Information on EC 1.21.3.3 - reticuline oxidase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
1.21.3.3
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RECOMMENDED NAME
GeneOntology No.
reticuline oxidase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
(S)-reticuline + O2 = (S)-scoulerine + H2O2
show the reaction diagram
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-
-
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
oxidative cyclization
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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
berberine biosynthesis
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dehydroscoulerine biosynthesis
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noscapine biosynthesis
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sanguinarine and macarpine biosynthesis
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Isoquinoline alkaloid biosynthesis
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Metabolic pathways
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Biosynthesis of secondary metabolites
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SYSTEMATIC NAME
IUBMB Comments
(S)-reticuline:oxygen oxidoreductase (methylene-bridge-forming)
Contains FAD. The enzyme from the plant Eschscholtzia californica binds the cofactor covalently [3]. Acts on (S)-reticuline and related compounds, converting the N-methyl group into the methylene bridge ('berberine bridge') of (S)-tetrahydroprotoberberines. The product of the reaction, (S)-scoulerine, is a precursor of protopine, protoberberine and benzophenanthridine alkaloid biosynthesis in plants.
CAS REGISTRY NUMBER
COMMENTARY hide
152232-28-5
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
Berberis beaniana
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-
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
overview of occurence of BBE in Berberidaceae, Ranunculaceae, Menispermaceae, Papaveraceae and Fumariaceae
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Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(R,S)-6-O-methyllaudanosoline + O2
? + H2O2
show the reaction diagram
-
-
-
-
?
(R,S)-crassifoline + O2
? + H2O2
show the reaction diagram
-
-
-
-
?
(R,S)-laudanosoline + O2
? + H2O2
show the reaction diagram
(S)-coreximine + O2
(13aS)-2,11-dihydroxy-3,10-dimethoxy-5,8,13,13a-tetrahydroisoquinolino[3,2-a]isoquinolin-7-ium + H2O2
show the reaction diagram
-
-
-
-
?
(S)-laudanosine + H2O2
? + O2
show the reaction diagram
-
-
-
-
?
(S)-N-methylcoclaurine + O2
(S)-coclaurine + H2O2
show the reaction diagram
-
-
-
-
?
(S)-norsteponine + H2O2
? + O2
show the reaction diagram
-
-
-
-
?
(S)-protosinomenine + O2
? + H2O2
show the reaction diagram
(S)-reticuline + O2
(S)-scoulerine + H2O2
show the reaction diagram
(S)-reticuline + O2
dehydroscoulerine + H2O2
show the reaction diagram
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(S)-scoulerine is further oxidized to dehydroscoulerine in a second oxidation step
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-
?
(S)-scoulerine + H2O2
(S)-reticuline + O2
show the reaction diagram
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-
-
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r
1-(2-fluoro-3-hydroxybenzyl)-6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinolin-7-ol + O2
(13aS)-12-fluoro-3-methoxy-5,8,13,13a-tetrahydro-6H-isoquinolino[3,2-a]isoquinoline-2,11-diol + H2O
show the reaction diagram
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49% conversion, more than 99% of product (13aS)-12-fluoro-3-methoxy-5,8,13,13a-tetrahydro-6H-isoquinolino[3,2-a]isoquinoline-2,11-diol
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?
1-(2-fluoro-3-hydroxybenzyl)-7-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinolin-6-ol + O2
(13aS)-12-fluoro-2-methoxy-5,8,13,13a-tetrahydro-6H-isoquinolino[3,2-a]isoquinoline-3,11-diol + H2O
show the reaction diagram
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-
49% conversion, more than 99% of product (13aS)-12-fluoro-2-methoxy-5,8,13,13a-tetrahydro-6H-isoquinolino[3,2-a]isoquinoline-3,11-diol
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?
1-(3-hydroxy-4-methoxybenzyl)-7-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinolin-6-ol + O2
(13aS)-2,10-dimethoxy-5,8,13,13a-tetrahydro-6H-isoquinolino[3,2-a]isoquinoline-3,9-diol + isocoreximine + H2O2
show the reaction diagram
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53% conversion, ratio (13aS)-2,10-dimethoxy-5,8,13,13a-tetrahydro-6H-isoquinolino[3,2-a]isoquinoline-3,9-diol to isocoreximine is 98:2
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?
1-(3-hydroxybenzyl)-6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinolin-7-ol + O2
(13aS)-3-methoxy-5,8,13,13a-tetrahydro-6H-isoquinolino[3,2-a]isoquinoline-2,9-diol + (1R)-1-(3-hydroxybenzyl)-6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinolin-7-ol + H2O2
show the reaction diagram
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reaction leads to the (S)-enantiomer of the product and enantiomerically pure (R)-substrate. 22% yield of (13aS)-3-methoxy-5,8,13,13a-tetrahydro-6H-isoquinolino[3,2-a]isoquinoline-2,9-diol in more than 97% enantiomeric excess, 549% yield of + (1R)-1-(3-hydroxybenzyl)-6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinolin-7-olin more than 97% enantiomeric excess
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?
