Cinnamic acid 4-hydroxylase from the hornwort Anthoceros agrestis (AaC4H) was functionally expressed in the moss Physcomitrella patens and characterized at biochemical and molecular levels. Cinnamic acid 4-hydroxylase (C4H), a cytochrome P450-dependent hydroxylase, catalyzes the formation of 4-coumaric acid (=4-hydroxycinnamic acid) from trans-cinnamic acid. In the hornwort Anthoceros agrestis (Aa), this enzyme is supposed to be involved in the biosynthesis of rosmarinic acid (a caffeic acid ester of 3-(3,4-dihydroxyphenyl)lactic acid) and other related compounds. The coding sequence of AaC4H (CYP73A260) was expressed in the moss Physcomitrella patens (Pp_AaC4H). Protein extracts from the transformed moss showed considerably increased C4H activity driven by NADPH:cytochrome P450 reductase of the moss. Since Physcomitrella has own putative cinnamic acid 4-hydroxylases, enzyme characterization was carried out in parallel with the untransformed Physcomitrella wild type (Pp_WT). Apparent Km-values for cinnamic acid and NADPH were determined to be at 17.3 µM and 88.0 µM for Pp_AaC4H and 25.1 µM and 92.3 µM for Pp_WT, respectively. Expression levels of AaC4H as well as two Physcomitrella patens C4H isoforms were analyzed by quantitative real-time PCR. While PpC4H_1 displayed constantly low levels of expression during the whole 21-day culture period, AaC4H and PpC4H_2 increased their expression during the first 6-8 days of the culture period and then decreased again. This work describes the biochemical in vitro characterization of a cytochrome P450-dependent enzyme, namely C4H, heterologously expressed in the haploid model plant Physcomitrella patens.

中文翻译：

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金丝桃肉桂酸 4-羟化酶在立球菌中的功能表达和表征。

来自金缕梅 Anthoceros agrestis (AaC4H) 的肉桂酸 4-羟化酶在苔藓立球藻中功能性表达，并在生化和分子水平上进行了表征。肉桂酸 4-羟化酶 (C4H) 是一种细胞色素 P450 依赖性羟化酶，可催化反式肉桂酸形成 4-香豆酸 (=4-羟基肉桂酸)。在金银花 Anthoceros agrestis (Aa) 中，这种酶被认为参与了迷迭香酸（一种 3-(3,4-二羟基苯基)乳酸的咖啡酸酯）和其他相关化合物的生物合成。AaC4H（CYP73A260）的编码序列在苔藓Physcommitrella patens（Pp_AaC4H）中表达。来自转化苔藓的蛋白质提取物显示出由苔藓的 NADPH：细胞色素 P450 还原酶驱动的 C4H 活性显着增加。由于立球藻具有自己推定的肉桂酸 4-羟化酶，酶表征与未转化的立球菌野生型 (Pp_WT) 平行进行。Pp_AaC4H 的肉桂酸和 NADPH 的表观 Km 值分别为 17.3 µM 和 88.0 µM，Pp_WT 的表观 Km 值分别为 25.1 µM 和 92.3 µM。通过定量实时 PCR 分析 AaC4H 以及两种立球菌 C4H 同种型的表达水平。虽然 PpC4H_1 在整个 21 天的培养期间一直表现出低水平的表达，但 AaC4H 和 PpC4H_2 在培养的前 6-8 天增加了它们的表达，然后又下降了。这项工作描述了一种细胞色素 P450 依赖性酶，即 C4H，在单倍体模型植物立球藻中异源表达的体外生化表征。