Structure/Function Studies on Flavoproteins

黄素蛋白的结构/功能研究

• 批准号: 7106444
• 负责人: Irina F Sevrioukova
• 金额: $ 22.34万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7106444
• 关键词: NAD(P)H oxidoreductase; X ray crystallography; apoptosis; catalyst; conformation; cysteine; electron density; electron transport; enzyme mechanism; flavoproteins; genetic manipulation; nephelometry; protein protein interaction; protein structure function; site directed mutagenesis

DESCRIPTION (provided by applicant): The aim of the proposed work is to use a combination of structural, biochemical and genetic approaches to study structure/function relationships in flavoproteins. The work will focus on two structurally homologous FAD-containing enzymes, putidaredoxin reductase from Pseudomonas putida and apoptosis inducing factor from mice. Putidaredoxin reductase catalyzes electron transfer from NADH to an iron-sulfur protein, putidaredoxin, in cytochrome P450cam monooxygenase. The mechanism of complex formation and electron transfer between putidaredoxin reductase and putidaredoxin is not well understood. We have recently demonstrated that putidaredoxin reductase has redox active cysteines and can function as dithiol/disulfide oxidoreductase. Since there are no putative disulfide redox centers encoded by CysXXCys or CysXXXXCys motifs characteristic for traditional disulfide reductases, the mechanism of dithiol/disulfide oxidoreduction catalyzed by putidaredoxin reductase seems to be unique and needs to be elucidated. Apoptosis inducing factor is a phylogenetically old mammalian, caspase-independent death effector which, upon apoptosis induction, translocates from its normal localization, the mitochondrial intermembrane space, to the nucleus where it causes chromatin condensation and DNA fragmentation. Neither physiological function nor mechanism of apoptosis induced by this protein is known. Our research will address the question of how the structures of putidaredoxin reductase and apoptosis inducing factor are related to their catalytic function. Crystal structures of putidaredoxin reductase and apoptosis inducing factor will be solved, compared, and related to the catalytic properties of the enzymes. From structural interpretations, these relationships will be further probed and modified by genetic engineering and the effect of specific structural changes on catalytic function of the proteins will be assessed. The study will explain the mechanisms of electron transfer and dithiol/disulfide oxidoreduction catalyzed by putidaredoxin reductase and will provide an insight into the mechanism and function of apoptosis inducing factor.

描述（由申请人提供）：拟议工作的目的是结合使用结构、生化和遗传学方法来研究黄素蛋白的结构/功能关系。这项工作将重点关注两种结构同源的含有 FAD 的酶，即来自恶臭假单胞菌的恶臭氧还蛋白还原酶和来自小鼠的凋亡诱导因子。 Putidaredoxin 还原酶催化电子从 NADH 转移到细胞色素 P450cam 单加氧酶中的铁硫蛋白 Putidaredoxin。恶臭还蛋白还原酶和恶臭恶臭之间的复合物形成和电子转移机制尚不清楚。我们最近证明，putidaredoxin 还原酶具有氧化还原活性半胱氨酸，可以起到二硫醇/二硫键氧化还原酶的作用。由于不存在传统二硫键还原酶特有的由CysXXCys或CysXXXXCys基序编码的推定二硫键氧化还原中心，因此由恶臭氧还蛋白还原酶催化的二硫醇/二硫键氧化还原机制似乎是独特的并且需要阐明。细胞凋亡诱导因子是一种系统发育古老的哺乳动物、不依赖半胱天冬酶的死亡效应物，在诱导细胞凋亡时，它从其正常定位（线粒体膜间隙）转移到细胞核，在细胞核中引起染色质浓缩和 DNA 片段化。该蛋白诱导细胞凋亡的生理功能和机制尚不清楚。 我们的研究将解决腐臭氧还蛋白还原酶的结构如何与 细胞凋亡诱导因子与其催化功能有关。腐胺氧还蛋白还原酶和凋亡诱导因子的晶体结构将被解析、比较，并与酶的催化特性相关。从结构解释来看，这些关系将通过基因工程进一步探讨和修改，并将评估特定结构变化对蛋白质催化功能的影响。该研究将解释恶臭氧还蛋白还原酶催化的电子转移和二硫醇/二硫化物氧化还原的机制，并将深入了解细胞凋亡诱导因子的机制和功能。