Information on EC 4.2.1.78 - (S)-norcoclaurine synthase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
4.2.1.78
-
RECOMMENDED NAME
GeneOntology No.
(S)-norcoclaurine synthase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
4-hydroxyphenylacetaldehyde + dopamine = (S)-norcoclaurine + H2O
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
condensation
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
(S)-reticuline biosynthesis I
-
-
Biosynthesis of secondary metabolites
-
-
Isoquinoline alkaloid biosynthesis
-
-
Metabolic pathways
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-
SYSTEMATIC NAME
IUBMB Comments
4-hydroxyphenylacetaldehyde hydro-lyase [adding dopamine; (S)-norcoclaurine-forming]
The reaction makes a six-membered ring by forming a bond between C-6 of the 3,4-dihydroxyphenyl group of the dopamine and C-1 of the aldehyde in the imine formed between the substrates. The product is the precursor of the benzylisoquinoline alkaloids in plants. The enzyme, formerly known as (S)-norlaudanosoline synthase, will also catalyse the reaction of 4-(2-aminoethyl)benzene-1,2-diol + (3,4-dihydroxyphenyl)acetaldehyde to form (S)-norlaudanosoline, but this alkaloid has not been found to occur in plants.
CAS REGISTRY NUMBER
COMMENTARY hide
389139-02-0
-
79122-01-3
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
ecotype Col-0
-
-
Manually annotated by BRENDA team
Berberis regeliana
-
-
-
Manually annotated by BRENDA team
Eschscholtzia tenuifolia
-
-
-
Manually annotated by BRENDA team
Eschscholzia pulchella
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
Glaucium rubrum
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
no activity in Catharanthus roseus
-
-
-
Manually annotated by BRENDA team
no activity in Nicotiana tabacum
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
Thalictrum flavum ssp. glaucum
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
metabolism
physiological function
-
key steps in the biotransformation consist of the oxidative decarboxylation of tyrosine by stoichiometric amounts of sodium hypochlorite in order to generate 4-hydroxyphenylacetadehyde, followed by the addition of enzyme and dopamine substrate in the presence of ascorbate, a necessary ingredient in order to avoid oxidation of the catechol moiety
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(2-fluorophenyl)acetaldehyde + dopamine
(1S)-1-[2-(2-fluorophenyl)ethyl]-1,2,3,4-tetrahydroisoquinoline-6,7-diol + H2O
show the reaction diagram
-
65% conversion after 3 h
-
-
?
(2-methylphenyl)acetaldehyde + dopamine
(1S)-1-[2-(2-methylphenyl)ethyl]-1,2,3,4-tetrahydroisoquinoline-6,7-diol + H2O
show the reaction diagram
-
66% conversion after 3 h
-
-
?
(3,4-dimethoxyphenyl)acetaldehyde + dopamine
(1S)-1-[2-(3,4-dimethoxyphenyl)ethyl]-1,2,3,4-tetrahydroisoquinoline-6,7-diol + H2O
show the reaction diagram
-
69% conversion after 3 h
-
-
?
(3-fluorophenyl)acetaldehyde + dopamine
(1S)-1-[2-(3-fluorophenyl)ethyl]-1,2,3,4-tetrahydroisoquinoline-6,7-diol + H2O
show the reaction diagram
-
66% conversion after 3 h
-
-
?
(3-methylphenyl)acetaldehyde + dopamine
(1S)-1-[2-(3-methylphenyl)ethyl]-1,2,3,4-tetrahydroisoquinoline-6,7-diol + H2O
show the reaction diagram
-
58% conversion after 3 h
-
-
?
(4-fluorophenyl)acetaldehyde + dopamine
(1S)-1-[2-(4-fluorophenyl)ethyl]-1,2,3,4-tetrahydroisoquinoline-6,7-diol + H2O
show the reaction diagram
-
71% conversion after 3 h
-
-
?
(4-methoxyphenyl)acetaldehyde + dopamine
(1S)-1-[2-(4-methoxyphenyl)ethyl]-1,2,3,4-tetrahydroisoquinoline-6,7-diol + H2O
show the reaction diagram
-
69% conversion after 3 h
-
-
?
(4-methylphenyl)acetaldehyde + dopamine
(1S)-1-[2-(4-fluorophenyl)ethyl]-1,2,3,4-tetrahydroisoquinoline-6,7-diol + H2O
show the reaction diagram
-
57% conversion after 3 h
-
-
?
