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）：拟议工作的目的是将结构，生化和遗传方法的结合结合起来，研究黄素蛋白中的结构/功能关系。这项工作将集中于两种结构上同源的含FAD的酶，pseudomonas putida的putidaredoxin还原酶以及小鼠诱导的凋亡因子。 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。不太了解putidaredoxin还原酶和putidaredoxin之间复杂形成和电子转移的机制。我们最近证明，putidaredoxin还原酶具有氧化还原活性半胱氨酸，并且可以用作二硫醇/二硫化物氧化还原酶。由于没有推定的二硫键氧化还原中心，该中心由cysxxcys或Cysxxxxcys序列序列特征是传统的二硫化物还原酶的特征，因此二硫醇/二硫化物氧化剂的机制是由putidaredredoxin还原酶催化的，似乎是独一无二的，并且需要阐明。凋亡诱导因子是一种神质上的旧哺乳动物，caspase独立的死亡效应子，在凋亡诱导后，它会从其正常定位（线粒体膜间间空间）转移到导致染色质缩合和DNA碎片的细胞核。该蛋白诱导的生理功能和凋亡的机制均不知道。 我们的研究将解决一个问题，即putidaredoxin还原酶和 诱导因子与其催化功能有关。将解决，比较，比较并与酶的催化特性有关，putid氧蛋白还原酶和凋亡诱导因子的晶体结构将得到解决。从结构解释中，将评估这些关系的进一步探讨和修改，并评估特定结构变化对蛋白质催化功能的影响。该研究将解释通过putidaredoxin还原酶催化的电子转移和二硫醇/二硫化物氧化的机制，并将深入了解诱导凋亡因子的机制和功能。