cDNA cloning and biochemical characterization of S-adenosyl-L-methionine:: 2,7,4′-trihydroxyisoflavanone 4′-O-methyltransferase, a critical enzyme of the legume isoflavonoid phytoalexin pathway
cDNA cloning and biochemical characterization of S-adenosyl-L-methionine:: 2,7,4′-trihydroxyisoflavanone 4′-O-methyltransferase, a critical enzyme of the legume isoflavonoid phytoalexin pathway
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DOI：
10.1093/pcp/pcg034
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发表时间：
2003-02-01
影响因子：
4.9
通讯作者：
Ayabe, S
中科院分区：
文献类型：
--
作者：
Akashi, T;Sawada, Y;Ayabe, S
芒柄花素(7-羟基-4‘-甲氧基异黄酮，又称4’-O-甲基大豆苷元)是具有生态生理活性的豆科异黄酮类化合物的重要中间体。芒柄花素4‘-甲氧基的生物合成途径一直不清楚，因为在任何植物中都没有检测到从S-腺苷-L-蛋氨酸到大豆苷元4’-羟基的甲基转移。最近有一种假说认为，SAM：大豆苷元7-O-甲基转移酶(D7OMT)是一种具有不同区域特异性的酶，它通过与其他酶的胞内隔室参与芒柄花素的生物合成，但没有直接的证据。我们提出了一种以2，7，4‘-三羟基异黄酮为甲基受体，然后脱水合成芒柄花素的新方案。现在，我们通过筛选功能表达的棘果甘草cDNA克隆了一个编码SAM：2，7，4-三羟基异黄酮4‘-O-甲基转移酶(HI4’OMT)的基因。确定了反应产物为2，7-二羟基-4-甲氧基异黄酮类化合物。重组棘球绦虫D7OMT不显示HI4‘OMT活性，不含D7OMT的棘球绦虫HI4’OMT蛋白得到部分纯化。因此，HI4‘OMT与D7OMT是不同的，并进一步提出了它们的系统发育关系。HI4‘OMT可能与豌豆的(+)-6a-羟甲基-3-OMT在功能上相同。在几种豆科植物中发现了同源的cDNA，验证了莲花HI4‘OMT的催化功能，表明HI4’OMT是一般豆科植物中芒柄花素生物合成的酶。
Formononetin (7-hydroxy-4'-methoxyisoflavone, also known as 4'-O-methyldaidzein) is an essential intermediate of ecophysiologically active leguminous isoflavonoids. The biosynthetic pathway to produce 4'-methoxyl of formononetin has been unknown because the methyl transfer from S-adenosyl-L-methionine (SAM) to 4'-hydroxyl of daidzein has never been detected in any plants. A hypothesis that SAM: daidzein 7-O-methyltransferase (D7OMT), an enzyme with a different regiospecificity, is involved in formononetin biosynthesis through its intracellular compartmentation with other enzymes recently prevails, but no direct evidence has been presented. We proposed a new scheme of formononetin biosynthesis involving 2,7,4'-trihydroxyisoflavanone as the methyl acceptor and subsequent dehydration. We now cloned a cDNA encoding SAM: 2,7,4-trihydroxyisoflavanone 4'-O-methyltransferase (HI4'OMT) through the screening of functionally expressed Glycyrrhiza echinata (Fabaceae) cDNAs. The reaction product, 2,7-dihydroxy-4-methoxyisoflavanone, was unambiguously identified. Recombinant G. echinata D7OMT did not show HI4'OMT activity, and G. echinata HI4'OMT protein free from D7OMT was partially purified. HI4'OMT is thus concluded to be distinct from D7OMT, and their distant phylogenetic relationship was further presented. HI4'OMT may be functionally identical to (+)-6a-hydroxymaackiain 3-OMT of pea. Homologous cDNAs were found in several legumes, and the catalytic function of the Lotus japonicus HI4'OMT was verified, indicating that HI4'OMT is the enzyme of formononetin biosynthesis in general legumes.