cDNA cloning and functional characterisation of CYP98A14 and NADPH:cytochrome P450 reductase from Coleus blumei involved in rosmarinic acid biosynthesis
cDNA cloning and functional characterisation of CYP98A14 and NADPH:cytochrome P450 reductase from Coleus blumei involved in rosmarinic acid biosynthesis
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DOI：
10.1007/s11103-008-9420-7
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发表时间：
2009-02-01
影响因子：
5.1
通讯作者：
Petersen, Maike
中科院分区：
文献类型：
--
作者：
Eberle, David;Ullmann, Pascaline;Petersen, Maike
迷迭香酸生物合成的最终反应，即芳香族3-和3 '-羟基的引入，由细胞色素P450依赖性羟化酶催化。编码CYP 98 A14以及NADPH：细胞色素P450还原酶（CPR）的cDNA从彩叶草中分离并在酿酒酵母中活性表达。CYP 98 A14-cDNA的开放阅读框为1521个核苷酸，与4-香豆酰莽草酸/奎尼酸3-羟化酶具有高度的相似性。携带CYP 98 A14蛋白的酵母微粒体催化4-香豆酰-3 '，4'-二羟基苯基乳酸的3-羟基化和咖啡酰-4 '-羟基苯基乳酸的3'-羟基化，在两种情况下均形成迷迭香酸。测定4-香豆酰-3 '，4'-二羟基苯基乳酸酯和咖啡酰-4 '-羟基苯基乳酸酯的表观K（m）值分别为5 μ M和40 μ M。CYP 98 A14不同于来自其他植物的CYP 98，因为4-香豆酰莽草酸或-奎尼酸不被接受为底物。在相同的酵母细胞中共表达彩叶草CPR和CYP 98 A14使羟基化活性增加了7倍。彩叶草CYP 98 A14是一种新的双功能细胞色素P450，专门用于迷迭香酸的生物合成。
The final reactions of rosmarinic acid biosynthesis, the introduction of the aromatic 3- and 3'-hydroxyl groups, are catalysed by cytochrome P450-dependent hydroxylases. The cDNAs encoding CYP98A14 as well as a NADPH:cytochrome P450 reductase (CPR) were isolated from Coleus blumei and actively expressed in Saccharomyces cerevisiae. The CYP98A14-cDNA showed an open reading frame of 1521 nucleotides with high similarities to 4-coumaroylshikimate/quinate 3-hydroxylases. Yeast microsomes harbouring the CYP98A14 protein catalysed the 3-hydroxylation of 4-coumaroyl-3',4'-dihydroxyphenyllactate and the 3'-hydroxylation of caffeoyl-4'-hydroxyphenyllactate, in both cases forming rosmarinic acid. Apparent K (m)-values for 4-coumaroyl-3',4'-dihydroxyphenyllactate and caffeoyl-4'-hydroxyphenyllactate were determined to be at 5 mu M and 40 mu M, respectively. CYP98A14 differs from CYP98s from other plants, since 4-coumaroylshikimate or -quinate were not accepted as substrates. Coexpression of the Coleus blumei CPR and CYP98A14 in the same yeast cells increased the hydroxylation activity up to sevenfold. CYP98A14 from Coleus blumei is a novel bifunctional cytochrome P450 specialised for rosmarinic acid biosynthesis.