
Organism
Organism
UniProt
Commentary
Textmining
Anisodus acutangulus
A2IBI0
-
-
Substrates and Products (Substrate)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
Substrate Product ID
L-hyoscyamine + 2-oxoglutarate + O2
-
742949
Anisodus acutangulus
(6S)-hydroxyhyoscyamine + succinate + CO2
-
-
-
?
L-hyoscyamine + 2-oxoglutarate + O2
the enzyme catalyzes the hydroxylation of (-)-hyoscyamine and the subsequent epoxidation of 6beta-hydroxyhyoscyamine to form scopolamine, a natural alkaloid
742949
Anisodus acutangulus
(6S)-hydroxyhyoscyamine + succinate + CO2
-
-
-
?
Synonyms
Synonyms
Commentary
Organism
AaH6H
-
Anisodus acutangulus
hyoscyamine 6beta-hydroxylase
-
Anisodus acutangulus
Temperature Optimum [°C]
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
35
-
assay at
Anisodus acutangulus
Turnover Number [1/s]
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
1
-
L-hyoscyamine
pH 8.0, 35°C, wild-type enzyme
Anisodus acutangulus
3.9
-
L-hyoscyamine
pH 8.0, 35°C, mutant enzyme S14P/K97A
Anisodus acutangulus
pH Optimum
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
8
-
assay at
Anisodus acutangulus
Cloned(Commentary) (protein specific)
Commentary
Organism
expression of wild-type and mutant enzymes in Escherichia coli
Anisodus acutangulus
Engineering (protein specific)
Protein Variants
Commentary
Organism
K97A
the shows a 3.3fold improved hydroxylation activity compared with the wild-type enzyme
Anisodus acutangulus
K97E
the shows a 2.7fold improved hydroxylation activity compared with the wild-type enzyme
Anisodus acutangulus
K97G
the shows a 1.7fold improved hydroxylation activity compared with the wild-type enzyme
Anisodus acutangulus
S14H
the shows a 2.4fold improved hydroxylation activity compared with the wild-type enzyme
Anisodus acutangulus
S14P
the shows a 3.7fold improved hydroxylation activity compared with the wild-type enzyme
Anisodus acutangulus
S14P/K97A
double mutant shows a 4.4fold improved hydroxylation activity compared with the wild-type enzyme, and the in vivo epoxidation activity is also improved by 2.3times. With this mutant, in a 5 l bioreactor with a working volume of 3 l, scopolamine is produced via a single-enzyme-mediated two-step transformation from hyoscyamine of 500 mg/l in 97% yield (1.068 g)
Anisodus acutangulus
KM Value [mM] (protein specific)
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
0.0348
-
L-hyoscyamine
pH 8.0, 35°C, mutant enzyme S14P/K97A
Anisodus acutangulus
0.0416
-
L-hyoscyamine
pH 8.0, 35°C
Anisodus acutangulus
Natural Substrates/ Products (Substrates) (protein specific)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
ID
L-hyoscyamine + 2-oxoglutarate + O2
Anisodus acutangulus
the enzyme catalyzes the hydroxylation of (-)-hyoscyamine and the subsequent epoxidation of 6beta-hydroxyhyoscyamine to form scopolamine, a natural alkaloid
(6S)-hydroxyhyoscyamine + succinate + CO2
-
-
?
Substrates and Products (Substrate) (protein specific)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
ID
L-hyoscyamine + 2-oxoglutarate + O2
-
742949
Anisodus acutangulus
(6S)-hydroxyhyoscyamine + succinate + CO2
-
-
-
?
L-hyoscyamine + 2-oxoglutarate + O2
the enzyme catalyzes the hydroxylation of (-)-hyoscyamine and the subsequent epoxidation of 6beta-hydroxyhyoscyamine to form scopolamine, a natural alkaloid
742949
Anisodus acutangulus
(6S)-hydroxyhyoscyamine + succinate + CO2
-
-
-
?
