Enzymatic synthesis of polymethylated flavonols in Chrysosplenium americanum. III. Purification and kinetic analysis of S-adenosyl-L-methionine:3-methylquercetin 7-O-methyltransferase.
An O-methyltransferase (OMT) which catalyzes the methylation of 3-methylquercetin to 3,7-dimethylquercetin, the second step of methyl transfers toward the biosynthesis of polymethylated flavonol glucosides, has been isolated from Chrysosplenium americanum shoot tips. The 7-OMT was purified by ammonium sulfate precipitation, gel filtration, chromatofocusing and ion-exchange chromatography using a fast protein liquid chromatography system. Compared with previously reported methods [1985) Arch. Biochem. Biophys. 238, 596-605), this protocol resulted in a highly purified enzyme preparation, free from other OMT activities, which allowed the study of its kinetic mechanism. Substrate interaction and product inhibition patterns obtained were consistent with an ordered bi bi mechanism, where S-adenosyl-L-methionine is the first substrate to bind to the enzyme and S-adenosyl-L-homocysteine is the last product released. However, the results obtained did not exclude the formation of one or more dead-end complex. The similarity in kinetic characteristics of this enzyme to those of the other Chrysosplenium OMTs suggests that methyltransferases of this tissue may have evolved from a common precursor.
Department of Biology, Concordia University, Montreal, Quebec, Canada.
H E
HE
Khouri
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V
De Luca
R K
RK
Ibrahim
D005419:Flavonoids / Q000378:metabolism*; D044948:Flavonols; D007700:Kinetics; D008780:Methyltransferases / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism; D010944:Plants / Q000201:enzymology*; D011794:Quercetin / Q000031:analogs & derivatives / Q000378:metabolism*; D012436:S-Adenosylmethionine / Q000378:metabolism*; D013379:Substrate Specificity
D005419:Flavonoids; D044948:Flavonols; C047368:3-methylquercetin; D012436:S-Adenosylmethionine; D011794:Quercetin; C045146:3-methylquercetin 0-7-methyltransferase; D008780:Methyltransferases