Cloned (Comment) | Organism |
---|---|
expressed in Escherichia coli | Triticum aestivum |
expression of His-tagged TaOMT2 mutant enzymes in Escherichia coli strain BL21 (DE3) | Triticum aestivum |
Protein Variants | Comment | Organism |
---|---|---|
D263E | severe loss of activity is due to a conflict between the catalytic His262-imidazole group and Glu-CH2 | Triticum aestivum |
D263E | site-directed mutagenesis, severe loss of activity is due to a conflict between the catalytic His262-imidazole group and Glu-CH2 | Triticum aestivum |
D263I | no activity, Ile263 can not form a H-bond with 3'-OH group | Triticum aestivum |
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 |
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 |
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 |
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 |
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 |
E290Q | no activity. This mutation results in a more extensive H-bonding that hinders charge transfer and affects B-ring flexibility | Triticum aestivum |
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 |
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 |
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 |
E322Q | no activity | Triticum aestivum |
E322Q | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Triticum aestivum |
G305A | Loss of activity due to loss of H-bonding with the amide group of the neighboring Asn348 | Triticum aestivum |
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 |
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 |
G305S | site-directed mutagenesis, change in polarity is less effective than chain length on catalytic activity | Triticum aestivum |
H262F | no activity | Triticum aestivum |
H262F | site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme | Triticum aestivum |
H262L | no activity | Triticum aestivum |
H262L | site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme | Triticum aestivum |
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 |
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 |
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 |
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 |
N124Q | site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme | Triticum aestivum |
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 |
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 |
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 |
W259Y | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Triticum aestivum |
W259Y | Km increased compared to wild-type, kcat/Km decreased compared to wild-type | Triticum aestivum |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.0595 | - |
tricetin | pH and temperature not specified in the publication, wild-type | Triticum aestivum | |
0.118 | - |
tricetin | pH and temperature not specified in the publication, mutant G305S | Triticum aestivum | |
0.1282 | - |
tricetin | pH and temperature not specified in the publication, mutant D263N | Triticum aestivum | |
0.131 | - |
tricetin | pH and temperature not specified in the publication, mutant W259A | Triticum aestivum | |
0.17 | - |
tricetin | pH and temperature not specified in the publication, mutant W259Y | Triticum aestivum | |
0.193 | - |
tricetin | pH and temperature not specified in the publication, mutant E322I | Triticum aestivum |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
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 | ? | - |
? | |
S-adenosyl-L-methionine + 3',5'-O-dimethyltricetin | Triticum aestivum | - |
S-adenosyl-L-homocysteine + 3',4',5'-O-trimethyltricetin | - |
? | |
S-adenosyl-L-methionine + 3'-O-methyltricetin | Triticum aestivum | - |
S-adenosyl-L-homocysteine + 3',5'-O-dimethyltricetin | - |
? | |
S-adenosyl-L-methionine + tricetin | Triticum aestivum | - |
S-adenosyl-L-homocysteine + 3'-O-methyltricetin | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Triticum aestivum | Q38J50 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged TaOMT2 mutant enzymes from Escherichia coli strain BL21 (DE3) by nickel affinity chromatography | Triticum aestivum |
using affinity chromatography | Triticum aestivum |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
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 | |
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 | |
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 | |
S-adenosyl-L-methionine + tricetin = S-adenosyl-L-homocysteine + 3'-O-methyltricetin | mechanism for the sequential methylation of tricetin, overview | Triticum aestivum |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
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 | ? | - |
? | |
additional information | structure-function relationships, homology modeling and molecular docking using MsCOMT crystal structure, PDB ID 1KYZ, overview | Triticum aestivum | ? | - |
? | |
S-adenosyl-L-methionine + 3',5'-O-dimethyltricetin | - |
Triticum aestivum | S-adenosyl-L-homocysteine + 3',4',5'-O-trimethyltricetin | - |
? | |
S-adenosyl-L-methionine + 3'-O-methyltricetin | - |
Triticum aestivum | S-adenosyl-L-homocysteine + 3',5'-O-dimethyltricetin | - |
? | |
S-adenosyl-L-methionine + tricetin | - |
Triticum aestivum | S-adenosyl-L-homocysteine + 3'-O-methyltricetin | - |
? | |
S-adenosyl-L-methionine + tricetin | - |
Triticum aestivum | S-adenosyl-L-homocysteine + 3',4',5'-O-trimethyltricetin | - |
? |
Subunits | Comment | Organism |
---|---|---|
dimer | - |
Triticum aestivum |
More | structure-function relationships, three-dimensional enzyme modelling, homology modeling and molecular docking using MsCOMT crystal structure, PDB ID 1KYZ, overview | Triticum aestivum |
Synonyms | Comment | Organism |
---|---|---|
O-methyltransferase | - |
Triticum aestivum |
TaOMT2 | - |
Triticum aestivum |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
S-adenosyl-L-methionine | - |
Triticum aestivum |
General Information | Comment | Organism |
---|---|---|
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 |
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 Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
2300000 | - |
tricetin | pH and temperature not specified in the publication, mutant W259Y | Triticum aestivum | |
5200000 | - |
tricetin | pH and temperature not specified in the publication, mutant W259A | Triticum aestivum | |
6330000 | - |
tricetin | pH and temperature not specified in the publication, mutant E322I | Triticum aestivum | |
7310000 | - |
tricetin | pH and temperature not specified in the publication, mutant G305S | Triticum aestivum | |
63000000 | - |
tricetin | pH and temperature not specified in the publication, mutant D263N | Triticum aestivum | |
74000000 | - |
tricetin | pH and temperature not specified in the publication | Triticum aestivum |