Information on EC 2.1.1.128 - (RS)-norcoclaurine 6-O-methyltransferase

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The enzyme appears in viruses and cellular organisms

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EC NUMBER
COMMENTARY hide
2.1.1.128
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RECOMMENDED NAME
GeneOntology No.
(RS)-norcoclaurine 6-O-methyltransferase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
S-adenosyl-L-methionine + (RS)-norcoclaurine = S-adenosyl-L-homocysteine + (RS)-coclaurine
show the reaction diagram
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
methyl group transfer
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
(S)-reticuline biosynthesis I
-
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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-adenosyl-L-methionine:(RS)-norcoclaurine 6-O-methyltransferase
The enzyme will also catalyse the 6-O-methylation of (RS)-norlaudanosoline to form 6-O-methyl-norlaudanosoline, but this alkaloid has not been found to occur in plants.
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CAS REGISTRY NUMBER
COMMENTARY hide
167398-06-3
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89700-33-4
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
-
-
Manually annotated by BRENDA team
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
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suppression of norcoclaurine 6-O-methyltransferase transcript levels significantly suppresses total alkaloid accumulation in opium poppy (73% compared to the controls). However, the relative abundance of morphine increased to 55% of the total alkaloid content. Suppression of the enzyme does not significantly affect (S)-3'-hydroxy-N-methylcoclaurine 1 or (S)-3'-hydroxy-N-methylcoclaurine 2 transcript levels
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SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
S-adenosyl-L-homocysteine + (S)-reticuline
S-adenosyl-L-methionine + 3'-hydroxy-N-methyl-(S)-coclaurine
show the reaction diagram
-
-
-
?
S-Adenosyl-L-methionine + (R)-norlaudanosoline
S-Adenosyl-L-homocysteine + 6-O-methylnorlaudanosoline + 7-O-methylnorlaudanosoline
show the reaction diagram
S-Adenosyl-L-methionine + (R,S)-2,3-dihydroxy-9,10-dimethoxytetrahydroprotoberberine
S-Adenosyl-L-homocysteine + ?
show the reaction diagram
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5% of the activity with (S)-norlaudanosoline
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-
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S-Adenosyl-L-methionine + (R,S)-4'-O-methylnorlaudanosoline
S-Adenosyl-L-homocysteine + norprotosinomenine
show the reaction diagram
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34% of the activity with (S)-norlaudanosoline
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S-Adenosyl-L-methionine + (R,S)-5'-O-methylnorlaudanosoline
S-Adenosyl-L-homocysteine + 6-O-methyllaudanosoline
show the reaction diagram
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81% of the activity with (S)-norlaudanosoline
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S-adenosyl-L-methionine + (RS)-norcoclaurine
S-adenosyl-L-homocysteine + (RS)-coclaurine
show the reaction diagram
S-adenosyl-L-methionine + (S)-norcoclaurine
S-adenosyl-L-homocysteine + (S)-coclaurine
show the reaction diagram
S-adenosyl-L-methionine + (S)-norlaudanosoline
S-adenosyl-L-homocysteine + 6-O-methylnorlaudanosoline
show the reaction diagram
S-adenosyl-L-methionine + (S)-norlaudanosoline
S-adenosyl-L-homocysteine + 6-O-methylnorlaudanosoline + 7-O-methylnorlaudanosoline
show the reaction diagram
S-Adenosyl-L-methionine + (S)-scoulerine
S-Adenosyl-L-homocysteine + jatrorrhizine
show the reaction diagram
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1% of the activity with (S)-norlaudanosoline
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S-Adenosyl-L-methionine + 2,3-dihydroxy-9,10-dimethoxyprotoberberine
S-Adenosyl-L-homocysteine + tetrahydrojatrorrhizine
show the reaction diagram
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7% of the activity with (S)-norlaudanosoline
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S-adenosyl-L-methionine + laudanosoline
?
show the reaction diagram
S-Adenosyl-L-methionine + laudanosoline
S-Adenosyl-L-homocysteine + tetrahydrocolumbamine
show the reaction diagram
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79% of the activity with (S)-norlaudanosoline
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S-Adenosyl-L-methionine + norcoclaurine
S-Adenosyl-L-homocysteine + ?
show the reaction diagram
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(S)-norcoclaurine and (R)-norcoclaurine
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-
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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-adenosyl-L-homocysteine + (S)-reticuline
S-adenosyl-L-methionine + 3'-hydroxy-N-methyl-(S)-coclaurine
show the reaction diagram
Q9LEL6
-
-
-
?
S-adenosyl-L-methionine + (RS)-norcoclaurine
S-adenosyl-L-homocysteine + (RS)-coclaurine
show the reaction diagram
S-adenosyl-L-methionine + (S)-norcoclaurine
S-adenosyl-L-homocysteine + (S)-coclaurine
show the reaction diagram
S-adenosyl-L-methionine + (S)-norlaudanosoline
S-adenosyl-L-homocysteine + 6-O-methylnorlaudanosoline
show the reaction diagram
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putative rate-limiting step enzymes in benzylisoquinoline alkaloid biosynthesis
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-
?
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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the enzyme requires divalent cations for activity
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INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
5,6-Dihydro-9,10-dimethoxybenzo[g]-1,3-benzodioxolo[5,6-a]quinolizium
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10 mM, 50% inhibition
Iodobenzoate
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S-adenosyl-L-homocysteine
additional information
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not inhibited by chloromercuribenzenesulfonate and iodoacetamide
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
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LITERATURE
IMAGE
WRKY1
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CjWRKY1 is a necessary regulator to control overall gene expression in berberine biosynthesis
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.1
(R,S)-4'-O-methylnorlaudanosoline
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0.3
(R,S)-laudanosoline
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0.2 - 2.23
(R,S)-norlaudanosoline
3.95
S-adenosyl-L-methionine
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0.05
S-adenosylmethionine
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with (S)-norlaudanosoline as cosubstrate
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
10
5,6-Dihydro-9,10-dimethoxybenzo[g]-1,3-benzodioxolo[5,6-a]quinolizium
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IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.18
S-adenosyl-L-homocysteine
Coptis japonica;
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using (S)-norlaudanosoline as cosubstrate, in 0.3 M Tris-HCl (pH 7.5), 25mM sodium ascorbate, at 30°C
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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-
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7 - 9.5
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about 50% of maximal activity at pH 7.0 and 9.5
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25
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culture condition for Coptis japonica
30
OMT enzyme assay
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SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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low enzyme level
Manually annotated by BRENDA team
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low enzyme level
Manually annotated by BRENDA team
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sieve elements of the phloem adjacent or proximal to laticifers
Manually annotated by BRENDA team
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constant enzyme level over 16 days of germination
Manually annotated by BRENDA team
additional information
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gene transcripts detected in all organs with highest levels in root and stem and lowest in leaf
Manually annotated by BRENDA team
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LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
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immunogold labelling studies show the strict association with electron dense regions of the peripheral cytoplasm of sieve elements
Manually annotated by BRENDA team
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PDB
SCOP
CATH
UNIPROT
ORGANISM
Thalictrum flavum subsp. glaucum;
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
38500
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x * 38500, immunoblotting
47000
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gel filtration
95000
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gel filtration
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SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30
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half-life: 8 h
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GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
complete loss of activity after freezing in 30% glycerol solution
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4°C, 50% loss of activity after 4 weeks
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Q-Sepharose column chromatography and Bio-Gel HTP column chromatography
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recombinant protein using His-tag
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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
expressed as His-tag fusion protein in Escherichia coli ER2566pLys S
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expressed in Escherichia coli
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expressed in Escherichia coli BL21(DE3) cells
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expressed in Eschscholzia californica; full-length cDNA of Coptis japonica 6OMT is cloned into the binary vector pBITXEl2 for introduction into Agrobacterium tumefaciens, for infection Eschscholzia californica seedlings are co-cultured with Agrobacterium tumefaciens; into the pET-21d vector for expression in Escherichia coli BL21DE3 cells
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
enzyme expression is upregulated in the high papaverine mutant pap1
the over-expression of regulatory factors AP2G, AN1-like, ERF2, GARP, MDB025 and WRKY1 increases the levels of codeinone reductase, (S-adenosyl-L-methionine:3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase) and (R,S)-norcoclaurine 6-O-methyltransferase transcripts by 10- to more than 100fold. The transcriptional activations translate into an enhancement of alkaloid production in opium poppy of up to at least 10fold
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine
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
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LINKS TO OTHER DATABASES (specific for EC-Number 2.1.1.128)
ExplorEnz
ExPASy
KEGG
MetaCyc
SABIO-RK
NCBI: PubMed, Protein, Nucleotide, Structure, Genome, OMIM
IUBMB Enzyme Nomenclature
PROSITE Database of protein families and domains
InterPro (database of protein families, domains and functional sites)