Division of Bioscience and Biotechnology, BMIC, RCD, Konkuk University, Seoul 143-701, Korea.
J Microbiol Biotechnol. 2010 Sep;20(9):1359-66. doi: 10.4014/jmb.1005.05012.
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.
从阿维链霉菌基因组中搜索发现，其最接近的同源物是几种 O-甲基转移酶。其中，一个基因（即 saomt5）通过聚合酶链反应使用序列特异性寡核苷酸引物被克隆到 pET-15b 表达载体中。用重组蛋白进行的生化特性分析表明，SaOMT5 是 S-腺苷-L-甲硫氨酸依赖性 O-甲基转移酶。几种化合物被测试为 SaOMT5 的底物。结果表明，SaOMT5 催化黄酮类化合物如 6,7-二羟基黄酮、2',3'-二羟基黄酮、3',4'-二羟基黄酮、槲皮素和 7,8-二羟基黄酮以及酚类化合物如咖啡酸和咖啡酰辅酶 A 的 O-甲基化。这些反应产物通过 TLC、HPLC、LC/MS 和 NMR 光谱进行分析。此外，SaOMT5 可以将含有邻二羟基基团的酚类化合物转化为 O-甲基化化合物，并且已知 6,7-二羟基黄酮是最好的底物。SaOMT5 将 6,7-二羟基黄酮转化为 6-羟基-7-甲氧基黄酮和 7-羟基-6-甲氧基黄酮，将咖啡酸分别转化为阿魏酸和异阿魏酸。此外，SaOMT5 被证明是一种依赖于 Mg2+的 OMT，并且 Mg2+离子对其活性的影响比 Ca2+、Fe2+和 Cu2+离子、EDTA 和无金属介质大五倍。
