Identification, purification, and characterization of S-adenosyl-L-methionine: isoliquiritigenin 2'-O-methyltransferase from alfalfa (Medicago sativa L.).
An O-methyltransferase (OMT) which methylates the 2'-hydroxyl of isoliquiritigenin (2',4,4'-trihydroxychalcone) was identified in alfalfa (Medicago sativa L.) seedlings and cell cultures. The OMT activity increased during early stages of seedling development and was predominantly located in roots. Treatment of alfalfa cell cultures with an elicitor from yeast resulted in a fivefold increase in chalcone OMT activity, whereas treatment of seedlings with CuCl2 caused a reduction in activity. The chalcone OMT was purified to near homogeneity from elicited alfalfa cell cultures. Only one form of the enzyme was found. It consisted of an active monomer of subunit Mr 43,000 which could be photoaffinity labeled with S-adenosyl-L-[methyl-3H]methionine. The purified OMT had a pH optimum of 9.0, pI of 4.7, and was highly specific for the 2'-hydroxyl of 2',4,4'-trihydroxychalcone, with essentially no activity toward narigenin chalcone, caffeic acid, or daidzein. Kinetic analysis indicated a sequential bi bi mechanism with Km values of 2.2 and 17.7 microM for 2',4,4'-trihydroxychalcone and S-adenosyl-L-methionine, respectively. S-Adenosyl-L-homocysteine was a potent inhibitor. The chalcone OMT represents the third distinct OMT isolated from alfalfa cell cultures.
Maxwell
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CA
Plant Biology Division, Samuel Roberts Noble Foundation, Ardmore, Oklahoma 73402.
Edwards
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R
Dixon
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RA
D000345:Affinity Labels; D002599:Chalcone / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D005419:Flavonoids / Q000378:metabolism; D007535:Isomerases / Q000378:metabolism; D007700:Kinetics; D000455:Medicago sativa / Q000201:enzymology*; D008780:Methyltransferases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D010940:Plant Proteins / Q000302:isolation & purification; D013379:Substrate Specificity
D000345:Affinity Labels; D005419:Flavonoids; D010940:Plant Proteins; D002599:Chalcone; D008780:Methyltransferases; C072283:isoliquiritigen 2'-O-methyltransferase; D007535:Isomerases