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

线粒体维生素 D 和维生素 A 代谢细胞色素 P450 的结构调节

基本信息

批准号：
10630084
负责人：
David Fernando Estrada
金额：
$ 39.76万
依托单位：
STATE UNIVERSITY OF NEW YORK AT BUFFALO
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-09-01 至 2025-05-31
项目状态：
未结题

项目摘要

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.

项目摘要和摘要细胞色素P450（CYPS）构成了一类含有介导的酶的血红素类催化剂的一系列反应。所有CYP介导的反应的共同特征是缓解辅助蛋白作为电子供体，变构调节剂或两者兼而有之。小血红素蛋白细胞色素B5，例如，是微粒体CYP活性的特征良好的调节剂。但是，线粒体中的P450系统目前尚不理解B5（其中b5不可用），从而代表了这些系统如何工作的知识中的根本差距。因此，该研究计划的总体目标是概述如何活动的结构框架线粒体CYPS酶受到调节。作为朝着这一目标的重要第一步，该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。 CYP27B1通过维生素D前激素的1α-羟基化激活维生素D，而CYP24A1执行通过通过碳23或碳24维生素D侧链的碳化而停用的效果。在音乐会，他们表现出严格的调节控制，该控制控制了维生素D生物利用度。封闭的初步数据表明，CYP-ADX电子传输复合物的形成在CYP24A1结构中产生远距离变化，该结构使酶结合维生素D和这也与关闭CYP活动站点的关闭一致。这些数据构成了整体的基础底物结合和电子转移（CYP催化中的步骤1和2）参与的工作假设线粒体中CYP功能的变构调节。在接下来的五年中，埃斯特拉达实验室计划测试该假设的要素是利用多学科工具套件的一组项目目标其他技术，核磁共振光谱，X射线晶体学，化学交联，分子动力学模拟和功能测定。这项努力的目的是解决基本与线粒体CYP功能，结构和功能调节有关的问题。工作时这里提出的概述了该研究计划的短期目标，完成这些目标将是研究其他线粒体CYP的研究基金会，导致可持续的长期研究程序。