前胡香豆素生物合成中关键异戊烯基转移酶的功能验证及催化机制研究

Coumarins are the main medicinal active ingredients of Traditional Chinese Medicine “Qianhu”. The characteristic compounds in Peucedanum praeruptorum and Angelica decursiva are angular coumarins and linear coumarins, respectively, which is also the reason for their classification and different pharmacological activities. However, the biosynthesis mechanism that determines their differences is not yet clear. Prenyltransferases transfer prenyl moiety to 6- or 8-position of the coumarins skeleton, which is a key step in forming angular or linear coumarins. In our previous work, nine candidate genes have been screened and amplified in P. praeruptorum and A. decursiva by gene differential expression analysis, structural domain prediction and transcriptome- metabolome analysis. In this program, the candidate genes in P. praeruptorum and A. decursiva were functionally characterized and further confirmed by transgenosis/RNAi/gene silencing. Furthermore, physiological and biochemical differences, such as gene transcription, product specificity, enzyme kinetics and so on, were employed to elucidate their role in determining the biosynthesis of angular or linear coumarins. At last, protein crystallization and site-specific mutagenesis were conducted to deeply reveal the catalytic mechanism and illuminate the reason in formation of different products of two kinds of protein. Through this study, the mechanism of prenyltransferases in the formation of angular and linear coumarins was fully elucidated, which will laid a scientific basis for exploring coumarins biosynthesis pathway, synthetic biology researching and improving the quality of “Qianhu”.

香豆素是传统中药前胡中的主要药用活性成分。角型与线型香豆素分别是白花与紫花前胡中的特征化合物，也是两者分类、药效差异的关键，但决定其不同类型的生物合成机制尚不明确。异戊烯基转移酶在香豆素母核6位或8位引入异戊烯基，是决定角型或线型香豆素的关键步骤。在前期工作中，申请人通过基因差异表达分析、结构域预测及组学方法，在白花与紫花前胡中筛选并扩增得到9条可能行使该功能的基因。本项目拟分别对两类前胡中的候选基因进行功能验证，并根据转基因/RNAi/基因沉默等实验进一步确定目的基因；进而，比较两者转录、产物特异性、酶动力学等生理生化差异，分别阐明两者在决定角型或线型香豆素中的作用；最后，通过蛋白结晶与定点突变实验，深入揭示两类蛋白的催化机制并阐明两者形成不同类型产物的原因。本研究将全面阐明异戊烯基转移酶在角型与线型香豆素形成中的机制，为探究香豆素生源合成途径、合成生物学研究及前胡品质改良提供科学依据。

香豆素是传统中药前胡中的主要药用活性成分。角型与线型香豆素分别是白花与紫花前胡中的特征化合物，也是两者分类、药效差异的关键，但决定其不同类型的生物合成机制尚不明确。异戊烯基转移酶在香豆素母核6位或8位引入异戊烯基，是决定角型或线型香豆素的关键步骤。在前期工作中，申请人通过基因差异表达分析、结构域预测及组学方法，在白花与紫花前胡中筛选并扩增得到9条可能行使该功能的基因。本项目拟分别对两类前胡中的候选基因进行功能验证，并根据转基因/RNAi/基因沉默等实验进一步确定目的基因；进而，比较两者转录、产物特异性、酶动力学等生理生化差异，分别阐明两者在决定角型或线型香豆素中的作用；最后，通过蛋白结晶与定点突变实验，深入揭示两类蛋白的催化机制并阐明两者形成不同类型产物的原因。经过三年的工作，（1）我们克隆并功能验证了4条来源于白花前胡，紫花前胡的基因，并研究了他们的生理生化特征，催化机制及产物特异性的机制。此外，（2）我们还验证了前胡苯丙氨酸裂解酶，（3）补骨脂素合成酶，异补骨脂素合成酶及(4)咖啡酸氧甲基转移酶的活性。本研究全面阐明了前胡香豆素形成的生物合成机制，为探究香豆素生源合成途径、合成生物学研究及前胡品质改良提供科学依据。