Mechanistic Studies of Functional Switching in the PutA Flavoprotein

PutA 黄素蛋白功能转换的机制研究

• 批准号: 8668068
• 负责人: Donald F Becker
• 金额: $ 29.7万
• 依托单位: UNIVERSITY OF NEBRASKA LINCOLN
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-01 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8668068
• 关键词: Active Sites; Affinity; African Trypanosomiasis; Amino Acids; Binding; Binding Sites; Bioenergetics; Biological Assay; C-terminal; Catabolism; Connective Tissue Diseases; Coupled; Couples; Crystallography; DNA Binding; DNA Binding Domain; Data; Electron Transport; Electrons; Engineering; Enzymatic Biochemistry; Enzymes; Escherichia coli; Family; Feeds; Flavins; Flavoproteins; Funding; Gene Expression Regulation; Genes; Glutamates; Goals; Gram-Negative Bacteria; Health; Helicobacter pylori; Human; Inborn Genetic Diseases; Incidence; Investigation; Kinetics; Knowledge; Length; Link; Location; Malignant Neoplasms; Membrane; Metabolic; Metabolism; Modeling; Molecular; Molecular Conformation; Neurologic Dysfunctions; Osteopenia; Outcome; Oxidases; Oxidation-Reduction; Oxidoreductase; Pathway interactions; Peptic Ulcer; Peptides; Positioning Attribute; Predisposition; Premature aging syndrome; Prokaryotic Cells; Proline; Proline Dehydrogenase; Proteins; Reaction; Resolution; Respiratory Chain; Role; Schizophrenia; Seizures; Signal Transduction; Site; Site-Directed Mutagenesis; Solutions; Source; Stimulus; Structural Models; Structure; Thermodynamics; Transcription Repressor/Corepressor; Trypanosoma brucei brucei; Tumor Suppressor Proteins; Ubiquinone; Work; X-Ray Crystallography; base; carboxylate; cofactor; genetic regulatory protein; in vivo; insight; mutant; novel; oxidation; pathogen; prevent; proline permease; pyrroline; response; sensor; structural biology

DESCRIPTION (provided by applicant): The overall goal of this study is to provide molecular and structural understanding for the redox based functional switching of a multifunctional enzyme involved in regulating and catalyzing proline metabolism. The two-step conversion of proline to glutamate in Gram-negative bacteria is catalyzed by PutA (proline utilization A), a large membrane-associated flavoenzyme. PutA catalyzes the four-electron oxidation of proline to glutamate by coordinating the activities of separate flavin-dependent proline dehydrogenase (PRODH) and NAD+-dependent 1-pyrroline- 5-carboxylate dehydrogenase (P5CDH) domains. In certain prokaryotes such as Escherichia coli, PutA also contains a ribbon-helix-helix (RHH) DNA-binding domain and is an autogenous transcriptional repressor of the proline utilization genes putA and putP (encodes a high affinity proline transporter). To fulfill its mutually exclusiv functions as a transcriptional repressor and membrane-bound enzyme, PutA undergoes proline-dependent functional switching. Thus, PutAs with DNA binding activity are unique trifunctional flavoproteins that act as sensors of cellular metabolism by responding to proline availability. Earlier studies have established that proline reduction of the flavin activates PutA membrane- binding thereby triggering PutA switching from a transcriptional repressor to a membrane-bound enzyme. The principal hypothesis of this proposal is that redox signals in the flavin active site control the conformation, subcellular localization, and function of PutA. The goal of this study is to further examine this hypothesis by building a structural and dynamic model for how reduction of the flavin cofactor drives PutA functional switching. Several major milestones achieved in the previous funding period form the basis for the proposed studies. In particular, conformational changes in the flavin itself and surrounding active site residues were identified and shown to be critical for initiating functional switching. The thermodynamic and structural basis of the PutA repressor function was elucidated. The first crystal structure of a full-length bifunctional PutA was determined. The solution structure of a trifunctional PutA was modeled using SAXS data and crystal structures of domains. And most recently, the elusive membrane-binding domain of PutA was identified. These results provide an outstanding framework for understanding, at unprecedented detail, the molecular mechanisms whereby PutA transforms from a gene regulatory protein to a membrane-bound enzyme. A new direction integrated into this study is to understand how proline catabolism is coupled to reduction of the respiratory chain in vivo. The specific aims are the following: 1. Determine the organization and structure of trifunctional PutA. 2. Characterize the bioenergetics of proline metabolism. 3. Elucidate the mechanism of functional switching in PutA.

描述(申请人提供)：这项研究的总体目标是提供基于氧化还原的多功能酶的功能转换的分子和结构了解，该多功能酶参与调节和催化脯氨酸代谢。在革兰氏阴性菌中，脯氨酸转化为谷氨酸的两步反应是由一种大的膜相关黄素酶PUTA(Pro利用A)催化的。PUTA通过调节依赖黄素的脯氨酸脱氢酶(PROH)和依赖NAD+的1-吡咯烷-5-羧酸脱氢酶(P5CDH)结构域的活性来催化四电子氧化脯氨酸生成谷氨酸。在某些原核生物中，如大肠杆菌中，putA还含有一个带状-螺旋-螺旋(RHH)DNA结合域，是Pro利用基因putA和putP(编码一种高亲和力的脯氨酸转运体)的自体转录抑制因子。PUTA作为一种转录抑制因子和膜结合酶，为了实现其相互排斥的功能，需要进行依赖于脯氨酸的功能转换。因此，具有DNA结合活性的PUTA是一种独特的三功能黄素蛋白，它通过响应脯氨酸的可获得性来作为细胞新陈代谢的传感器。早期的研究已经证实，黄素的Pro还原激活了Puta膜结合，从而触发了Puta从转录抑制物到膜结合酶的转换。这一设想的基本假设是，黄素活性部位的氧化还原信号控制PUTA的构象、亚细胞定位和功能。这项研究的目标是 为了进一步检验这一假设，建立了一个结构和动态模型，说明黄素辅因子的减少如何驱动PUTA功能转换。在上一个供资期间取得的几个主要里程碑构成了拟议研究的基础。特别是，黄素自身和周围活性部位残基的构象变化被发现并被证明是启动功能转换的关键。阐明了普塔阻滞剂作用的热力学和结构基础。测定了全长双功能PUTA的第一个晶体结构。利用SAXS数据和结构域的晶体结构模拟了三官能团PUTA的溶液结构。最近，人们发现了Puta难以捉摸的膜结合区。这些结果为以前所未有的细节理解PuTA从基因调控蛋白转化为膜结合酶的分子机制提供了一个出色的框架。这项研究的一个新方向是了解体内脯氨酸分解代谢如何与呼吸链的减少相耦合。具体目标如下：1.确定三大职能部门的组织结构。 2.研究了脯氨酸代谢的生物能量学特征。3.阐明PUTA的功能转换机制。