Structure and Function of P450 Enzymes in Steroid Hormone Biosynthesis

描述(申请人提供)：大型细胞色素P450(P450，CYP)超家族分布于所有生物界。P450反应的两大类是异源代谢(如药物、致癌物)和内源性底物生物合成(如激素、二十烷类化合物)。在17,000个P450超家族成员中，约50个亚家族成员的高分辨率结构是已知的(所有成员都显示了共同的结构折叠)，但大多数单个结构元素的确切作用尚不清楚。我们建议通过研究参与类固醇激素生物合成的两个P450来解决这个问题：哺乳动物的P450 21A2已知存在影响其类固醇21-羟化活性的&gt；100氨基酸突变，以及人的P450 17A1和斑马鱼的P450 17A1和17A2，后两种是不同的酶，它们的类固醇17α-羟化/17，20-裂解酶活性有很大的变化。通过详细分析这些酶(结构和动力学)，我们将能够建立一个比目前可用的更精确的P450结构/功能的观点。我们预计这些研究将详细阐明这两个重要的P450如何在类固醇激素生物合成中发挥作用，然后这些结果可以应用于理解其他P450的结构/功能。P450 21A2和17A1的突变是一组被称为先天性肾上腺增生症的遗传病的主要原因，而P450 17A1是治疗前列腺癌的重要药物靶点。这些研究的结果将使人们更好地了解这两种疾病。利用确定氨基酸变化如何改变这些酶的功能属性的优势，我们将确定特定结构区域所起的作用。该方法将涉及异源蛋白质的表达/纯化/突变、X射线结晶学、详细的动力学分析和其他生化研究(例如底物和其他蛋白质的结合)。预计这些创新研究将对P450的结构/功能产生新的见解和意想不到的新理解。

DESCRIPTION (provided by applicant): The large cytochrome P450 (P450, CYP) superfamily is distributed throughout all biological kingdoms. The two general classes of P450 reactions are xenobiotic metabolism (e.g., drugs, carcinogens) and endogenous substrate biosynthesis (e.g., hormones, eicosanoids). High-resolution structures of members of ~50 subfamilies among the >17,000 P450 superfamily members are known (all showing a common structural fold), but the precise roles of most individual structural elements are not clearly established. We propose to address this topic by investigating two P450s involved in steroid hormone biosynthesis: mammalian P450 21A2, which is known to have >100 amino acid mutations that influence its steroid 21-hydroxylation activity, and human P450 17A1 and zebrafish P450s 17A1 and 17A2, the latter two being distinct enzymes with dramatic variations in their steroid 17α-hydroxylation/17,20-lyase activities. By analyzing these enzymes in detail (structure and kinetics), we will be able to establish a much more precise view of P450 structure/function than is currently available. We anticipate that these studies will clarify in detail how these two important P450s can function in steroid hormone biosynthesis, and the results can then be applied to understanding the structure/function of other P450s. Mutations in both P450 21A2 and 17A1 are the major causes of a genetic group of diseases known as congenital adrenal hyperplasia, and P450 17A1 is an important drug target for treatment of prostate cancer. The results from these studies will allow a better understanding of both diseases. Taking advantage of determining how amino acid alterations modify functional properties of these enzymes, we will identify the roles that specific structural regions play. The methodology will involve heterologous protein expression/purification/mutagenesis, X-ray crystallography, detailed kinetic analysis, and other biochemical studies (e.g., binding of substrates and other proteins). It is expected that these innovative studies will produce novel insights into and unexpected new understanding of P450 structure/function.