CYP76B74 Catalyzes the 3 ''-Hydroxylation of Geranylhydroquinone in Shikonin Biosynthesis
CYP76B74 Catalyzes the 3 ''-Hydroxylation of Geranylhydroquinone in Shikonin Biosynthesis
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CYP76B74 催化紫草素生物合成中香叶基氢醌的 3'-羟基化
DOI：
10.1104/pp.18.01056
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发表时间：
2019
期刊：
影响因子：
7.4
通讯作者：
Huang Luqi
中科院分区：
文献类型：
--
作者：
Wang Sheng;Wang Ruishan;Liu Tan;Lv Chaogeng;Liang Jiuwen;Kang Chuanzhi;Zhou Liangyun;Guo Juan;Cui Guanghong;Zhang Yan;Werck Reichhart Daniele;Guo Lanping;Huang Luqi
紫草素及其衍生物是紫草科植物中含量最丰富的萘醌类色素。紫草素生物合成途径中的一个关键步骤，即异戊烯化的酚类中间体香叶基氢醌的C-3′′羟基化，预计由细胞色素P450催化。为了鉴定与已知参与紫草素生物合成的基因转录谱相似的细胞色素P450候选基因，我们对紫草素熟练和紫草素缺陷细胞系的转录组数据集进行了共表达分析，并研究了候选基因在新疆紫草不同器官中的空间表达。在以香叶基对苯二酚为底物的生化试验中，CYP 76 B74表现出香叶基对苯二酚3′′-羟化酶活性并产生3′′-羟基香叶基对苯二酚。InCYP 76 B74 RNA干扰A.常染色毛状根中，紫草素衍生物的积累显著减少，表明CYP 76 B74是紫草素生物合成所必需的。系统发育分析证实，CYP 76 B74属于CYP 76 B亚家族，最有可能来自祖先的香叶醇10-羟化酶。在亚细胞定位分析中，GFP-CYP 76 B74融合定位于内质网膜。我们的研究结果表明，CYP 76 B74高效催化紫草素生物合成中的关键羟基化步骤。本文描述的CYP 76 B74的表征为进一步探索生成萘醌骨架的闭环反应以及香叶基氢醌3′′-羟化酶向二氢棘呋喃的替代代谢铺平了道路。
Shikonin and its derivatives are the most abundant naphthoquinone pigments formed in species of the medicinally and economically valuable Boraginaceae. A key step in the shikonin biosynthetic pathway, namely the C-3′′ hydroxylation of the prenylated phenolic intermediate geranylhydroquinone, is expected to be catalyzed by a cytochrome P450. To identify cytochrome P450 candidates with transcription profiles similar to those of genes known to be involved in shikonin biosynthesis, we carried out coexpression analysis of transcriptome data sets of shikonin-proficient and shikonin-deficient cell lines and examined the spatial expression of candidate genes in different organs ofArnebia euchroma. In biochemical assays using geranylhydroquinone as the substrate, CYP76B74 exhibited geranylhydroquinone 3′′-hydroxylase activity and produced 3′′-hydroxy-geranylhydroquinone. InCYP76B74RNA interferenceA. euchromahairy roots, shikonin derivative accumulation decreased dramatically, which demonstrated that CYP76B74 is required for shikonin biosynthesis in the plant. Phylogenetic analysis confirmed that CYP76B74 belonged to the CYP76B subfamily and was most likely derived from an ancestral geraniol 10-hydroxylase. In a subcellular localization analysis, a GFP-CYP76B74 fusion localized to endoplasmic reticulum membranes. Our results demonstrate that CYP76B74 catalyzes the key hydroxylation step in shikonin biosynthesis with high efficiency. The characterization of the CYP76B74 described here paves the way for further exploration of the ring closure reactions generating the naphthoquinone skeleton as well as for the alternative metabolism of geranylhydroquinone 3′′-hydroxylase to dihydroechinofuran.