西红花苷生物合成两个关键酶的催化机制一致性研究

西红花苷是素有“红色黄金”之称名贵中药西红花的主要活性成分，其生物合成的关键酶类胡萝卜素裂解双加氧酶CsCCD2催化机制不清楚，成为制约西红花苷合成生物学生产的难点。与西红花亲缘关系很远的栀子果实也含西红花苷，课题组研究发现其生物合成的关键酶是GjCCD4a，催化机制亦不清楚。本项目提出“关键酶CsCCD2和GjCCD4a催化机制的一致性是西红花苷生物合成的决定因素”学术假设。课题组拟从关键酶的活性部位、三维实体结构、与底物诱导契合等方面研究两个关键酶对底物番茄红素，β-胡萝卜素，玉米黄质催化产生产物西红花酸二醛（一个西红花苷合成的关键前体物质）的作用机制。进而比较两个关键酶催化作用，验证二者催化机制的一致性，获得具有优化催化能力的CsCCD2或GjCCD4a用于西红花苷的合成生物学生产。本项目为西红花苷合成生物学生产提供理论依据，对中药西红花和栀子的资源开发具有重要理论价值和应用前景。

Crocins are apocarotenoid glycosides, responsible for the pigmentation of the stigmas of Crocus sativus and of the mature fruits of Gardenia jasminoides. Crocin biosynthesis in saffron stigmas is initiated by carotenoid cleavage dioxygenase 2 (CsCCD2) and one in G. jasminoides does by GjCCD4a, which cleave zeaxanthin to produce crocetin dialdehyde. Up to date, their consistency and catalytic mechanisms of two key enzymes, CsCCD2 and GjCCD4a, are still uncharacterized. Here, crocin biosynthetic genes, CsCCD2 and GjCCD4a, would be functionally characterized in E. coli, in order to confirm that the protein products of the CsCCD2 and GjCCD4a genes would be able to cleave lycopene, β-carotene and zeaxanthin in bacterio, yielding crocetin dialdehyde, the first intermediate in crocin biosynthesis. Then, we will investigate its capacity to bind all three carotenoid substrates using protein structural modeling and docking analysis of CsCCD2 and GjCCD4a. Next, the active sites and induced fits of two key enzymes for substrates access will be revealed by crystal structures of these enzymes. Finally, we should compare their consistency between catalytic mechanisms of two key enzymes, CsCCD2 and GjCCD4a. Taken together, our investigation would provide significant insights into the evolution and molecular mechanism of crocin biosynthesis in crocin-producing plants. Especially, it would benefit to produce crocins using synthetic biology technology.