Coumarins exhibit many biological activities and are the main specialised metabolites of Peucedanum praeruptorum Dunn, an important plant used in traditional Chinese medicine. In preliminary studies, we cloned several genes involved in coumarin biosynthesis in P. praeruptorum, such as 4-coumarate: CoA ligase (4CL), p-coumaroyl CoA 2'-hydroxylase (C2'H), feruloyl CoA 6'-hydroxylase (F6'H) and bergaptol O-methyltransferase (BMT). However, phenylalanine ammonia-lyase (PAL) in P. praeruptorum (PpPAL) has not yet been studied. In the present study, we cloned one novel PpPAL gene. Subsequently, the relationship between gene and compounds was studied using quantitative real-time PCR (qRT-PCR) and High Performance Liquid Chromatography (HPLC) analysis. Then, enzyme function was analyzed with L-phenylalanine (L-Phe) as substrate. These experiments showed that the coumarin content could be upregulated by methyl jasmonate (MeJA), UV irradiation and cold, which was consistent with increased expression levels of PpPAL. In addition, correlation analysis indicated that coumarins were partially related to PpPAL. And the recombinant protein could catalyze the conversion of L-Phe to trans-cinnamic acid (t-CA) with a Km of 120 ± 33 μM and a Kcat of 117 ± 32 min-1. Besides, Tyr110, Phe116, Gly117, Ser206, Leu209, Leu259, Tyr354, Arg357, Asn387 and Phe403 were essential for enzymatic activity based on three-dimensional modeling and site-directed mutagenesis experiments. Altogether these results highlight the importance of PpPAL in abiotically induced coumarin biosynthesis and provide further insights regarding the structure-function relationships of this protein.

中文翻译：

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前胡前胡苯丙氨酸解氨酶（PAL）在香豆素生物合成中的功能表征及相关性分析

香豆素具有多种生物活性，是重要的中药植物前胡的主要特化代谢产物。在初步研究中，我们克隆了几个参与 P. praeruptorum 香豆素生物合成的基因，例如 4-香豆酸：CoA 连接酶 (4CL)、对香豆酰 CoA 2'-羟化酶 (C2'H)、阿魏酰 CoA 6'-羟化酶 ( F6'H) 和香柠檬醇 O-甲基转移酶 (BMT)。然而，P. praeruptorum (PpPAL) 中的苯丙氨酸解氨酶 (PAL) 尚未被研究。在本研究中，我们克隆了一个新的 PpPAL 基因。随后，使用实时定量 PCR (qRT-PCR) 和高效液相色谱 (HPLC) 分析研究了基因与化合物之间的关系。然后，以 L-苯丙氨酸 (L-Phe) 作为底物分析酶功能。这些实验表明香豆素含量可以通过茉莉酸甲酯 (MeJA)、紫外线照射和寒冷上调，这与 PpPAL 表达水平的增加一致。此外，相关性分析表明香豆素与 PpPAL 部分相关。重组蛋白可以催化 L-Phe 转化为反式肉桂酸 (t-CA)，Km 为 120 ± 33 μM，Kcat 为 117 ± 32 min-1。此外，基于三维建模和定点诱变实验，Tyr110、Phe116、Gly117、Ser206、Leu209、Leu259、Tyr354、Arg357、Asn387 和 Phe403 对酶活性至关重要。总之，这些结果突出了 PpPAL 在非生物诱导的香豆素生物合成中的重要性，并提供了有关该蛋白质结构-功能关系的进一步见解。