Characterization of an O-methyltransferase from Streptomyces avermitilis MA-4680.
A search of the Streptomyces avermitilis genome reveals that its closest homologs are several O-methyltransferases. Among them, one gene (viz., saomt5) was cloned into the pET-15b expression vector by polymerase chain reaction using sequence-specific oligonucleotide primers. Biochemical characterization with the recombinant protein showed that SaOMT5 was S-adenosyl-L-methionine-dependent Omethyltransferase. Several compounds were tested as substrates of SaOMT5. As a result, SaOMT5 catalyzed Omethylation of flavonoids such as 6,7-dihydroxyflavone, 2',3'-dihydroxyflavone, 3',4'-dihydroxyflavone, quercetin, and 7,8-dihydroxyflavone, and phenolic compounds such as caffeic acid and caffeoyl Co-A. These reaction products were analyzed by TLC, HPLC, LC/MS, and NMR spectroscopy. In addition, SaOMT5 could convert phenolic compounds containing ortho-dihydroxy groups into Omethylated compounds, and 6,7-dihydroxyflavone was known to be the best substrate. SaOMT5 converted 6,7- dihydroxyflavone into 6-hydroxy-7-methoxyflavone and 7-hydroxy-6-methoxyflavone, and caffeic acid into ferulic acid and isoferulic acid, respectively. Moreover, SaOMT5 turned out to be a Mg2+-dependent OMT, and the effect of Mg2+ ion on its activity was five times greater than those of Ca2+, Fe2+, and Cu2+ ions, EDTA, and metal-free medium.
Yoon
Youngdae
Y
Division of Bioscience and Biotechnology, BMIC, RCD, Konkuk University, Seoul 143-701, Korea.
Park
Younghee
Y
Lee
Youngshim
Y
Yi
Yong Sub
YS
Jo
Geunhyeong
G
Park
Jun Cheol
JC
Ahn
Joong-Hoon
JH
Lim
Yoongho
Y
D000214:Acyl Coenzyme A / Q000378:metabolism; D000595:Amino Acid Sequence; D002109:Caffeic Acids / Q000378:metabolism; D047309:Flavones / Q000378:metabolism; D005419:Flavonoids / Q000378:metabolism; D008274:Magnesium / Q000502:physiology; D008780:Methyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
D000214:Acyl Coenzyme A; D002109:Caffeic Acids; D047309:Flavones; D005419:Flavonoids; D011994:Recombinant Proteins; C058645:caffeoyl-coenzyme A; D008780:Methyltransferases; D008274:Magnesium; C040048:caffeic acid