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Information on EC 4.2.1.78 - (S)-norcoclaurine synthase and Organism(s) Thalictrum flavum and UniProt Accession Q67A25

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EC Tree
     4 Lyases
         4.2 Carbon-oxygen lyases
             4.2.1 Hydro-lyases
                4.2.1.78 (S)-norcoclaurine synthase
IUBMB Comments
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.
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Thalictrum flavum
UNIPROT: Q67A25
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The taxonomic range for the selected organisms is: Thalictrum flavum
The enzyme appears in selected viruses and cellular organisms
Synonyms
ncs, pbncs, norcoclaurine synthase, csncs, pr10a, (s)-norcoclaurine synthase, cjncs1, cjpr10a, tfncs, (s)-norlaudanosoline synthase, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
norcoclaurine synthase
-
TFLNCSDELTA25
-
(S)-norcoclaurine synthase
-
-
(S)-Norlaudanosoline synthase
-
-
-
-
norcoclaurine synthase
-
-
Synthase, (S)-norlaudanosoline
-
-
-
-
additional information
-
norcoclaurine synthase is a member of the pathogenesis-related 10/Bet v1 protein family
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
condensation
-
-
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
-
SUBSTRATE
PRODUCT                       
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-hydroxyphenylacetaldehyde + dopamine
(S)-norcoclaurine + H2O
show the reaction diagram
4-hydroxyphenylacetone + dopamine
1-(4-hydroxybenzyl)-1-methyl-1,2,3,4-tetrahydroisoquinoline-6,7-diol
show the reaction diagram
-
-
-
?
(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 + 4-hydroxyphenylacetaldehyde
(S)-Norcoclaurine + H2O
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
-
60% conversion after 3 h
-
-
?
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
-
-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
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-hydroxyphenylacetaldehyde + dopamine
(S)-norcoclaurine + H2O
show the reaction diagram
the enzyme catalyzes the enantioselective Pictet-Spengler condensation of dopamine and 4-hydroxyphenylacetaldehyde as a step in benzylisoquinoline alkaloid biosynthesis
-
-
?
4-(2-Aminoethyl)benzene-1,2-diol + 4-hydroxyphenylacetaldehyde
(S)-Norcoclaurine + H2O
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
-
60% conversion after 3 h
-
-
?
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
-
immunoprecipitation of NCS from total protein extracts of Thalictrum flavum cells results in a nearly complete attenuation of NCS activity
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.33 - 0.505
4-hydroxyphenylacetaldehyde
335
(3,4-dihydroxyphenyl)acetaldehyde
-
-
0.288
4-hydroxyphenylacetaldehyde
-
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.8 - 4.5
4-hydroxyphenylacetaldehyde
7
(3,4-dihydroxyphenyl)acetaldehyde
-
cosubstrate: 4-(2-aminoethyl)benzene-1,2-diol
6
3-(2-aminoethyl)phenol
-
cosubstrate: 4-(2-aminoethyl)benzene-1,2-diol
6.4 - 7
4-(2-Aminoethyl)benzene-1,2-diol
6 - 6.4
4-hydroxyphenylacetaldehyde
6
5-(2-aminoethyl)-2-methoxyphenol
-
cosubstrate: 4-(2-aminoethyl)benzene-1,2-diol
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7
-
assay at
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37
-
assay at
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
-
very low expression level
Manually annotated by BRENDA team
additional information
-
not in leaves
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
metabolism
metabolism
-
norcoclaurine synthase, NCS, catalyzes the first committed step in the biosynthesis of benzylisoquinoline alkaloids, exclusive role of PR10/Bet v1-type NCS enzymes, overview
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
-
amino acid sequence comparisons of NCSs from different species, phylogenetic tree, overview
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
21180
-
mass spectrometry of recombinant Delta29NCS (coding for amino acids 30–210 of NCS)
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
tetramer
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
high-resolution X-ray crystallography. The structure supports two features of the dopamine-first mechanism: the binding of dopamine catechol to Lys-122 and the position of the carbonyl substrate binding site at the active site entrance. The catalytically vital residue Glu-110 occupies a ligand-bound conformation that may be catalytically significant
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°
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
-
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A79F
improved turnover of ketones, increase in side-chain steric interactions
A79I
improved turnover of ketones, increase in side-chain steric interactions
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
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
HisTrap Fast Flow column
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
nickel resin column chromatography
-
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
expression in Escherichia coli and in Saccharomyces cereviseae
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
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
expressed in Escherichia coli BL21(DE3) cells
-
expression in Escherichia coli is optimized
-
overexpressed in Escherichia coli as a recombinant His-tagged enzyme
-
recombinant expression of C-terminally His-tagged NCS in Eschericha coli strain BL21(DE3)
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unlabelled and 15N-labelled delta 29NCS is expressed using the expression system Escherichia coli Rosetta(DE3)/pET29b-delta 29NCS
-
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
synthesis
synthesis
-
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
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Luk, L.Y.; Bunn, S.; Liscombe, D.K.; Facchini, P.J.; Tanner, M.E.
