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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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
oxidative cyclization
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redox reaction
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reduction
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
berberine biosynthesis
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chelerythrine 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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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
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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
cultivar K326
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
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
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-
-
-
?
(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
(S)-tetrahydropalmatine + O2
palmatine + H2O2
show the reaction diagram
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-
-
-
?
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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?
1-[(4-chlorophenyl)methyl]-2-ethyl-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline + O2
(1S)-1-[(4-chlorophenyl)methyl]-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline + (1R)-1-[(4-chlorophenyl)methyl]-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline + H2O2
show the reaction diagram
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-
-
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?
2-ethyl-6,7-dimethoxy-1-[(3-methoxyphenyl)methyl]-1,2,3,4-tetrahydroisoquinoline + O2
(1S)-6,7-dimethoxy-1-[(3-methoxyphenyl)methyl]-1,2,3,4-tetrahydroisoquinoline + (1R)-6,7-dimethoxy-1-[(3-methoxyphenyl)methyl]-1,2,3,4-tetrahydroisoquinoline + H2O2
show the reaction diagram
-
-
-
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?
2-ethyl-6,7-dimethoxy-1-[(4-methoxyphenyl)methyl]-1,2,3,4-tetrahydroisoquinoline + O2
(1S)-6,7-dimethoxy-1-[(4-methoxyphenyl)methyl]-1,2,3,4-tetrahydroisoquinoline + (1R)-2-ethyl-6,7-dimethoxy-1-[(4-methoxyphenyl)methyl]-1,2,3,4-tetrahydroisoquinoline + H2O2
show the reaction diagram
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-
-
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?
3-[(2-ethyl-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinolin-1-yl)methyl]phenol + O2
3-[[(1S)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinolin-1-yl]methyl]phenol + 3-[[(1R)-2-ethyl-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinolin-1-yl]methyl]phenol + H2O2
show the reaction diagram
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-
-
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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 + 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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?
4-coumaryl alcohol + O2
4-coumaryl aldehyde + H2O2
show the reaction diagram
-
-
-
?
6,7-dimethoxy-2-methyl-1,2,3,4-tetrahydroisoquinoline + H2O2
? + O2
show the reaction diagram
-
-
-
-
?
6-ethyl-5-[(4-methoxyphenyl)methyl]-5,6,7,8-tetrahydro-2H-[1,3]dioxolo[4,5-g]isoquinoline + O2
(5S)-5-[(4-methoxyphenyl)methyl]-5,6,7,8-tetrahydro-2H-[1,3]dioxolo[4,5-g]isoquinoline + (5R)-5-[(4-methoxyphenyl)methyl]-5,6,7,8-tetrahydro-2H-[1,3]dioxolo[4,5-g]isoquinoline + H2O2
show the reaction diagram
-
-
-
-
?
cannabigerolic-acid + O2
annabidiolic-acid + H2O2
show the reaction diagram
-
-
-
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?
cinnamyl alcohol + O2
cinnamyl aldehyde + H2O2
show the reaction diagram
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-
-
?
coniferyl alcohol + O2
coniferyl aldehyde + H2O2
show the reaction diagram
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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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?
sinapyl alcohol + O2
sinapyl aldehyde + H2O2
show the reaction diagram
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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
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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
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10.5
O2
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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
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wild-type, 25C, pH 9.0
37
O2
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wild-type, 25C, pH 9.0
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
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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
54000
Berberis beaniana
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1 * 54000, SDS-PAGE
59599
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x * 59599, calculated, including FAD cofactor
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
hanging drop vapor diffusion method, using 0.2 M sodium thiocyanate and 20% (w/v) PEG 3350; hanging drop vapor diffusion method, using 0.2 M sodium thiocyanate and 20% (w/v) PEG 3350
microbatch method, using 0.1 M HEPES buffer pH 7.0 containing 30% (v/v) Jeffamine ED 2001, pH 7.0
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
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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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Ni Sepharose column chromatography and Superdex 200 gel filtration
nickel-Sepharose 6 column chromatography and Superdex 200 gel filtration; nickel-Sepharose 6 column chromatography and Superdex 200 gel filtration
the secreted enzymes are purified by gel filtration and anion exchange chromatography
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
canadine-producing Saccharomyces cerevisiae strain harbors expression cassettes for seven heterologous enzymes: Papaper somniferum norcoclaurine 6-O-methyltransferase (Ps6OMT), Papaver somniferum 3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase 2 (Ps4'OMT), Papapver somniferum coclaurine N-methyltransferase (PsCNMT), Papaver somniferum berberine bridge enzyme (PsBBE), Thalictrum flavum scoulerine 9-O-methyltransferase (TfS9OMT), Thalictrum flavum canadine synthase (TfCAS), and Arabidopsis thaliana cytochrome P450 reductase 1 (CPR). The expression cassettes for the methyltransferases Ps6OMT, PsCNMT, and Ps4'OMT and the cytochrome P450 reductase CPR were chromosomally integrated, TfS9OMT and TfCAS are expressed from a high-copy plasmid, and PsBBE is expressed from a second high-copy plasmid
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expressed in Escherichia coli BL21
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expressed in Komagataella pastoris
expressed in Komagataella pastoris; expressed in Komagataella pastoris
expressed in Pichia pastoris strain GS115
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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
-
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
-
mutant, based on structural information, Tyr106 do not directly interact with the substrate, bicovalent flavin linkage is not affected by the mutation
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine
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a Saccharomyces cerevisiae strain is engineered to express seven heterologous enzymes (Papaper somniferum norcoclaurine 6-O-methyltransferase (Ps6OMT), Papaver somniferum 3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase 2 (Ps4'OMT), Papapver somniferum coclaurine N-methyltransferase (PsCNMT), Papaver somniferum berberine bridge enzyme (PsBBE), Thalictrum flavum scoulerine 9-O-methyltransferase (TfS9OMT), Thalictrum flavum canadine synthase (TfCAS), and Arabidopsis thaliana cytochrome P450 reductase 1 (CPR)), resulting in protoberberine alkaloid production from a simple benzylisoquinoline alkaloid precursor. A number of strategies are implemented to improve flux through the pathway, including enzyme variant screening, genetic copy number variation, and culture optimization. This leads to an over 70-fold increase in canadine titer up to 1.8 mg/l. Increased canadine titers enable extension of the pathway to produce berberine, a major constituent of several traditional medicines in a microbial host. This strain is viable at pilot scale
synthesis