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: 7586623
• 负责人: Donald F Becker
• 金额: $ 28.87万
• 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 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7586623
• 关键词: Active Sites; Affinity; Architecture; Binding; Bioenergetics; Biological Assay; Cell membrane; Charge; Chimera organism; DNA Binding; Deuterium; Disease; Dissection; Electrons; Enzymatic Biochemistry; Enzymes; Escherichia coli; Family; Flavins; Flavoproteins; Genes; Genetic Transcription; Glutamates; Goals; Gram-Negative Bacteria; Homologous Gene; Hydrogen; Ligands; Location; Mass Spectrum Analysis; Membrane; Metabolism; Molecular; Molecular Conformation; Mutagenesis; N-terminal; Organism; Outcome; Oxidation-Reduction; Oxidoreductase; Predisposition; Prokaryotic Cells; Proline; Proline Dehydrogenase; Proteins; Research Personnel; Schizophrenia; Screening procedure; Signal Transduction; Site-Directed Mutagenesis; Structural Models; Structure; Surface; Surface Plasmon Resonance; Work; X-Ray Crystallography; analog; base; carboxylate; cofactor; genetic regulatory protein; insight; malignant stomach neoplasm; member; novel; oxidation; polypeptide; programs; proline permease; pyrroline; reconstitution; three dimensional structure

DESCRIPTION (provided by applicant): Proline utilization A (PutA) from Escherichia coli is a large multifunctional protein that uniquely combines enzymatic and transcriptional regulatory activities within a single polypeptide. As an enzyme, PutA peripherally associates with the inner cytoplasmic membrane to catalyze the four-electron oxidation of proline to glutamate via the coordinated actions of separate flavin-dependent proline dehydrogenase (PRODH) and NAD-dependent A1-pyrroline-5-carboxylate dehydrogenase (P5CDH) domains. An N-terminal ribbon-helix-helix motif endows PutA with DMA-binding activity enabling PutA to function also as a cytosolic autogenous transcriptional represser of the proline utilization (put) genes putA and putP (encodes a high affinity proline transporter). To fulfill its mutually exclusive functions as a transcriptional represser and membrane-bound proline catabolic enzyme, PutA undergoes proline-dependent functional switching. The central hypothesis of this proposal is that flavin redox signals generated in the PRODH active site control the global conformation, subcellular location and function of PutA. This idea is supported by recent work demonstrating that reduction of the flavin cofactor drives PutA-membrane association and induces structural changes in the PRODH active site. To further explore this hypothesis, a dynamic and structural model for how the flavin cofactor controls functional switching of PutA will be developed using a wide variety of approaches, including spectroelectrochemistry, site-directed mutagenesis, surface plasmon resonance, X- ray crystallography, hydrophobic photolabeling and hydrogen-deuterium exchange mass spectrometry. The major goals of this study are to uncover the novel redox-based mechanism whereby PutA transforms from a gene regulatory protein into a membrane-bound enzyme and to provide a structural understanding of how PutA integrates catalytic, membrane-binding and DMA-binding activities within a single polypeptide. The specific aims to achieve this are the following: 1. Identify flavin-protein interactions that direct the functional switching of PutA. 2. Elucidate the global three-dimensional architecture of trifunctional PutA. 3. Identify membrane-binding domains of PutA. 4. Characterize proline-dependent conformational changes in PutA. This work will generate mechanistic insights into how proteins perform multiple tasks. Project outcomes will also further the understanding of proline bioenergetics in gastric cancer, trypanosomal diseases, type I hyperprolinemia and schizophrenia susceptibility.

描述（由申请人提供）：来自大肠杆菌的脯氨酸利用A（PutA）是一种大的多功能蛋白质，其在单一多肽内独特地结合了酶促和转录调节活性。作为一种酶，PutA与细胞内膜外周结合，通过单独的黄素依赖性脯氨酸脱氢酶（PRODH）和NAD依赖性A1-吡咯啉-5-羧酸脱氢酶（P5 CDH）结构域的协调作用催化脯氨酸的四电子氧化为谷氨酸。一个N-末端的带状-螺旋-螺旋基序赋予PutA DMA结合活性，使PutA也能作为脯氨酸利用（put）基因putA和putP（编码高亲和力脯氨酸转运蛋白）的胞质自体转录抑制因子发挥作用。为了实现其相互排斥的功能，作为一个转录抑制因子和膜结合脯氨酸分解代谢酶，PutA经历脯氨酸依赖性功能转换。该提议的中心假设是，在PRODH活性位点产生的黄素氧化还原信号控制PutA的全局构象、亚细胞位置和功能。最近的研究表明，黄素辅因子的减少驱动PutA-膜缔合并诱导PRODH活性位点的结构变化，这一观点得到了支持。为了进一步探索这一假设，将使用多种方法开发黄素辅因子如何控制PutA功能转换的动态和结构模型，包括光谱电化学、定点诱变、表面等离子体共振、X射线晶体学、疏水光标记和氢-氘交换质谱。本研究的主要目标是揭示新的氧化还原为基础的机制，使PutA从基因调控蛋白转化为膜结合酶，并提供一个结构的理解PutA如何整合催化，膜结合和DMA结合活性在一个单一的多肽。实现这一目标的具体目标如下：1.确定黄素蛋白质的相互作用，指导PutA的功能转换。2.阐明三功能PutA的整体三维结构。3.鉴定PutA的膜结合结构域。4.表征PutA中脯氨酸依赖性构象变化。这项工作将产生对蛋白质如何执行多种任务的机械见解。项目成果也将进一步了解脯氨酸生物能量学在胃癌，锥虫病，I型高脯氨酸血症和精神分裂症的易感性。