Mechanistic studies on norcoclaurine synthase of benzylisoquinoline alkaloid biosynthesis: an enzymatic Pictet-Spengler reaction
Biochemistry
46
10153-10161
2007
Thalictrum flavum
Manually annotated by BRENDA team
Berkner, H.; Engelhorn, J.; Liscombe, D.K.; Schweimer, K.; Woehrl, B.M.; Facchini, P.J.; Roesch, P.; Matecko, I.
High-yield expression and purification of isotopically labeled norcoclaurine synthase, a Bet v 1-homologous enzyme, from Thalictrum flavum for NMR studies
Protein Expr. Purif.
56
197-204
2007
Thalictrum flavum
Manually annotated by BRENDA team
Pasquo, A.; Bonamore, A.; Franceschini, S.; Macone, A.; Boffi, A.; Ilari, A.
Cloning, expression, crystallization and preliminary X-ray data analysis of norcoclaurine synthase from Thalictrum flavum
Acta Crystallogr. Sect. F
64
281-283
2008
Thalictrum flavum (Q67A25), Thalictrum flavum
Manually annotated by BRENDA team
Berkner, H.; Schweimer, K.; Matecko, I.; Roesch, P.
Conformation, catalytic site, and enzymatic mechanism of the PR10 allergen-related enzyme norcoclaurine synthase
Biochem. J.
413
281-290
2008
Thalictrum flavum
Manually annotated by BRENDA team
Ilari, A.; Franceschini, S.; Bonamore, A.; Arenghi, F.; Botta, B.; Macone, A.; Pasquo, A.; Bellucci, L.; Boffi, A.
Structural basis of enzymatic (S)-norcoclaurine biosynthesis
J. Biol. Chem.
284
897-904
2009
Thalictrum flavum (Q67A25), Thalictrum flavum
Manually annotated by BRENDA team
Bonamore, A.; Rovardi, I.; Gasparrini, F.; Baiocco, P.; Barba, M.; Molinaro, C.; Botta, B.; Boffi, A.; MacOne, A.
An enzymatic, stereoselective synthesis of (S)-norcoclaurine
Green Chem.
12
1623-1627
2010
Thalictrum flavum
-
Manually annotated by BRENDA team
Lee, E.J.; Facchini, P.
Norcoclaurine synthase is a member of the pathogenesis-related 10/Bet v1 protein family
Plant Cell
22
3489-3503
2010
Coptis japonica, Papaver somniferum, Thalictrum flavum
Manually annotated by BRENDA team
Ruff, B.M.; Braese, S.; O'Connor, S.E.
Biocatalytic production of tetrahydroisoquinolines
Tetrahedron Lett.
53
1071-1074
2012
Thalictrum flavum
Manually annotated by BRENDA team
Lichman, B.R.; Sula, A.; Pesnot, T.; Hailes, H.C.; Ward, J.M.; Keep, N.H.
Structural evidence for the dopamine-first mechanism of norcoclaurine synthase
Biochemistry
56
5274-5277
2017
Thalictrum flavum (Q67A25), Thalictrum flavum
Manually annotated by BRENDA team
Lichman, B.R.; Zhao, J.; Hailes, H.C.; Ward, J.M.
Enzyme catalysed Pictet-Spengler formation of chiral 1,1-disubstituted- and spiro-tetrahydroisoquinolines
Nat. Commun.
8
14883
2017
Thalictrum flavum (Q67A25), Thalictrum flavum
Manually annotated by BRENDA team
Li, J.; Lee, E.J.; Chang, L.; Facchini, P.J.
Genes encoding norcoclaurine synthase occur as tandem fusions in the Papaveraceae
Sci. Rep.
6
39256
2016
Nandina domestica, Corydalis chinensis, Papaver somniferum (Q4QTJ1), Papaver somniferum (Q4QTJ2), Papaver somniferum, Thalictrum flavum (Q67A25)
Manually annotated by BRENDA team