Structure and Function of P450 Enzymes in Steroid Hormone Biosynthesis

类固醇激素生物合成中 P450 酶的结构和功能

• 批准号: 8740504
• 负责人: MARTIN EGLI
• 金额: $ 41.13万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-30 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8740504
• 关键词: Active Sites; Address; Adrenal Cortex; Aldosterone; Amino Acids; Anabolism; Androstenedione; Androsterone; Aryl Hydrocarbon Hydroxylases; Beryllium; Binding; Biochemical; Biological; Carbon; Carcinogens; Cattle; Cholesterol; Clinical; Congenital adrenal hyperplasia; Cortodoxone; Cytochrome P450; Cytochromes; Cytochromes b; Disease; Drug Targeting; Eicosanoids; Elements; Endocrine System Diseases; Enzymes; Genes; Genetic; Glucocorticoids; Goals; Gonadal Steroid Hormones; Gonadal structure; Heme; Hormones; Human; Human Activities; Hydrocortisone; Hydroxylation; Indium; Individual; Kinetics; Lead; Location; Malignant neoplasm of prostate; Methodology; Mineralocorticoids; Mixed Function Oxygenases; Mutagenesis; Mutation; Oxidoreductase; Pathway interactions; Pharmaceutical Preparations; Play; Positioning Attribute; Pregnenolone; Progesterone; Property; Proteins; Reaction; Reporting; Resolution; Roentgen Rays; Role; Scheme; Series; Site; Site-Directed Mutagenesis; Steroid 17-alpha-monooxygenase; Steroid 21-Monooxygenase; Steroid Hydroxylases; Steroid biosynthesis; Steroids; Structure; Structure-Activity Relationship; Time; Tissues; Variant; X-Ray Crystallography; Xenobiotic Metabolism; Zebrafish; base; cell type; drug candidate; hormone biosynthesis; inhibitor/antagonist; innovation; insight; interest; member; mutant; novel; protein expression; protein function; protein structure; public health relevance; research study; steroid hormone; steroid hormone biosynthesis

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 17?-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.

描述（由申请人提供）：大细胞色素P450（P450，P450）超家族分布于所有生物界。P450反应的两大类是异生物质代谢（例如，药物，致癌物）和内源性底物生物合成（例如，激素、类花生酸）。在超过17，000个P450超家族成员中，约有50个亚家族成员的高分辨率结构是已知的（都显示出共同的结构折叠），但大多数单个结构元件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种P450来解决这个问题：哺乳动物P450 21 A2，已知其具有影响其类固醇21-羟基化活性的>100个氨基酸突变，以及人类P450 17 A1和斑马鱼P450 17 A1和17 A2，后两者是不同的酶，其类固醇17？羟基化/17，20-裂解酶活性。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/功能视图。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，其结果可用于了解其他P450的结构/功能。P450 21 A2和17 A1的突变是一组称为先天性肾上腺增生的遗传疾病的主要原因，P450 17 A1是治疗前列腺癌的重要药物靶点。这些研究的结果将有助于更好地了解这两种疾病。利用确定氨基酸改变如何改变这些酶的功能特性，我们将确定特定结构区域发挥的作用。该方法将涉及异源蛋白表达/纯化/诱变、X射线晶体学、详细的动力学分析和其他生物化学研究（例如，底物和其它蛋白质的结合）。预计这些创新的研究将产生新的见解和意想不到的新的理解P450的结构/功能。