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.

项目概要和摘要细胞色素 P450 (CYP) 是一类广泛的含血红素的酶，可介导所有 CYP 介导的反应的共同特征是对多种反应的催化作用。作为电子供体、变构调节剂或两者的辅助蛋白小血红素蛋白细胞色素 b5，例如，是微粒体 CYP 活性的一种充分表征的调节剂。目前尚不清楚线粒体中的 P450 系统（其中 b5 不能作为调节剂），从而代表了对这些系统如何工作的了解的根本差距。该研究计划的总体目标是概述如何开展活动的结构框架作为实现这一目标的重要第一步，该提案概述了线粒体 CYP 酶。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-羟基化来激活维生素 D，而 CYP24A1 则发挥通过维生素 D 侧链的碳 23 或碳 24 羟基化而产生相反的作用。同时，它们表现出严格的监管控制，以控制维生素 D 的生物利用度。随附的初步数据表明 CYP-Adx 电子转移复合物的形成产生 CYP24A1 结构的长程变化，扰乱酶结合维生素 D 的能力这也与 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 催化的步骤 1 和 2）参与的工作假设 Estrada 实验室计划在未来五年内测试线粒体中 CYP 功能的变构调节。该假设的要素作为一组项目目标，利用多学科工具包，其中包括其他技术、核磁共振波谱、X射线晶体学、化学交联、这项工作的目标是解决基本问题。有关线粒体 CYP 功能、结构和功能调节的问题。这里提出的概述了该研究计划的短期目标，完成这些目标将奠定为研究其他线粒体 CYP 奠定基础，从而进行可持续的长期研究程序。