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

• 批准号: 10401867
• 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
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10401867
• 关键词: Active Sites; Address; Allosteric Regulation; Binding; Biological Assay; Biological Availability; CYP27B1 gene; Carbon; Catalysis; Chemicals; Complex; Cytochrome P450; Cytochromes b5; Data; Electron Transport; Elements; Enzymes; Exhibits; Foundations; Goals; Heme; Hemeproteins; Hormones; Hydroxylation; Knowledge; Mediating; Metabolism; Mitochondria; NMR Spectroscopy; Proteins; Reaction; Regulation; Research; Scheme; Side; Structure; System; Techniques; Testing; Vitamin A; Vitamin D; Work; X-Ray Crystallography; crosslink; electron donor; molecular dynamics; multidisciplinary; programs; tool

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.

项目总结和摘要 细胞色素P450（CYP）构成一类广泛的含血红素的酶，其介导 催化多种反应。所有的酶介导的反应的共同特征是依赖于 辅助蛋白作为电子供体、变构调节剂或两者。小血红素蛋白细胞色素b5， 例如是微粒体α-淀粉酶活性的充分表征的调节剂。然而， 线粒体中的P450系统（其中b5不能用作调节剂）目前还不清楚， 从而代表了在这些系统如何工作的知识方面的根本差距。因此 这项研究计划的总体目标是概述如何活动的结构框架， 线粒体CYP酶受到调节。作为实现这一目标的重要的第一步，该提案概述了 维生素D代谢线粒体酶CYP 27 B1和CYP 24 A1结构和功能研究 虽然CYP 27 B1通过维生素D前激素的1α-羟基化激活维生素D，但CYP 24 A1通过维生素D前激素的1α-羟基化激活维生素D。 通过维生素D侧链的碳23或碳24羟基化而失活的相反效果。在 因此，它们表现出对维生素D生物利用度的严格调控。 所附的初步数据表明，CYP-Adx电子转移复合物的形成 产生CYP 24 A1结构的长程变化，干扰酶结合维生素D的能力， 这也与封闭的生物活性位点相一致。这些数据构成了整体的基础。 工作假设，底物结合和电子转移（步骤1和2的催化）参与 线粒体中对线粒体功能的变构调节。在接下来的五年里，埃斯特拉达实验室计划测试 这一假设的要素作为一套项目目标，利用多学科工具包，其中包括， 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分析。这项工作的目标是解决基本的 与线粒体的功能、结构和功能调节有关的问题。虽然工作 这里提出的概述了这项研究计划的短期目标，完成这些目标将奠定 研究额外的线粒体CYP的基础，导致可持续的长期研究 程序.