Cytochrome P450-reductase interactions in P450 1A subfamily

描述（由申请人提供）：细胞色素P450 （P450）是一种含血红素的单加氧酶，在化学致癌物的代谢激活和药物代谢中起关键作用。P450 1A亚家族的成员，P450 1A1和1A2，都被认为在癌变中很重要。人类肺中存在的P450 1A1被认为与肺癌有关，因为这种酶能够将环境污染物，如多环芳烃（PAHs）代谢为其致癌衍生物。P450 1A2是一种典型的肝脏P450，可氧化多种药物，并在杂环胺代谢激活中发挥重要作用，产生遗传毒性产物。因此，阐明这些酶的催化机制和活性的结构基础将有助于我们对p450相关的致癌作用有一些了解，并对抗癌药物的合理设计具有重要意义。由于P450在催化过程中需要氧化还原伙伴细胞色素P450还原酶（CPR），因此我们打算重点研究P450-CPR相互作用。本文提出的研究重点是酶-底物/抑制剂的相互作用以及P450与更大的配体，即P450还原酶（CPR）的相互作用，P450氧化还原伙伴。核心假设是活性位点的底物动力学和P450-CPR相互作用决定了酶的特异性。这些相互作用将受到CPR结合位点上P450残基的特性的影响，从而定义给定P450的区域特异性和立体特异性。这将通过理论技术（包括同源性建模和分子动力学）和实验方法（包括核磁共振、定点诱变和功能分析）的结合来研究。模型P450将是人类P450 1A1和1A2，目前正在我们实验室进行调查。由于这些酶具有较高的序列相似性（72%），但不同的底物特异性，因此构成了这些研究的理想系统。预计的结果将有助于我们解释异种生物的代谢，特别是致癌物质的激活。研究结果将广泛传播，并可用于学术界和工业界的药物发现目的。由此产生的计算方法将显著减少时间，节省材料，并限制药物代谢和设计领域的动物和/或人类研究。这些研究还将为博士后和研究生提供高级建模、生物物理和生化技术方面的培训。由于这个项目的跨学科性质，他们将接触到生物学研究的各个方面，并将学习一个全面的方法来解决生物学问题。此外，该项目的工作将促进参与不同研究方面的学生和博士后之间的合作和协作。他们还将接受稿件准备和提交程序方面的培训。这种类型的经验将有助于学生和博士后在未来的研究事业中取得成功。此外，该项目还将为本科生提供暑期研究机会，作为WV-BRIN和WV-INBRE项目的一部分。

DESCRIPTION (provided by applicant): Cytochromes P450 (P450s) are heme-containing monooxygenases that play a key role in the metabolic activation of chemical carcinogens and in the metabolism of drugs. The members of the P450 1A subfamily, P450 1A1 and 1A2, are both considered important in carcinogenesis. Human P450 1A1, which is present in lungs, is thought to be linked to lung cancer because this enzyme is able to metabolize environmental pollutants, such as polycyclic aromatic hydrocarbons (PAHs), to their carcinogenic derivatives. P450 1A2, a typical hepatic P450, oxidizes a number of drugs and plays a major role in the metabolic activation of heterocyclic amines to genotoxic products. Therefore, the elucidation of the catalytic mechanism of these enzymes and the structural basis for activity would give us some insight into P450-linked carcinogenesis and have important implications for the rational design of anticancer drugs. Since P450s require a redox partner, cytochrome P450 reductase (CPR), during catalysis, we intend to focus on the investigation of P450-CPR interactions. The studies proposed here focus both on the investigation of enzyme-substrate/inhibitor interactions and the interactions of P450s with a larger ligand, namely P450 reductase (CPR), a P450 redox partner. The central hypothesis is that both substrate dynamics in the active site and P450-CPR interactions determine enzyme specificity. These interactions will be affected by the identity of P450 residues at the CPR binding site, defining regio- and stereospecificity of a given P450. This will be studied by a combination of theoretical techniques including homology modeling and molecular dynamics, and experimental approaches, including NMR, site-directed mutagenesis and functional analyses. The model P450s will be human P450 1A1 and 1A2, currently under investigation in our laboratory. These enzymes constitute an ideal system for these studies due to their high sequence similarity (72%), but different substrate specificities. The projected outcomes will help us to explain the metabolism of xenobiotics, in particular carcinogen activation. The findings will be widely disseminated and available for drug discovery purposes both in academia and industry. The resulting computational methodology will significantly reduce time, save materials, and limit animal and/or human research in the area of drug metabolism and design. These studies will also provide training in advanced modeling, biophysical and biochemical techniques to postdoctoral fellows and graduate students. Due to the interdisciplinary nature of this project, they will be exposed to various facets of biological research and will learn a comprehensive approach to biological problems. Moreover, the work on this project will foster cooperation and collaboration among students and postdocs involved in different aspects of the studies. They will be also trained in manuscript preparation and submission procedures. This type of experience will help students and postdocs to succeed in their future research careers. Furthermore, this project will also provide opportunities for summer research by undergraduate students, as part of WV-BRIN and WV-INBRE programs.