Flavonoid 3'-O-methyltransferase from rice: cDNA cloning, characterization and functional expression.
Plant O-methyltransferases (OMTs) are known to be involved in methylation of plant secondary metabolites, especially phenylpropanoid and flavonoid compounds. An OMT, ROMT-9, was cloned and characterized from rice using a reverse transcriptase polymerase chain reaction (RT-PCR). The blast results for ROMT-9 showed a 73% identity with caffeic acid OMTs from maize and Triticum aestivum. ROMT-9 was expressed in Escherichia coli and its recombinant protein was purified using affinity chromatography. It was then tested for its ability to transfer the methyl group of S-adenosyl-l-methionine to the flavonoid substrates, eriodictyol, luteolin, quercetin, and taxifolin, all of which have a 3'-hydroxyl functional group. The reaction products were analyzed using TLC, HPLC, HPLC/MS, and NMR spectroscopy. The NMR analysis showed that ROMT-9 transferred the methyl group specifically to the 3'-hydroxyl group of quercetin, resulting in the formation of its methoxy derivative. Furthermore, ROMT-9 converted flavonoids containing the 3'-hydroxy functional group such as eriodictyol, luteolin, quercetin and taxifolin into the corresponding methoxy derivatives, suggesting that ROMT-9 is an OMT with strict specificity for the 3'-hydroxy group of flavonoids.
Kim
Bong-Gyu
BG
Bio/Molecular Informatics Center, Department of Molecular Biotechnology, Konkuk University, 1 Hwayang-dong, Kwangjin-gu, Seoul 143-701, Republic of Korea.
Lee
Youngshim
Y
Hur
Hor-Gil
HG
Lim
Yoongho
Y
Ahn
Joong-Hoon
JH
D000595:Amino Acid Sequence; D002109:Caffeic Acids / Q000737:chemistry / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000032:analysis*; D004926:Escherichia coli / Q000235:genetics; D044950:Flavanones / Q000378:metabolism; D005419:Flavonoids / Q000378:metabolism*; D044948:Flavonols / Q000378:metabolism; D005786:Gene Expression Regulation; D047311:Luteolin / Q000378:metabolism; D008969:Molecular Sequence Data; D012275:Oryza / Q000737:chemistry / Q000201:enzymology*; D011497:Protein O-Methyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011794:Quercetin / Q000031:analogs & derivatives / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000378:metabolism; D013379:Substrate Specificity; D014908:Triticum / Q000737:chemistry; D003313:Zea mays / Q000737:chemistry
D002109:Caffeic Acids; D018076:DNA, Complementary; D044950:Flavanones; D005419:Flavonoids; D044948:Flavonols; D011994:Recombinant Proteins; D012436:S-Adenosylmethionine; D011794:Quercetin; C003377:taxifolin; D011497:Protein O-Methyltransferase; D047311:Luteolin; C007619:eriodictyol; C040048:caffeic acid