3-[(2-methyl-1,2,3,4-tetrahydroisoquinolin-1-yl)methyl]phenol + O2
(13aS)-5,8,13,13a-tetrahydro-6H-isoquinolino[3,2-a]isoquinolin-9-ol + 3-[[(1R)-2-methyl-1,2,3,4-tetrahydroisoquinolin-1-yl]methyl]phenol + H2O2
show the reaction diagram
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reaction leads to the (S)-enantiomer of the product and enantiomerically pure (R)-substrate. 46% yield of (13aS)-5,8,13,13a-tetrahydro-6H-isoquinolino[3,2-a]isoquinolin-9-ol in more than 97% enantiomeric excess, 49% yield of + 3-[[(1R)-2-methyl-1,2,3,4-tetrahydroisoquinolin-1-yl]methyl]phenol in more than 97% enantiomeric excess
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?
3-[(6,7-dimethoxy-2-methyl-1,2,3,4-tetrahydroisoquinolin-1-yl)methyl]-2-fluorophenol + O2
(13aS)-12-fluoro-2,3-dimethoxy-5,8,13,13a-tetrahydro-6H-isoquinolino[3,2-a]isoquinolin-11-ol + H2O
show the reaction diagram
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48% conversion, more than 99% of product (13aS)-12-fluoro-2,3-dimethoxy-5,8,13,13a-tetrahydro-6H-isoquinolino[3,2-a]isoquinolin-11-ol
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?
3-[(6,7-dimethoxy-2-methyl-1,2,3,4-tetrahydroisoquinolin-1-yl)methyl]phenol + O2
(13aS)-2,3-dimethoxy-5,8,13,13a-tetrahydro-6H-isoquinolino[3,2-a]isoquinolin-9-ol + 1_3-[[(1R)-6,7-dimethoxy-2-methyl-1,2,3,4-tetrahydroisoquinolin-1-yl]methyl]phenol + H2O2
show the reaction diagram
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reaction leads to the (S)-enantiomer of the product and enantiomerically pure (R)-substrate. 42% yield of (13aS)-2,3-dimethoxy-5,8,13,13a-tetrahydro-6H-isoquinolino[3,2-a]isoquinolin-9-ol in more than 97% enantiomeric excess, 50% yield of + 3-[[(1R)-6,7-dimethoxy-2-methyl-1,2,3,4-tetrahydroisoquinolin-1-yl]methyl]phenol in more than 97% enantiomeric excess
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?
6,7-dimethoxy-2-methyl-1,2,3,4-tetrahydroisoquinoline + H2O2
? + O2
show the reaction diagram
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-
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-
?
reticuline + O2
(S)-scoulerine + (S)-coreximine + H2O2
show the reaction diagram
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50% conversion, ratio (S)-scoulerine to (S)-coreximine is >99 to <1
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?
additional information
?
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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
(S)-reticuline + O2
(S)-scoulerine + H2O2
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(R)-norreticuline
Berberis beaniana
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50% inhibition at 0.02 mM
(S)-coreximine
Berberis beaniana
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50% inhibition at 0.2 mM
(S)-norreticuline
Berberis beaniana
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50% inhibition at 0.001 mM
(S)-scoulerine
Berberine
Berberis beaniana
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50% inhibition at 0.004 mM
diethyldithiocarbamate
Berberis beaniana
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50% inhibition at 0.4 mM
dithioerythritol
Berberis beaniana
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50% inhibition at 4 mM
H2O2
Berberis beaniana
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50% inhibition at 0.7 M
Jatrorrhizine
Berberis beaniana
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50% inhibition at 0.03 mM
Na2EDTA
Berberis beaniana
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50% inhibition at 6 mM
o-phenanthroline
Berberis beaniana
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50% inhibition at 0.006 mM
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
expression of protein is enhanced by treatment with Botrytis cinerea homogenate
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00014 - 0.003
(S)-reticuline
0.035
(S)-scoulerine
-
-
0.28
O2
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wild-type, 25C, pH 9.0
additional information
additional information
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steady-state kinetic analysis, and redox potentials for both wild type and C166A mutant enzyme are +132 mV and +53 mV, respectively, rapid reaction stopped-flow experiments, overview
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.054 - 103
(S)-reticuline
0.0025
(S)-scoulerine
Eschscholzia californica
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10.5
O2
Eschscholzia californica
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wild-type, 25C, pH 9.0
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
20000
(S)-reticuline
Eschscholzia californica
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wild-type, 25C, pH 9.0
2631
37
O2
Eschscholzia californica
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wild-type, 25C, pH 9.0
9
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8 - 11
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high conversion rates with this range
9 - 10
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30 - 50
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high conversion rates with this range
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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recombinant enzyme
Manually annotated by BRENDA team
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of seedling, localization in the sieve elelments
Manually annotated by BRENDA team
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gene transcripts are restricted to the protoderm of leaf primordia. Cell-type specific localization of protoberberine alkaloid biosynthesis and accumulation are temporally and spatially separated in roots and rhizomes, resp.
