Abstract
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.
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