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Literature summary extracted from

  • Zhou, J.M.; Lee, E.; Kanapathy-Sinnaiaha, F.; Park, Y.; Kornblatt, J.A.; Lim, Y.; Ibrahim, R.K.
    Structure-function relationships of wheat flavone O-methyltransferase: homology modeling and site-directed mutagenesis (2010), BMC Plant Biol., 10, 156.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.1.1.169 expressed in Escherichia coli Triticum aestivum
2.1.1.169 expression of His-tagged TaOMT2 mutant enzymes in Escherichia coli strain BL21 (DE3) Triticum aestivum

Protein Variants

EC Number Protein Variants Comment Organism
2.1.1.169 D263E severe loss of activity is due to a conflict between the catalytic His262-imidazole group and Glu-CH2 Triticum aestivum
2.1.1.169 D263E site-directed mutagenesis, severe loss of activity is due to a conflict between the catalytic His262-imidazole group and Glu-CH2 Triticum aestivum
2.1.1.169 D263I no activity, Ile263 can not form a H-bond with 3'-OH group Triticum aestivum
2.1.1.169 D263I site-directed mutagenesis, Ile263 can not form a H-bond with 3'-OH group, the mutant shows almost complete loss in activity Triticum aestivum
2.1.1.169 D263N 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 Triticum aestivum
2.1.1.169 D263N slight decrease in activity due to a decreased electronegativity of Asn-N compared to Asp-O, that affects charge transfer to tricetin-OH groups Triticum aestivum
2.1.1.169 E290I loss of activity is due to the fact that Ile can not form a H-bond with the 4'-OH of tricetin Triticum aestivum
2.1.1.169 E290I 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 Triticum aestivum
2.1.1.169 E290Q no activity. This mutation results in a more extensive H-bonding that hinders charge transfer and affects B-ring flexibility Triticum aestivum
2.1.1.169 E290Q 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 Triticum aestivum
2.1.1.169 E322I 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 Triticum aestivum
2.1.1.169 E322I 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 Triticum aestivum
2.1.1.169 E322Q no activity Triticum aestivum
2.1.1.169 E322Q site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Triticum aestivum
2.1.1.169 G305A Loss of activity due to loss of H-bonding with the amide group of the neighboring Asn348 Triticum aestivum
2.1.1.169 G305A 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 Triticum aestivum
2.1.1.169 G305S 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 Triticum aestivum
2.1.1.169 G305S site-directed mutagenesis, change in polarity is less effective than chain length on catalytic activity Triticum aestivum
2.1.1.169 H262F no activity Triticum aestivum
2.1.1.169 H262F site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme Triticum aestivum
2.1.1.169 H262L no activity Triticum aestivum
2.1.1.169 H262L site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme Triticum aestivum
2.1.1.169 H262R 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 Triticum aestivum
2.1.1.169 H262R 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 Triticum aestivum
2.1.1.169 N124I no activity. Mutation results in a decreased substrate binding but not protein folding. Mutations disrupt H-bonding with 5-OH group of tricetin Triticum aestivum
2.1.1.169 N124I 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 Triticum aestivum
2.1.1.169 N124Q site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme Triticum aestivum
2.1.1.169 N124Q no activity. Mutation results in a decreased substrate binding but not protein folding. Mutations disrupt H-bonding with 5-OH group of tricetin Triticum aestivum
2.1.1.169 W259A 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 Triticum aestivum
2.1.1.169 W259A 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 Triticum aestivum
2.1.1.169 W259Y site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Triticum aestivum
2.1.1.169 W259Y Km increased compared to wild-type, kcat/Km decreased compared to wild-type Triticum aestivum

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
2.1.1.169 0.0595
-
 tricetin pH and temperature not specified in the publication, wild-type Triticum aestivum
2.1.1.169 0.118
-
 tricetin pH and temperature not specified in the publication, mutant G305S Triticum aestivum
2.1.1.169 0.1282
-
 tricetin pH and temperature not specified in the publication, mutant D263N Triticum aestivum
2.1.1.169 0.131
-
 tricetin pH and temperature not specified in the publication, mutant W259A Triticum aestivum
2.1.1.169 0.17
-
 tricetin pH and temperature not specified in the publication, mutant W259Y Triticum aestivum
2.1.1.169 0.193
-
 tricetin pH and temperature not specified in the publication, mutant E322I Triticum aestivum