Temperature Optimum [°C] (protein specific)
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
35
-
assay at
Anisodus acutangulus
Turnover Number [1/s] (protein specific)
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
1
-
L-hyoscyamine
pH 8.0, 35°C, wild-type enzyme
Anisodus acutangulus
3.9
-
L-hyoscyamine
pH 8.0, 35°C, mutant enzyme S14P/K97A
Anisodus acutangulus
pH Optimum (protein specific)
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
8
-
assay at
Anisodus acutangulus
General Information
General Information
Commentary
Organism
metabolism
the enzyme catalyzes the hydroxylation of (-)-hyoscyamine and the subsequent epoxidation of 6beta-hydroxyhyoscyamine to form scopolamine, a natural alkaloid
Anisodus acutangulus
General Information (protein specific)
General Information
Commentary
Organism
metabolism
the enzyme catalyzes the hydroxylation of (-)-hyoscyamine and the subsequent epoxidation of 6beta-hydroxyhyoscyamine to form scopolamine, a natural alkaloid
Anisodus acutangulus
KCat/KM [mM/s]
kcat/KM Value [1/mMs-1]
kcat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
24.04
-
L-hyoscyamine
pH 8.0, 35°C
Anisodus acutangulus
112.1
-
L-hyoscyamine
pH 8.0, 35°C, mutant enzyme S14P/K97A
Anisodus acutangulus
KCat/KM [mM/s] (protein specific)
KCat/KM Value [1/mMs-1]
KCat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
24.04
-
L-hyoscyamine
pH 8.0, 35°C
Anisodus acutangulus
112.1
-
L-hyoscyamine
pH 8.0, 35°C, mutant enzyme S14P/K97A
Anisodus acutangulus
Other publictions for EC 1.14.11.11
No.
1st author
Pub Med
title
organims
journal
volume
pages
year
Activating Compound
Application
Cloned(Commentary)
Crystallization (Commentary)
Engineering
General Stability
Inhibitors
KM Value [mM]
Localization
Metals/Ions
Molecular Weight [Da]
Natural Substrates/ Products (Substrates)
Organic Solvent Stability
Organism
Oxidation Stability
Posttranslational Modification
Purification (Commentary)
Reaction
Renatured (Commentary)
Source Tissue
Specific Activity [micromol/min/mg]
Storage Stability
Substrates and Products (Substrate)
Subunits
Synonyms
Temperature Optimum [°C]
Temperature Range [°C]
Temperature Stability [°C]
Turnover Number [1/s]
pH Optimum
pH Range
pH Stability
Cofactor
Ki Value [mM]
pI Value
IC50 Value
Activating Compound (protein specific)
Application (protein specific)
Cloned(Commentary) (protein specific)
Cofactor (protein specific)
Crystallization (Commentary) (protein specific)
Engineering (protein specific)
General Stability (protein specific)
IC50 Value (protein specific)
Inhibitors (protein specific)
Ki Value [mM] (protein specific)
KM Value [mM] (protein specific)
Localization (protein specific)
Metals/Ions (protein specific)
Molecular Weight [Da] (protein specific)
Natural Substrates/ Products (Substrates) (protein specific)
Organic Solvent Stability (protein specific)
Oxidation Stability (protein specific)
Posttranslational Modification (protein specific)
Purification (Commentary) (protein specific)
Renatured (Commentary) (protein specific)
Source Tissue (protein specific)
Specific Activity [micromol/min/mg] (protein specific)
Storage Stability (protein specific)
Substrates and Products (Substrate) (protein specific)
Subunits (protein specific)
Temperature Optimum [°C] (protein specific)
Temperature Range [°C] (protein specific)
Temperature Stability [°C] (protein specific)
Turnover Number [1/s] (protein specific)
pH Optimum (protein specific)
pH Range (protein specific)
pH Stability (protein specific)
pI Value (protein specific)
Expression
General Information
General Information (protein specific)
Expression (protein specific)
KCat/KM [mM/s]
KCat/KM [mM/s] (protein specific)
741836
Cardillo
-
Production of tropane alkaloi ...
Brugmansia x candida
Biochem. Eng. J.
125
180-189
2017
-
1
1
-
-
-
-
-
-
-
-
-
-
1
-
-
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1
1
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-
-
-
-
-
-
-
-
-
-
-
-
743509
Xia
Promoting scopolamine biosynt ...
Hyoscyamus niger
Plant Physiol. Biochem.
106
46-53
2016
-
-
1
-
-
-
-
-
-
-
-
-
-
5
-
-
-
-
-
-
-
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-
1
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1
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-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
742949
Cao
Efficient biosynthesis of rar ...
Anisodus acutangulus
J. Biotechnol.
211
123-129
2015
-
-
1
-
6
-
-
2
-
-
-
1
-
6
-
-
-
-
-
-
-
-
2
-
2
1
-
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2
1
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1
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6
-
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2
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1
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-
-
-
-
2
-
1
-
-
2
1
-
-
-
-
1
1
-
2
2
726207
Dehghan
An atypical pattern of accumul ...
Hyoscyamus senecionis
Plant Physiol. Biochem.
70
188-194
2013
-
-
1
-
-
-
-
-
-
-
1
1
-
7
-
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6
-
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2
1
2
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1
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1
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1
1
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6
-
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2
1
-
-
-
-
-
-
-
1
-
2
2
-
-
-
724556
Li
Functional characterization of ...