(5,5-dimethyl-1,3-dioxan-2-yl)acetaldehyde + dopamine
(1S)-1-[2-(5,5-dimethyl-1,3-dioxan-2-yl)ethyl]-1,2,3,4-tetrahydroisoquinoline-6,7-diol + H2O
show the reaction diagram
-
42% conversion after 3 h
-
-
?
3,3-dimethylbutanal + dopamine
(1S)-1-(3,3-dimethylbutyl)-1,2,3,4-tetrahydroisoquinoline-6,7-diol + H2O
show the reaction diagram
-
52% conversion after 3 h
-
-
?
3-(2-aminoethyl)phenol + 4-hydroxyphenylacetaldehyde
?
show the reaction diagram
-
-
-
-
?
4-(2-aminoethyl)benzene-1,2-diol + (3,4-dihydroxyphenyl)acetaldehyde
?
show the reaction diagram
-
-
-
-
?
4-(2-aminoethyl)benzene-1,2-diol + 3,4-dihydroxyphenylacetaldehyde
(S)-norlaudanosoline + H2O
show the reaction diagram
4-(2-Aminoethyl)benzene-1,2-diol + 4-hydroxyphenylacetaldehyde
(S)-Norcoclaurine + H2O
show the reaction diagram
4-(2-Aminoethyl)benzene-1,2-diol + 4-hydroxyphenylacetaldehyde
?
show the reaction diagram
4-hydroxyphenylacetaldehyde + 4-(2-aminoethyl)benzene-1,2-diol
(S)-norcoclaurine + H2O
show the reaction diagram
4-hydroxyphenylacetaldehyde + dopamine
(S)-norcoclaurine + H2O
show the reaction diagram
5-(2-aminoethyl)-2-methoxyphenol + 4-hydroxyphenylacetaldehyde
?
show the reaction diagram
-
-
-
-
?
cyclohexylacetaldehyde + dopamine
(1S)-1-(2-cyclohexylethyl)-1,2,3,4-tetrahydroisoquinoline-6,7-diol + H2O
show the reaction diagram
-
71% conversion after 3 h
-
-
?
naphthalen-1-ylacetaldehyde + dopamine
(1S)-1-[2-(naphthalen-1-yl)ethyl]-1,2,3,4-tetrahydroisoquinoline-6,7-diol + H2O
show the reaction diagram
-
61% conversion after 3 h
-
-
?
phenylacetaldehyde + dopamine
(1S)-1-(2-phenylethyl)-1,2,3,4-tetrahydroisoquinoline-6,7-diol + H2O
show the reaction diagram
-
51% conversion after 3 h
-
-
?
thiophen-3-ylacetaldehyde + dopamine
(1S)-1-[2-(thiophen-3-yl)ethyl]-1,2,3,4-tetrahydroisoquinoline-6,7-diol + H2O
show the reaction diagram
-
68% conversion after 3 h
-
-
?
[4-(trifluoromethoxy)phenyl]acetaldehyde + dopamine
(1S)-1-[2-[4-(trifluoromethoxy)phenyl]ethyl]-1,2,3,4-tetrahydroisoquinoline-6,7-diol + H2O
show the reaction diagram
-
65% conversion after 3 h
-
-
?
additional information
?
-
-
isozyme NCS1 of Coptis japonica does not catalyze the formation of (S)-norcoclaurine from dopamine and 4-hydroxyphenylacetaldehyde
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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
4-(2-Aminoethyl)benzene-1,2-diol + 4-hydroxyphenylacetaldehyde
(S)-Norcoclaurine + H2O
show the reaction diagram
4-(2-Aminoethyl)benzene-1,2-diol + 4-hydroxyphenylacetaldehyde
?
show the reaction diagram
4-hydroxyphenylacetaldehyde + 4-(2-aminoethyl)benzene-1,2-diol
(S)-norcoclaurine + H2O
show the reaction diagram
4-hydroxyphenylacetaldehyde + dopamine
(S)-norcoclaurine + H2O
show the reaction diagram
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Fe2+
-
CjNCS1 contains a Fe2+-binding site
additional information
Thalictrum flavum ssp. glaucum
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the enzyme is not affected by divalent cations
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
norlaudanosoline
Thalictrum flavum ssp. glaucum
-
product inhibition, kinetics
additional information
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
fungal elicitor of Bortrytis sp.
