N-acetylglutamate Synthase: Structure, Function & Defects

N-乙酰谷氨酸合成酶：结构、功能

• 批准号: 8035600
• 负责人: Mendel Tuchman
• 金额: $ 9.98万
• 依托单位: CHILDREN'S RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8035600
• 关键词: Acetyl Coenzyme A; Address; Affect; Allosteric Site; Amino Acid Substitution; Amino Acids; Anabolism; Arginine; Award; Bacteria; Behavior Therapy; Binding; Biochemical; Biochemistry; Biological; Biology; Brain Injuries; Carbamyl Phosphate; Catalysis; Cessation of life; Citrulline; Clinical; Clinical Trials; Cloning; Coenzyme A; Commit; Computer Systems Development; Crystallization; DNA Footprint; Data; Defect; Developmental Disabilities; Diagnosis; Discipline; Disease; Engineering; Enzyme Inhibition; Enzymes; Escherichia coli; Evolution; Functional disorder; Gene Proteins; Genes; Genetic Transcription; Genotype; Glutamates; Gonorrhea; Health; Hepatic; Human; Hyperammonemia; Hypersensitivity; Inherited; Intestines; Knowledge; Laboratories; Lead; Learning; Ligands; Ligase; Liver; Mammals; Maps; Messenger RNA; Methods; Mitochondrial Matrix; Molecular; Mus; Mutation; N acetyl L glutamate; Neisseria gonorrhoeae; Organ; Organism; Phenotype; Phylogenetic Analysis; Phylogeny; Physiological; Physiology; Plants; Play; Primer Extension; Production; Property; Protein Kinase; Proteins; Proteobacteria; Race; Recombinant Proteins; Recombinants; Refractory; Regulation; Reporting; Reverse Transcriptase Polymerase Chain Reaction; Role; Site; Small Intestines; Structural Models; Structure; Surface; System; Tetraodontidae; Tissues; Transcription Initiation Site; Transcriptional Regulation; Urea; Vertebrates; X-Ray Crystallography; Xanthomonas; Xanthomonas campestris; Xylella; Zebrafish; base; chromatin immunoprecipitation; cofactor; density; electron density; expression cloning; fascinate; fungus; hepatic ureagenesis; human NAT7 protein; improved; insight; interest; microorganism; mutant; protein folding; protein structure; protein structure function; research study; structural biology; three dimensional structure; valproate

DESCRIPTION (provided by applicant): N-acetylglutamate synthase (NAGS) is an enzyme that produces the cognate cofactor N-acetylglutamate (NAG), an essential allosteric activator of the first and rate limiting enzyme of ureagenesis (CPS I) in mammals, and the first committed substrate for arginine biosynthesis in microorganisms. Our cloning and expression of the mouse and human NAGS genes and many other NAGS genes from various species, makes it now possible to gain structure/function insights into this interesting protein. We found in a number of proteobacteria species (X. campestris, M. maris, O. alexandrii, X. axonopodis, and X. fastidiosa Dixon) a gene for a bifunctional NAGS/NAGK that is similar to mammalian NAGS and for which we have obtained protein crystals. Recently, we obtained a high quality density map which will lead to the determination of the first three-dimensional structure of NAGS (from N. gonorrhoeae). Since NAGS is a likely regulator of ureagenesis and its function is allosterically affected by arginine, it is now possible to understand the mechanism(s) of the arginine effect and to compare the regulation of NAGS in hepatic vs. intestinal, tissues. The deficiency of NAG in inherited NAGS deficiency, organic acidemias and valproate treatment causes hyperammonemia that frequently leads to brain damage, developmental disabilities and death. Better understanding of the NAG/NAGS system will improve the diagnosis and treatment of these conditions. The specific aims of this project are 1) To solve the liganded and unliganded structures of NAGS and characterize mechanisms for catalysis and the effect of arginine; 2) To characterize the biochemical properties of NAGS proteins across phyla, focusing on the effect of arginine on structure and function; 3) To differentiate regulatory mechanisms that are specific to liver ureagenesis by characterizing and comparing the regulation of NAGS expression in liver and intestine; 4) To determine the functional effects of naturally-occurring mutations that cause inherited NAGS deficiency. Biochemical, crystallographic and molecular methods will be employed to gain an in depth understanding of the structural biology, biochemistry, pathophysiology, genotype/phenotype correlations of the NAGS genes and proteins in the context of evolutionary development of this system. After the first three- dimensional structure of NAGS has been solved, other structures of refractory NAGS proteins will become available. This will lead to constructing a structural model of mammalian NAGS, deriving at a catalytic mechanism, determining the mechanism of arginine effect, and the effects of mutations causing NAGS dysfunction and hyperammonemia.

描述（由申请人提供）： N-乙酰谷氨酸合酶 (NAGS) 是一种产生同源辅因子 N-乙酰谷氨酸 (NAG) 的酶，NAG 是哺乳动物尿素生成第一个限速酶 (CPS I) 的必需变构激活剂，也是微生物精氨酸生物合成的第一个关键底物。我们对小鼠和人类 NAGS 基因以及来自不同物种的许多其他 NAGS 基因进行克隆和表达，现在可以深入了解这种有趣的蛋白质的结构/功能。我们在许多变形菌物种（X.campestris、M.maris、O.alexandrii、X.axonopodis 和 X.fastidiosa Dixon）中发现了一个双功能 NAGS/NAGK 基因，该基因与哺乳动物 NAGS 相似，并且我们已经获得了该基因的蛋白质晶体。最近，我们获得了高质量的密度图，这将有助于确定 NAGS（来自淋病奈瑟菌）的第一个三维结构。由于 NAGS 可能是尿素生成的调节剂，并且其功能受到精氨酸的变构影响，因此现在可以了解精氨酸效应的机制并比较 NAGS 在肝脏组织和肠道组织中的调节。遗传性 NAGS 缺乏、有机酸血症和丙戊酸治疗中 NAG 的缺乏会导致高氨血症，常常导致脑损伤、发育障碍和死亡。更好地了解 NAG/NAGS 系统将改善这些疾病的诊断和治疗。该项目的具体目标是 1) 解决 NAGS 的配体和非配体结构，并表征催化机制和精氨酸的作用； 2) 表征跨门NAGS蛋白的生化特性，重点关注精氨酸对结构和功能的影响； 3）通过表征和比较肝脏和肠道中NAGS表达的调节来区分肝脏尿素生成特有的调节机制； 4) 确定导致遗传性 NAGS 缺陷的自然发生突变的功能影响。将采用生物化学、晶体学和分子方法来深入了解 NAGS 基因和蛋白质在该系统进化发展背景下的结构生物学、生物化学、病理生理学、基因型/表型相关性。在解决了 NAGS 的第一个三维结构之后，难处理的 NAGS 蛋白的其他结构将变得可用。这将导致构建哺乳动物 NAGS 的结构模型，推导催化机制，确定精氨酸作用的机制，以及导致 NAGS 功能障碍和高氨血症的突变的影响。