Shikonin is a natural naphthoquinone derivative that specifically occurs in boraginaceous plants, and the major active ingredient of the medicinal plant Lithospermum erythrorhizon. Previously, a cytochrome P450 oxygenase (CYP) CYP76B74 catalyzing 3″-hydroxylation of geranylhydroquinone (GHQ) - a key intermediate of shikonin biosynthesis, was identified from cultured cells of Arnebia euchroma. However, the enzymes catalyzing oxidation of the geranyl side-chain of GHQ from L. erythrorhizon remain unknown. In this study, we performed transcriptome analysis of different tissues (red roots and green leaves/stems) from L. erythrorhizon using RNA sequencing technology. Highly expressed CYP genes found in the roots were then heterologously expressed in Saccharomyces cerevisiae and functionally screened with GHQ as the substrate. As the result, two CYPs of CYP76B subfamily catalyzing the oxidation of GHQ were characterized. CYP76B100 catalyzed the hydroxylation of the geranyl side-chain of GHQ at the C-3″ position to form 3″-hydroxyl geranylhydroquinone (GHQ-3″-OH). The enzyme CYP76B101 carried out oxidation reaction of GHQ at the C-3″ position to produce a 3″-carboxylic acid derivative of GHQ (GHQ-3″-COOH) as well as GHQ-3″-OH. This enzyme-catalyzed oxidation reaction with GHQ as the substrate is reported for the first time. This study implicates CYP76B100 and CYP76B101 as having a potential role in shikonin biosynthesis in L. erythrorhizon.

中文翻译：

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从紫草中获得的两种细胞色素 P450 在紫草素生物合成过程中催化香叶基氢醌氧化的潜在作用

紫草素是一种天然存在于紫草科植物中的萘醌衍生物，是药用植物紫草的主要活性成分。以前，从 Arnebia euchroma 的培养细胞中鉴定出一种细胞色素 P450 加氧酶 (CYP) CYP76B74 催化香叶基氢醌 (GHQ) 的 3"-羟基化 - 紫草素生物合成的关键中间体。然而，催化来自 L. erythrorhizon 的 GHQ 香叶基侧链氧化的酶仍然未知。在这项研究中，我们使用 RNA 测序技术对来自 L. erythrorhizon 的不同组织（红根和绿叶/茎）进行了转录组分析。在根中发现的高表达 CYP 基因随后在酿酒酵母中异源表达，并以 GHQ 作为底物进行功能筛选。作为结果，表征了催化GHQ氧化的CYP76B亚家族的两个CYP。CYP76B100 催化 GHQ 的香叶基侧链在 C-3" 位置羟基化形成 3"-羟基香叶基氢醌 (GHQ-3"-OH)。酶CYP76B101在C-3″位置进行GHQ的氧化反应，产生GHQ的3″-羧酸衍生物(GHQ-3″-COOH)以及GHQ-3″-OH。这种以GHQ为底物的酶催化氧化反应是首次报道。该研究表明 CYP76B100 和 CYP76B101 在 L. erythrorhizon 中的紫草素生物合成中具有潜在作用。酶CYP76B101在C-3″位置进行GHQ的氧化反应，产生GHQ的3″-羧酸衍生物(GHQ-3″-COOH)以及GHQ-3″-OH。这种以GHQ为底物的酶催化氧化反应是首次报道。该研究表明 CYP76B100 和 CYP76B101 在 L. erythrorhizon 中的紫草素生物合成中具有潜在作用。酶CYP76B101在C-3″位置进行GHQ的氧化反应，产生GHQ的3″-羧酸衍生物(GHQ-3″-COOH)以及GHQ-3″-OH。这种以GHQ为底物的酶催化氧化反应是首次报道。该研究表明 CYP76B100 和 CYP76B101 在 L. erythrorhizon 中的紫草素生物合成中具有潜在作用。