Information on EC 2.1.1.169 - tricetin 3',4',5'-O-trimethyltransferase

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The expected taxonomic range for this enzyme is: Triticum aestivum

EC NUMBER
COMMENTARY hide
2.1.1.169
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RECOMMENDED NAME
GeneOntology No.
tricetin 3',4',5'-O-trimethyltransferase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
3 S-adenosyl-L-methionine + tricetin = 3 S-adenosyl-L-homocysteine + 3',4',5'-O-trimethyltricetin
show the reaction diagram
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S-adenosyl-L-methionine + 3',5'-O-dimethyltricetin = S-adenosyl-L-homocysteine + 3',4',5'-O-trimethyltricetin
show the reaction diagram
S-adenosyl-L-methionine + 3'-hydroxyflavone = S-adenosyl-L-homocysteine + 3'-methoxyflavone
show the reaction diagram
S-adenosyl-L-methionine + 3'-O-methyltricetin = S-adenosyl-L-homocysteine + 3',5'-O-dimethyltricetin
show the reaction diagram
S-adenosyl-L-methionine + tricetin = S-adenosyl-L-homocysteine + 3'-O-methyltricetin
show the reaction diagram
SYSTEMATIC NAME
IUBMB Comments
S-adenosyl-L-methionine:tricetin 3',4',5'-O-trimethyltransferase
The enzyme from Triticum aestivum catalyses the sequential O-methylation of tricetin via 3'-O-methyltricetin, 3',5'-O-methyltricetin to 3',4',5'-O-trimethyltricetin [2].
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
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residue Val309 determines substrate specificity for tricetin and may define the evolutionary differences between the two closely related proteins, COMT and flavone OMT
metabolism
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TaOMT2 catalyzes the sequential methylation of the flavone, tricetin, to its 3'-methyl-, 3',5'-dimethyl-, and 3',4',5'-trimethyl ether derivatives. Tricin, a potential multifunctional nutraceutical, is the major enzyme reaction product
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
3 S-adenosyl-L-methionine + tricetin
3 S-adenosyl-L-homocysteine + 3',4',5'-O-trimethyltricetin
show the reaction diagram
S-adenosyl-L-methionine + 3',5'-O-dimethyltricetin
S-adenosyl-L-homocysteine + 3',4',5'-O-trimethyltricetin
show the reaction diagram
S-adenosyl-L-methionine + 3'-O-methyltricetin
S-adenosyl-L-homocysteine + 3',5'-O-dimethyltricetin
show the reaction diagram
S-adenosyl-L-methionine + 5-hydroxyferulic acid
?
show the reaction diagram
second most preferred substrate with 78% activity compared to tricetin
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-
?
S-adenosyl-L-methionine + tricetin
S-adenosyl-L-homocysteine + 3',4',5'-O-trimethyltricetin
show the reaction diagram
S-adenosyl-L-methionine + tricetin
S-adenosyl-L-homocysteine + 3'-O-methyltricetin
show the reaction diagram
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-adenosyl-L-methionine + 3',5'-O-dimethyltricetin
S-adenosyl-L-homocysteine + 3',4',5'-O-trimethyltricetin
show the reaction diagram
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-
-
-
?
S-adenosyl-L-methionine + 3'-O-methyltricetin
S-adenosyl-L-homocysteine + 3',5'-O-dimethyltricetin
show the reaction diagram
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-
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?
S-adenosyl-L-methionine + tricetin
S-adenosyl-L-homocysteine + 3',4',5'-O-trimethyltricetin
show the reaction diagram
Q38J50
i.e. 5,7,3',4',5'-pentahydroxyflavone, most preferred substrate
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-
?
S-adenosyl-L-methionine + tricetin
S-adenosyl-L-homocysteine + 3'-O-methyltricetin
show the reaction diagram
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-
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?
additional information
?
