Structural regulation in mitochondrial vitamin-D and vitamin-A metabolizing cytochromes P450

项目摘要和摘要 细胞色素P450(Cyps)构成了一类广泛的血红素，其中含有介导 一系列反应的催化作用。所有CYP介导的反应的一个共同特征是依赖于 辅助蛋白作为电子供体或变构调节剂，或两者兼而有之。小的血红素蛋白细胞色素b5， 例如，是一种具有良好特性的微粒体CYP活性的调节剂。然而，监管计划 线粒体中的P450系统(其中B5不能用作调节器)目前还不清楚， 从而代表了对这些系统如何工作的知识的根本差距。因此， 这项研究计划的总体目标是勾勒出如何开展 线粒体的CYPS酶是受调控的。作为迈向这一目标的重要第一步，这项提案概述了 两种维生素D代谢线粒体酶CYP27B1和CYP24A1的结构和功能研究 虽然细胞色素P27B1通过维生素D前体激素的1α-羟基化来激活维生素D，而细胞色素P24A1则通过 相反，通过维生素D侧链的碳23或碳24羟化使其失活。在……里面 演唱会上，他们展示了严格的监管控制，管理维生素D的生物利用度。 所附的初步数据表明，CYP-ADX电子转移络合物的形成 导致CYP24A1结构的长期变化，扰乱该酶结合维生素D和 这也与CYP活性部位的关闭一致。这些数据构成了总体 底物结合和电子转移(CYP催化中的步骤1和2)参与的工作假说 线粒体细胞色素P450功能的变构调节。在接下来的五年里，Estrada实验室计划测试 这一假设的要素是一组利用多学科工具包的项目目标，其中包括 其他技术，核磁共振光谱学，X射线结晶学，化学交联， 分子动力学模拟和功能分析。这一努力的目标是解决根本问题 关于线粒体CYP的功能、结构和功能调节的问题。在工作的同时 这里提出了这项研究计划的短期目标，完成这些目标将为 为研究额外的线粒体Cyps奠定基础，导致可持续的长期研究 程序。

PROJECT SUMMARY AND ABSTRACT Cytochromes P450 (CYPs) constitute a wide-ranging class of heme containing enzymes that mediate catalysis for a diverse array of reactions. A common feature for all CYP mediated reactions is reliance on accessory proteins as electron donors, allosteric modulators, or both. The small heme protein cytochrome b5, for example, is a well-characterized modulator of microsomal CYP activity. However, the regulatory scheme for the P450 systems in the mitochondria (where b5 is not available as a modulator) is not currently understood, thereby representing a fundamental gap in the knowledge of how these systems work. Therefore, the overarching goal of this research program is to outline the structural framework for how the activity of mitochondrial CYPs enzymes are regulated. As an important first step toward this goal, this proposal outlines a structure and function study for two vitamin D metabolizing mitochondrial enzymes, CYP27B1 and CYP24A1. While CYP27B1 activates vitamin D via a 1α-hydroxylation of the vitamin D pre-hormone, CYP24A1 exerts the opposite effect by deactivating via a carbon 23 or carbon 24 hydroxylation of the vitamin D side chain. In concert, they exhibit tightly regulatory control that governs vitamin D bioavailability. The enclosed preliminary data demonstrates that formation of the CYP-Adx electron transfer complex produces a long-range change in CYP24A1 structure that perturbs the enzyme's ability to bind vitamin D and that is also consistent with a closing off of the CYP active site. These data form the basis for the overall working hypothesis that substrate binding and electron transfer (steps 1 and 2 in CYP catalysis) participate in an allosteric regulation of CYP function in mitochondria. Over the next five years, the Estrada lab plans to test elements of this hypothesis as a set of project goals that utilize a multidisciplinary tool kit including, among other techniques, nuclear magnetic resonance spectroscopy, X-ray crystallography, chemical cross-linking, molecular dynamics simulations, and functional assays. The goal of this effort is to address fundamental questions pertaining to mitochondrial CYP function, structure, and regulation of function. While the work proposed here outlines the short-term goals of this research program, completing these goals will lay the foundation for the study of additional mitochondrial CYPs, leading to a sustainable long-term research program.