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isolated elicitor induces the enzyme activity in cell suspension culture 20fold
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additional information
-
enzyme is not affected by Mg2+ and Ca2+
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
335
(3,4-dihydroxyphenyl)acetaldehyde
-
-
0.7 - 1
3,4-dihydroxyphenylacetaldehyde
1.5 - 5
4-(2-Aminoethyl)benzene-1,2-diol
0.288 - 1
4-hydroxyphenylacetaldehyde
additional information
additional information
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
7
(3,4-dihydroxyphenyl)acetaldehyde
Thalictrum flavum
-
cosubstrate: 4-(2-aminoethyl)benzene-1,2-diol
6
3-(2-aminoethyl)phenol
Thalictrum flavum
-
cosubstrate: 4-(2-aminoethyl)benzene-1,2-diol
6.4 - 7
4-(2-Aminoethyl)benzene-1,2-diol
0.8 - 6.4
4-hydroxyphenylacetaldehyde
6
5-(2-aminoethyl)-2-methoxyphenol
Thalictrum flavum
-
cosubstrate: 4-(2-aminoethyl)benzene-1,2-diol
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
4.7
norlaudanosoline
Thalictrum flavum ssp. glaucum
-
pH 7.5, 37°, with respect to dopamine
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.09
Thalictrum flavum ssp. glaucum
-
purified enzyme
additional information
Eschscholtzia tenuifolia
-
-
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.5 - 7
7
-
assay at
7.4
Eschscholtzia tenuifolia
-
substrate 4-hydroxyphenylacetaldehyde
7.8
Eschscholtzia tenuifolia
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substrate 3,4-dihydroxyphenylacetaldehyde
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.5 - 8.5
Eschscholtzia tenuifolia
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-
6.2 - 9
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50% of maximal activity at pH 6.2, 68% of maximal activity at pH 9.0
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37
-
assay at
40
Eschscholtzia tenuifolia
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substrate 3,4-dihydroxyphenylacetaldehyde
42 - 55
Thalictrum flavum ssp. glaucum
-
-
45 - 55
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37 - 57
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.5
Thalictrum flavum ssp. glaucum
-
isoelectric focusing, minor isoform
5.7
Thalictrum flavum ssp. glaucum
-
isoelectric focusing, major isoform
6
Thalictrum flavum ssp. glaucum
-
isoelectric focusing, major isoform
6.2
Thalictrum flavum ssp. glaucum
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isoelectric focusing, minor isoform
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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low level
Manually annotated by BRENDA team
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NCS2 shows cell type-specific occurrence in phloem sieve elements, colocalization with all other known benzylisoquinoline alkaloid biosynthetic enzymes
Manually annotated by BRENDA team
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all organs especially in root and stem
Manually annotated by BRENDA team
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germinating
Manually annotated by BRENDA team
additional information
-
not in leaves
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
Eschscholtzia tenuifolia
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-
Manually annotated by BRENDA team
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NCS2 has an N-terminal signal peptide and sorts to the vacuole
Manually annotated by BRENDA team
additional information
-
immunolocalization study of NCS, overview
-
Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
15000
Thalictrum flavum ssp. glaucum
-
2 * 15000, SDS-PAGE
15500
Eschscholtzia tenuifolia
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gel filtration
21180
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mass spectrometry of recombinant Delta29NCS (coding for amino acids 30–210 of NCS)
28000
Thalictrum flavum ssp. glaucum
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
tetramer
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composed of two asymmetric units, each containing a single dimer (A1-B1 or A2-B2)
additional information
-
size-exclusion chromatography (SEC) and NMR relaxation measurements, concentration-dependent oligomerization of delta 29NCS with an equilibrium of monomeric and oligomeric protein over a broad concentration range, at low protein concentrations in the range of 10 microM the enzyme is predominantly monomeric, no indication that substrate binding induces dimerization of delta 29NCS
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
at 294 K by the hanging-drop vapour-diffusion method using ammonium sulfate and sodium chloride as precipitant agents and diffract to better than 3.0 A resolution using a synchrotron-radiation source. The crystals belong to the trigonal space group P3121, with unit-cell parameters a = b = 86.31, c = 118.36 A. A selenomethionine derivate is crystallized in the same space group, 2.7 A resolution
