Molecular cloning and characterization of a flavonoid-O-methyltransferase with broad substrate specificity and regioselectivity from Citrus depressa.
Molecular cloning and characterization of a flavonoid-O-methyltransferase with broad substrate specificity and regioselectivity from Citrus depressa.
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DOI：
10.1186/s12870-016-0870-9
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发表时间：
2016-08-22
影响因子：
5.3
通讯作者：
Toda H
中科院分区：
文献类型：
--
作者：
Itoh N;Iwata C;Toda H
黄酮类化合物是植物细胞中重要的次生代谢产物。特别地，已经报道了聚甲氧基类黄酮（PMF），包括川陈皮素，表现出各种健康支持特性，例如抗癌、抗炎和抗病原特性。然而，很难将PMF用于医药和饮食用途，因为植物细胞含有少量的这些化合物。植物细胞中PMFs的生物合成是通过O-甲基转移酶（FOMT）对黄酮羟基的甲基化来实现的，多种具有不同底物特异性和区域选择性的FOMT协同参与了PMFs的生物合成。在这项研究中，我们分离出5个基因编码FOMT（CdFOMT 1，3，4，5，和6）从柑橘，这是已知的积累nobilopropylin果皮的水果。这些基因编码Mg 2+非依赖性O-甲基转移酶，并且与高等植物类黄酮O-甲基转移酶显示出高氨基酸序列相似性（60- 95%）。这些基因之一是CdFOMT 5，它在大肠杆菌中成功表达为可溶性同源二聚体酶。重组CdFOMT 5亚基的分子量为42.0 kDa，含有一个6×组氨酸标签。该酶以S-腺苷甲硫氨酸（SAM）为甲基供体，对槲皮素、柚皮素、（-）-表儿茶素和雌马酚具有O-甲基转移酶活性，最适pH为7.0，最适温度为45 °C。重组CdFOMT 5对黄酮的3-、5-、6-和7-羟基具有甲基化活性，并以槲皮素为终产物，经全细胞反应系统合成了3，3 ′，5，7-四-O-甲基化槲皮素。因此，CdFOMT 5是一种O-甲基转移酶，具有广泛的底物特异性和区域选择性的类黄酮。从C. depressa，并测定了它们的核苷酸序列。CdFOMT 5在大肠杆菌中成功表达。coli细胞中表达，并对重组蛋白的酶学性质进行了表征。重组CdFOMT 5表明O-甲基转移酶活性的许多黄酮类化合物和广泛的区域选择性槲皮素作为底物。利用在大肠杆菌中表达的CdFOMT 5进行全细胞生物催化。以槲皮素为底物，在大肠杆菌细胞中进行反应，最终得到3，3 ′，5，7-四甲基槲皮素。本文的在线版本（doi：10.1186/s12870-016-0870-9）包含补充材料，可供授权用户使用。
Flavonoids are secondary metabolites that play significant roles in plant cells. In particular, polymethoxy flavonoids (PMFs), including nobiletin, have been reported to exhibit various health-supporting properties such as anticancer, anti-inflammatory, and anti-pathogenic properties. However, it is difficult to utilize PMFs for medicinal and dietary use because plant cells contain small amounts of these compounds. Biosynthesis of PMFs in plant cells is carried out by the methylation of hydroxyl groups of flavonoids by O-methyltransferases (FOMT), and many kinds of FOMTs with different levels of substrate specificity and regioselectivity are cooperatively involved in this biosynthesis. In this study, we isolated five genes encoding FOMT (CdFOMT1, 3, 4, 5, and 6) from Citrus depressa, which is known to accumulate nobiletin in the peels of its fruits. The genes encoded Mg2+-independent O-methyltransferases and showed high amino acid sequence similarity (60–95 %) with higher plant flavonoid O-methyltransferases. One of these genes is CdFOMT5, which was successfully expressed as a soluble homodimer enzyme in Escherichia coli. The molecular mass of the recombinant CdFOMT5 subunit was 42.0 kDa including a 6× histidine tag. The enzyme exhibited O-methyltransferase activity for quercetin, naringenin, (-)-epicatechin, and equol using S-adenosyl-l-methionine (SAM) as a methyl donor, and its optimal pH and temperature were pH 7.0 and 45 °C, respectively. The recombinant CdFOMT5 demonstrated methylation activity for the 3-, 5-, 6-, and 7-hydroxyl groups of flavones, and 3,3′,5,7-tetra-O-methylated quercetin was synthesized from quercetin as a final product of the whole cell reaction system. Thus, CdFOMT5 is a O-methyltransferase possessing a broad range of substrate specificity and regioselectivity for flavonoids. Five FOMT genes were isolated from C. depressa, and their nucleotide sequences were determined. CdFOMT5 was successfully expressed in E. coli cells, and the enzymatic properties of the recombinant protein were characterized. Recombinant CdFOMT5 indicated O-methyltransferase activity for many flavonoids and a broad regioselectivity for quercetin as a substrate. Whole-cell biocatalysis using CdFOMT5 expressed in E. coli cells was performed using quercetin as a substrate, and 3,3′,5,7-tetramethylated quercetin was obtained as the final product. The online version of this article (doi:10.1186/s12870-016-0870-9) contains supplementary material, which is available to authorized users.