丹参酮生物合成途径中结构修饰关键酶的结构与功能研究

As the model for traditional Chinese medicines, Salvia miltiorrhiza (called danshen in China) is an important herb with high medicinal values. More than 40 tanshinone are lipophelic compounds isolated from danshen, derived from their precursor through structure modifications, have been widely used for treatment of coronary disease and many other diseases. The studies on genome, biosynthetic pathway of secondary metabolites of danshen have fully advanced in China. However, so far no scientific research on structural study of key enzyme of biosynthetic pathway of tanshinone is reported. CYP76AH1, CYP76AH3 and CYP76AK1 are key enzymes on the structure-modifying stage of the biosynthetic pathway of tanshinone. However, the catalytic mechanism of CYP76AH1 is not clear and the promiscuity of CYP76AH3 and CYP76AK1 leads to a supposed bifurcating biosynthetic pathway of tanshinone. We resolved the crystal structures of apo-protein of CYP76AH1 and its complex with a weak inhibitor 4-PI in our laboratory. We plan to start a project on structural studies to obtain the structures of CYP76AH1 complexed with its substrate or potential intermediate product to verify its catalytic mechanism, and the structures of CYP76AH3 and CYP76AK1 complexed with their substrates or products to explain their substrate specificity and diversity of catalytic activity, and help to verify the bifurcating biosynthetic pathway. And more, we will perform site-directed mutagenesis and activity assay on key residues of CYP76AH3 active center to develop biocatalysts.

丹参是有重要药用价值的模式中药，多达40种的丹参酮是其脂溶性有效成分，由前体化合物经过结构修饰生成，用于治疗冠心病等多种疾病。我国的丹参基因组学、丹参酮生物合成途径研究达到了国际先进水平，但针对该途径关键酶的晶体结构研究尚未见报道。CYP76AH1，CYP76AH3和CYP76AK1是丹参酮生物合成途径结构修饰阶段的关键酶。76AH1的催化机制尚存争议；76AH3和76AK1的催化功能错综复杂，只提出了假定的叉状合成途径。申请人解析了76AH1及其与弱抑制剂4-PI复合物的晶体结构。拟通过4-PI诱导配体与酶活性中心的结合，解析76AH1与底物及潜在中间产物复合物的晶体结构，验证76AH1的催化机制；解析76AH3和76AK1与底物、产物复合物的结构，阐释底物特异性及活性多样性，为叉状合成路径的确认提供结构生物学证据；对76AH3活性中心的关键氨基酸定点突变和活性分析，探索开发生物催化剂。

CYP76AH1，CYP76AH3和CYP76AK1是丹参酮生物合成途径中结构修饰关键酶。本研究解析了CYP76AH1及其与抑制剂4-P、底物次丹参酮二烯复合物的晶体结构，阐释了CYP76AH1催化机制，用GC-MS进行催化活性分析；成功培养了CYP76AH3与CYP76AK1的晶体；解析了CYP76AH3晶体结构，并与其三个底物铁锈醇、柳杉酚和11-羟基铁锈醇进行分子对接，通过点突变模拟预测与底物有氢键及疏水相互作用的氨基酸。本研究揭示了相关关键酶结构与功能的关系，将为基于关键酶三维结构的酶工程以及利用合成生物学手段生产丹参酮类天然药物奠定基础，为丹参资源的可持续利用提供有效策略。