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2.1.1.169 additional information Triticum aestivum 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 ?
-
 ?
2.1.1.169 S-adenosyl-L-methionine + 3',5'-O-dimethyltricetin Triticum aestivum
-
 S-adenosyl-L-homocysteine + 3',4',5'-O-trimethyltricetin
-
 ?
2.1.1.169 S-adenosyl-L-methionine + 3'-O-methyltricetin Triticum aestivum
-
 S-adenosyl-L-homocysteine + 3',5'-O-dimethyltricetin
-
 ?
2.1.1.169 S-adenosyl-L-methionine + tricetin Triticum aestivum
-
 S-adenosyl-L-homocysteine + 3'-O-methyltricetin
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
2.1.1.169 Triticum aestivum Q38J50
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.1.1.169 recombinant His-tagged TaOMT2 mutant enzymes from Escherichia coli strain BL21 (DE3) by nickel affinity chromatography Triticum aestivum
2.1.1.169 using affinity chromatography Triticum aestivum

Reaction

EC Number Reaction Comment Organism Reaction ID
2.1.1.169 S-adenosyl-L-methionine + 3',5'-O-dimethyltricetin = S-adenosyl-L-homocysteine + 3',4',5'-O-trimethyltricetin mechanism for the sequential methylation of tricetin, overview Triticum aestivum
a
2.1.1.169 S-adenosyl-L-methionine + 3'-hydroxyflavone = S-adenosyl-L-homocysteine + 3'-methoxyflavone unique structural features of TaOMT2 which permit the stepwise methylation of tricetin is disclosed. Substrate binding is mediated by an extensive network of H-bonds and van der Waals interactions. The partly buried tricetin active site, as well as proximity and orientation effects ensure sequential methylation of the substrate within the same pocket. Stepwise methylation of tricetin involves deprotonation of its hydroxyl groups by a His262-Asp263 pair followed by nucleophilic attack of SAM-methyl groups Triticum aestivum
a
2.1.1.169 S-adenosyl-L-methionine + 3'-O-methyltricetin = S-adenosyl-L-homocysteine + 3',5'-O-dimethyltricetin mechanism for the sequential methylation of tricetin, overview Triticum aestivum
a
2.1.1.169 S-adenosyl-L-methionine + tricetin = S-adenosyl-L-homocysteine + 3'-O-methyltricetin mechanism for the sequential methylation of tricetin, overview Triticum aestivum
a

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.1.1.169 additional information 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 Triticum aestivum ?
-
 ?
2.1.1.169 additional information structure-function relationships, homology modeling and molecular docking using MsCOMT crystal structure, PDB ID 1KYZ, overview Triticum aestivum ?
-
 ?
2.1.1.169 S-adenosyl-L-methionine + 3',5'-O-dimethyltricetin
-
 Triticum aestivum S-adenosyl-L-homocysteine + 3',4',5'-O-trimethyltricetin
-
 ?
2.1.1.169 S-adenosyl-L-methionine + 3'-O-methyltricetin
-
 Triticum aestivum S-adenosyl-L-homocysteine + 3',5'-O-dimethyltricetin
-
 ?
2.1.1.169 S-adenosyl-L-methionine + tricetin
-
 Triticum aestivum S-adenosyl-L-homocysteine + 3'-O-methyltricetin
-
 ?
2.1.1.169 S-adenosyl-L-methionine + tricetin
-
 Triticum aestivum S-adenosyl-L-homocysteine + 3',4',5'-O-trimethyltricetin
-
 ?

Subunits

EC Number Subunits Comment Organism
2.1.1.169 dimer
-
 Triticum aestivum
2.1.1.169 More structure-function relationships, three-dimensional enzyme modelling, homology modeling and molecular docking using MsCOMT crystal structure, PDB ID 1KYZ, overview Triticum aestivum

Synonyms

EC Number Synonyms Comment Organism
2.1.1.169 O-methyltransferase
-
 Triticum aestivum
2.1.1.169 TaOMT2
-
 Triticum aestivum

Cofactor

EC Number Cofactor Comment Organism Structure
2.1.1.169 S-adenosyl-L-methionine
-
 Triticum aestivum

General Information

EC Number General Information Comment Organism
2.1.1.169 evolution residue Val309 determines substrate specificity for tricetin and may define the evolutionary differences between the two closely related proteins, COMT and flavone OMT Triticum aestivum
2.1.1.169 metabolism 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 Triticum aestivum

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
2.1.1.169 2300000
-
 tricetin pH and temperature not specified in the publication, mutant W259Y Triticum aestivum
2.1.1.169 5200000
-
 tricetin pH and temperature not specified in the publication, mutant W259A Triticum aestivum
2.1.1.169 6330000
-
 tricetin pH and temperature not specified in the publication, mutant E322I Triticum aestivum
2.1.1.169 7310000
-
 tricetin pH and temperature not specified in the publication, mutant G305S Triticum aestivum
2.1.1.169 63000000
-
 tricetin pH and temperature not specified in the publication, mutant D263N Triticum aestivum
2.1.1.169 74000000
-
 tricetin pH and temperature not specified in the publication Triticum aestivum