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combining experimental NMR spectroscopic data with homology modelling using the homologous major birch pollen allergen Bet v 1 (PDB code 1BV1) as template structure a reliable model structure of delta 29NCS is built
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SeMet derivate of NCS unligated (2VNE) and substrate-bound (2VQ5), by the hanging-drop vapour-diffusion method using ammonium sulfate and sodium chloride as precipitant agents, SeMet NCS: 2.7A resolution, 298K, ammoniumsulfate 1.4-1.8 M, NaCl 0.2 M, acetate buffer pH 4.0-4.5, trigonal space group P3121 with unit cell dimensions a = b = 86.31 A, c = 118.36 A, alpha = beta = 90°, gamma = 120°
-
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
55
-
instable above 55°C
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-80°C, purified enzyme, 7 days, no loss of activity
4°C, purified enzyme, 7 days, 75% loss of activity
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
1590fold to homogeneity from cell suspension culture
Thalictrum flavum ssp. glaucum
-
4-isozymes, partial
Eschscholtzia tenuifolia
-
HisTrap Fast Flow column
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HisTrap Fast Flow column equilibrated with a 50 mM Tris HCl buffer at pH 7.5. A linear gradient of imidazole concentration from 0 to 0.5 M (buffered at pH 7.5) is applied
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Ni-NTA affinity column chromatography
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nickel resin column chromatography
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recombinant C-terminally His-tagged NCS from Eschericha coli strain BL21(DE3) by nickel affinity chromatography
-
recombinant His-tagged enzyme
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli BL21(DE3) cells
expression in Escherichia coli is optimized
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expression of CjNCS1 in Escherichia coli; expression of CjPR10A in Escherichia coli
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NCS2-green fluorescent protein fusion introduced by microprojectile bombardment into opium poppy cells initially localizes to the endoplasmic reticulum but subsequently sorts to the vacuole
-
overexpressed in Escherichia coli as a recombinant His-tagged enzyme
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recombinant expression of C-terminally His-tagged NCS in Eschericha coli strain BL21(DE3)
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the NCS protein truncated at the first 19 amino acids with an N-terminal MTGS sequence and a His tag at the C-terminus and its SeMet-substituted variant are subcloned into the NdeI and XhoI restriction sites of the vector pET22b. Transformation of chemically competent Escherichia coli strain BL21DE is performed and transformed cells are plated onto LB-ampicillin plates
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the NCS protein truncated at the first 19 amino acids, with a N-terminal MTGS sequence and a His tag at the C terminus is subcloned into the NdeI and XhoI restriction site of the vector pET22-b. Transformation of chemically competent Escherichia coli strain BL21DE3
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unlabelled and 15N-labelled delta 29NCS is expressed using the expression system Escherichia coli Rosetta(DE3)/pET29b-delta 29NCS
-
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
elicitor-induced cell cultures show increasing expression of NCS after 2 to 100 hours
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
E110A
-
site-directed mutagenesis, lower but significant activity
K122A
-
site-directed mutagenesis, lower but significant activity
Y108A
-
site-directed mutagenesis abolishes the stereoselective synthesis of (S)-norcoclaurine
Y108F
-
site-directed mutagenesis, reduced activity
additional information
-
virus-induced gene silencing of NCS in opium poppy profoundly reduces alkaloid levels compared to controls, a mixed culture of Agrobacterium tumefaciens harboring either pTRV2-PsNCS or pTRV1 is infiltrated into opium poppy seedlings tow weeks after germination. Levels of the five major alkaloids in opium poppy latex, i.e. morphine, codeine, thebaine, noscapine, and papaveine, are reduced by 75-82% in plants infiltrated with Agrobacterium tumefaciens harboring pTRV2-PsNCS
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
synthesis
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development of an efficient, stereoselective, green synthesis of (S)-norcoclaurine, i.e. higenamine, using the recombinant (S)-norcoclaurine synthase enzyme, starting from the cheap tyrosine and dopamine substrates in a one-pot, two step process, overview. The optimized process affords enantiomerically pure (S)-norcoclaurine (93%) in a yield higher than 80% and allows good recovery of the enzyme for recycling, by a green Pictet-Spengler synthesis