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stepwise methylation of tricetin involving deprotonation of its hydroxyl groups by a His262-Asp263 pair followed by nucleophilic attack of SAM-methyl groups, substrate binding is mediated by an extensive network of H-bonds and van der Waals interactions, residue Val309 determines substrate specificity for tricetin and may define the evolutionary differences between the two closely related proteins, COMT and flavone OMT, molecular modeling, overview
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
S-adenosyl-L-methionine
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
no requirement for Mg2+
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
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no loss of activity up to 0.4 M perchlorate
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0595 - 0.193
tricetin
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2300000 - 74000000
tricetin
2788
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30
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assay at
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.75
calculated from sequence
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
highest expression
Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
38900
x * 38900, calculated from sequence, SDS-PAGE
42616
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1 * 42616, characterisation of the dimer/monomer equilibrium, calculated from sequence; 2 * 42616, characterisation of the dimer/monomer equilibrium, calculated from sequence
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
x * 38900, calculated from sequence, SDS-PAGE
monomer
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1 * 42616, characterisation of the dimer/monomer equilibrium, calculated from sequence
additional information
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structure-function relationships, three-dimensional enzyme modelling, homology modeling and molecular docking using MsCOMT crystal structure, PDB ID 1KYZ, overview
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4°C, almost no loss of catalytic activity when stored for 3-4 weeks
4°C, the activity of the affinity-purified TaOMT2 is quite stable for up to 2 months when stored in buffer containing 10% glycerol, with a half-life of 10 to 11 weeks
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
recombinant His-tagged TaOMT2 mutant enzymes from Escherichia coli strain BL21 (DE3) by nickel affinity chromatography; using affinity chromatography
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli; expression of His-tagged TaOMT2 mutant enzymes in Escherichia coli strain BL21 (DE3)
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expression in Escherichia coli
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
the amount of enzyme increases during low temperature (4°C) acclimation while the specific activity of the enzyme remains constant. Wheat growth, especially in the floral parts, is associated with an increased expression and enzyme activity
the enzyme expression does not change during salinity and/or drought stresses
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D263E
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severe loss of activity is due to a conflict between the catalytic His262-imidazole group and Glu-CH2; site-directed mutagenesis, severe loss of activity is due to a conflict between the catalytic His262-imidazole group and Glu-CH2
D263I
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no activity, Ile263 can not form a H-bond with 3'-OH group; site-directed mutagenesis, Ile263 can not form a H-bond with 3'-OH group, the mutant shows almost complete loss in activity
D263N
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site-directed mutagenesis, slight decrease in activity due to a decreased electronegativity of Asn-N compared to Asp-O, that affects charge transfer to tricetin-OH groups; slight decrease in activity due to a decreased electronegativity of Asn-N compared to Asp-O, that affects charge transfer to tricetin-OH groups
E290I
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loss of activity is due to the fact that Ile can not form a H-bond with the 4'-OH of tricetin; site-directed mutagenesis, almost complete loss of activity is due to the fact that Ile can not form a H-bond with the 4'-OH of tricetin
E290Q
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no activity. This mutation results in a more extensive H-bonding that hinders charge transfer and affects B-ring flexibility; site-directed mutagenesis, the mutation results in a more extensive H-bonding that hinders charge transfer and affects B-ring flexibility and almost complete loss in activity
E322I
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Km increased compared to wild-type, kcat/Km decreased compared to wild-type. Loss of charge or a change in the side chain affects H-bonding with the neighboring residues, especially His262; site-directed mutagenesis, loss of charge or a change in the side chain affects H-bonding with the neighboring residues, especially His262, the mutant shows reduced activity and altered kinetics compared to the wild-type enzyme
E322Q
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no activity; site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
G305A
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Loss of activity due to loss of H-bonding with the amide group of the neighboring Asn348; site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme, the mutation results in loss of activity due to loss of H-bonding with the amide group of the neighboring Asn348
G305S
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Km increased compared to wild-type, kcat/Km decreased compared to wild-type. Change in polarity is less effective than chain length on catalytic activity; site-directed mutagenesis, change in polarity is less effective than chain length on catalytic activity
H262F
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no activity; site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme
H262L
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no activity; site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme
H262R
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results in almost complete loss of protein expression. All mutant proteins lack imidazole ring that is critical for proton flow among His262, Asp263 and the substrate; site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme, the mutation results in almost complete loss of protein expression, all mutant proteins lack imidazole ring that is critical for proton flow among His262, Asp263 and the substrate
N124I
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no activity. Mutation results in a decreased substrate binding but not protein folding. Mutations disrupt H-bonding with 5-OH group of tricetin; site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme, the mutation results in a decreased substrate binding but not protein folding. Both mutations disrupt H-bonding with 5-OH group of tricetin
N124Q
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no activity. Mutation results in a decreased substrate binding but not protein folding. Mutations disrupt H-bonding with 5-OH group of tricetin; site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme
W259A
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Km increased compared to wild-type, kcat/Km decreased compared to wild-type. Ala can maintain the H-bonding network between Trp259, Glu290 and His262, wheras Tyr cannot; site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme, Ala can maintain the H-bonding network between Trp259, Glu290 and His262, wheras Tyr cannot
W259Y
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Km increased compared to wild-type, kcat/Km decreased compared to wild-type; site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme