Literature summary for 2.1.1.169 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.

Search for more literature for 2.1.1.169

Cloned(Commentary)

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

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 [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
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/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.
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

Organism	UniProt	Comment	Textmining
Triticum aestivum	Q38J50	-	-

Purification (Commentary)

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

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	a
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
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
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)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
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

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

Synonyms	Comment	Organism
O-methyltransferase	-	Triticum aestivum
TaOMT2	-	Triticum aestivum

Cofactor

Cofactor	Comment	Organism	Structure
S-adenosyl-L-methionine	-	Triticum aestivum

General Information

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 [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
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