Atropa belladonna
Bioorg. Med. Chem.
20
4356-4363
2012
-
-
1
-
-
-
8
1
-
-
2
2
-
5
-
-
1
1
-
-
-
2
5
1
2
1
-
-
-
1
-
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-
1
-
-
-
-
-
9
-
1
-
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2
2
-
-
-
1
-
-
-
2
5
1
1
-
-
-
1
-
-
-
-
1
1
-
-
-
725946
Kai
Enhancing the production of tr ...
Anisodus acutangulus
Mol. Biosyst.
8
2883-2890
2012
-
1
1
-
1
-
-
-
-
-
-
1
-
6
-
-
-
-
-
1
-
-
2
-
2
-
-
-
-
-
-
-
-
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-
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1
1
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-
1
-
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1
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-
-
1
-
-
2
-
-
-
-
-
-
-
-
-
-
1
1
-
-
-
726202
Vakili
Chromium-induced tropane alkal ...
Atropa belladonna
Plant Physiol. Biochem.
52
98-103
2012
1
-
-
-
-
-
-
-
-
1
-
1
-
1
-
-
-
-
-
2
-
-
2
-
2
-
-
-
-
-
-
-
-
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-
-
1
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-
-
-
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1
-
1
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2
-
-
2
-
-
-
-
-
-
-
-
-
1
3
3
1
-
-
724637
Kai
Functional identification of h ...
Anisodus acutangulus
Biotechnol. Lett.
33
1361-1365
2011
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1
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2
1
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5
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1
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2
1
2
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1
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2
1
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1
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-
2
1
-
-
-
-
-
-
-
-
-
1
1
-
-
-
713300
Pramod
Biochemical and structural cha ...
Datura metel
Plant Physiol. Biochem.
48
966-970
2010
-
-
1
-
-
-
-
2
-
1
1
1
-
3
-
-
1
-
-
-
-
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2
1
2
1
-
-
-
1
-
-
-
-
-
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1
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-
-
-
-
2
-
1
1
1
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-
-
1
-
-
-
-
2
1
1
-
-
-
1
-
-
-
-
1
1
-
-
-
700788
Jaber-Vazdekis
Cloning, characterization and ...
Atropa baetica
Plant Physiol. Biochem.
47
20-25
2009
-
-
1
-
-
-
-
-
-
-
-
-
-
12
-
-
-
-
-
1
-
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2
-
2
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1
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1
-
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2
-
-
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-
-
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-
-
-
-
-
-
-
-
-
688836
Cardillo
Expression of Brugmansia candi ...
Brugmansia x candida
Microb. Cell Fact.
7
17
2008
-
-
1
-
1
-
-
-
-
-
-
-
-
1
-
-
-
-
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1
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1
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1
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-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
700783
Higa
Root tip-dependent, active rib ...
Hyoscyamus albus
Plant Physiol. Biochem.
46
452-460
2008
-
-
1
-
-
-
-
-
-
1
-
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-
1
-
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1
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1
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2
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1
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1
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1
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1
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-
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-
-
-
-
-
-
-
-
687475
Kai
Molecular cloning and characte ...
Anisodus acutangulus
J. Biochem. Mol. Biol.
40
715-722
2007
-
-
-
-
-
-
-
-
-
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1
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12
-
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4
-
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1
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1
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1
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4
-
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1
-
-
-
-
-
-
-
1
-
-
-
-
-
-
672740
Zarate
Tailoring tropane alkaloid acc ...
Hyoscyamus niger
Biotechnol. Lett.
28
1271-1277
2006
-
-
1
-
-
-
-
-
-
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1
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1
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1
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1
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1
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1
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-
-
-
-
-
-
-
-
-
673384
Rahman
-
Exogenous plant H6H but not ba ...
Hyoscyamus niger
Enzyme Microb. Technol.
39
1183-1189
2006
-
-
1
-
-
-
-
-
-
-
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1
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1
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2
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1
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1
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2
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-
660331
Liu
Molecular cloning, expression ...
Anisodus tanguticus
Planta Med.
71
249-253
2005
-
-
1
-
-
-
8
2
-
1
-
2
-
7
-
-
1
-
-
1
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5
-
1
1
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-
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1
1
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1
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8
-
2
-
1
-
2
-
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1
-
1
-
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5
-
1
-
-
-
1
1
-
-
-
-
-
-
-
-
675125
Haekkinen
Enhanced secretion of tropane ...
Hyoscyamus niger
J. Exp. Bot.
56
2611-2618
2005
-
-
1
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-
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13
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1
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1
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2
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1
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1
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1
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