【作者】 刘玲；

【导师】 许莲花；

【作者基本信息】 浙江大学 ， 海洋科学， 2016， 硕士

【摘要】 阿维链霉菌细胞色素P450是细菌体内生合成和氧化异源化学物质的重要催化剂,该类酶因其催化底物的区位特异性和空间立体特异性引起了化学家和生物学家的极大关注。CYP105D7作为该酶家族中的成员之一可以催化多种底物单加氧化生成新的化合物。已知二氢黄酮类化合物对治疗乳腺癌具有积极的作用效果,而美伐他汀是高效的降胆固醇药物,已有研究表明在该类底物分子中引入羟基均可提高化合物的活性。本文研究了阿维链霉菌细胞色素P450——CYP105D7对二氢黄酮类化合物柚皮素和松鼠素以及他汀类药物美伐他汀的生物转化和催化机制。对CYP105D7进行异源表达和纯化,通过紫外光谱分析检测酶与底物结合的亲和力大小,并分别对这些化合物进行了体外生物转化和全细胞生物转化,试图得到活性更高的羟基化产物。酶和底物的分子模拟实验进一步分析了CYP105D7的催化反应机制。紫外差光谱分析测得酶与柚皮素、松鼠素的解离常数分别为Kd=103±3μM和Kd=52.3±2.1μM,希尔常数分别为nH=1.25±0.03和nH=1.47±0.06。生物转化二氢黄酮类化合物的HPLC和LC-MS结果表明,CYP105D7可以对柚皮素和松鼠素等进行羟基化,并对柚皮素进行3’位羟基化生成圣草酚。一个酶分子结合两分子柚皮素并以一种略微协同的方式对底物进行催化,这种多分子结合的模式可能是CYP105D7对二氢黄酮类进行3’位羟基化所必需的。对于美伐他汀的生物转化,紫外差光谱分析和分子模拟实验表明CYP105D7对底物进行生物转化的可能性很高,进一步研究发现CYP105D7对美伐他汀的生物转化受美伐他汀的内酯和羧酸两种形式的影响。更多还原

【Abstract】 Cytochrome P450 of Streptomyces avermitilis are important catalysts for the biosynthesis and oxidation of xenobiotic chemicals in bacteria, such enzyme has attracted wide attention of the chemists and biologists due to their regio- and stereospecificity during the catalysis of substrates. As one of the enzyme family members, CYP105D7 is able to catalyze the monooxygenation of a variety of natural products into novel compounds. It is known that flavonones play positive roles in the treatment on breast cancer, and mevastatin is a kind of high efficient anticholesteremic agent, moreover, studies have indicated that the activities will all be enhanced when introducing hydroxyl groups into these compounds. In this thesis, the biotransfonnation and catalytic mechanism of flavonone naringenin and pinocembrin as well as mevastatin(a statin chemical)by CYP105D7 were investigated. By means of heterologous expression and purification of CYP105D7 and determination with UV spectroscopy, the binding affinity of enzyme with substrates were detected and determined, meanwhile, bioconversion in vitro and whole cell biotransformation in vivo of these compounds were carried out, trying to obtain the hydroxylated products with higher activities. The molecular dockings of CYP105D7 with these compounds were also conducted to analyze the catalytic reaction mechanism of the enzyme. The calculated results of UV difference spectroscopy indicated that the dissociation constants and Hill coefficients of naringenin and pinocembrin were Kd= 103±3μM, nH=1.25±0.03 and Kd= 52.3±2.1μM,nH= 1.47±0.06, respectively. HPLC and LC-MS analysis demonstrated that CYP105D7 had the ability to hydroxylate both naringeinin and pinocembrin, and the bioconversion product of naringenin was proved to be eriodictyol at the 3’site, moreover, the substrate-binding pocket of CYP105D7 was sufficiently wide to accommodate two naringenin molecules simultaneously. And multiple naringenin molecules are bound to CYP105D7 with weak cooperativity, which may be necessary to the 3’-hydroxylation of flavanone. For the bioconversion of mevastatin, the UV difference spectroscopy and molecular docking experiments showed that the possibility of substrates biotransfonnation by CYP105D7 was very high, and the subsequent investigation found that bioconversion of mevastatin was affected by the lactone and acid forms of this compound.更多还原