Manually annotated by BRENDA team
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sieve elements of root and hypocotyl, abundance of enzyme increases rapidly between 1 and 3 days of germination
Manually annotated by BRENDA team
additional information
-
not detectable in capsule and leaf
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
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vacuolar pH is below the functional range of BBE, it is active only before the entry into the vacuole, enters vacuole via a sorting determinant
Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
49000
Berberis beaniana
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
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x * 59599, calculated, including FAD cofactor
monomer
Berberis beaniana
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1 * 54000, SDS-PAGE
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
glycoprotein
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystal structure of the H174A variant shows significant structural rearrangements compared to wild-type enzyme. Residue H174 is part of a hydrogen bonding network that stabilizes the negative charge at the N1/C2=O locus via interaction with the hydroxyl group at C2 of the ribityl side chain of the flavin cofactor
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the crystal structure of Berberine bridge enzyme in complex with dehydroscoulerine is determined to 1.63 A resolution
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the crystal structures of berberine bridge enzyme in two different crystal forms, monoclinic and tetragonal, and in complex with (S)-reticuline are determined
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the crystal structures of the mutants H104A, C166A, C166A in complex with (S)-reticuline and of the wild-type enzyme in complex with (S)-scoulerine are determined
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ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
toluene
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reaction can be performed in 70% v/v toluene, allowing a substrate concentration of at least 20 g/l
additional information
-
enzyme tolerates a variety of organic solvents
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, 320 d, pH 7.4, 50% activity
Berberis beaniana
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25C, 18 d, pH 7.4, 50% activity
Berberis beaniana
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37C, 6 d, pH 7.4, 50% activity
Berberis beaniana
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4C, 150 d, pH 7.4, 50% activity
Berberis beaniana
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
-
Berberis beaniana
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expressed in Sf9 cells
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in a two step purification process
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the secreted enzymes are purified by gel filtration and anion exchange chromatography
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli BL21
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expressed in Spodoptera frugiperda (Sf9)
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expressed in Spodoptera frugiperda Sf9 cells
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expression in Pichia pastoris
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expression in Pichia pastoris strain KM71H
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for co-overexpression with disulfide isomerase in Pichia pastoris cells, for cloning of wild-type BBE, the mutant H104A and mutant H104A-C166A the vector pPICZalpha is used
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for expression in Pichia pastoris cells
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sense and antisense constructs expressed in root tissue
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
a transcriptomic approach discloses up-regulation of a BBE-like sequences in response to Penicillium digitatum infection which is confirmed by Northern blot and macroarray data
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
H104A
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mutant, lacking one of the covalent linkages to the cofactor FAD
H104T
-
no activity
H174A
-
mutation leads to substantial changes in all kinetic parameters and a decrease in midpoint potential. The crystal structure of the variant shows significant structural rearrangements compared to wild-type enzyme
H308S
-
5% activity of wild-type
H39G
-
40% activity of wild-type
H459A
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mutant, based on structural information, His459 do not directly interact with the substrate, bicovalent flavin linkage is not affected by the mutation
R100T
-
no activity
Y106F
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mutant, based on structural information, Tyr106 do not directly interact with the substrate, bicovalent flavin linkage is not affected by the mutation
additional information
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knockdown of berberine bridge enzyme by RNAi via Agrobacterium tumefaciens transfection leads to accumulation of (S)-reticuline and activates a silent pathway in cultured California poppy cells, they also produced a methylated derivative of reticuline, laudanine, which can scarcely be detected in control cells, analysis of reticuline metabolites, overview
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
synthesis
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substrate tuning by introducing a fluoro moiety at one potential reactive carbon center switches the reaction to the formation of exclusively one regioisomer with perfect enantioselectivity. The formation of 11-hydroxy-functionalized tetrahydroprotoberberines instead of the commonly formed 9-hydroxy-functionalized products from 1,2,3,4-tetrahydroisoquinolines can be successfully promoted