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-乙酰谷氨酸合成酶(NAG)是一种产生同源辅因子N-乙酰谷氨酸(NAG)的酶，是哺乳动物尿失禁第一限速酶(CPS I)的重要变构激活剂，也是微生物合成精氨酸的第一个固定底物。我们克隆和表达了小鼠和人类的NAG基因，以及来自不同物种的许多其他NAG基因，这使得现在有可能对这种有趣的蛋白质的结构/功能有更深入的了解。我们在许多变形杆菌(X.campestris，M.Maris，O.alexandrii，X.axonopodis，和X.fitidiosa Dixon)中发现了一个与哺乳动物NAG类似的双功能NAGS/NAGK基因，并对其进行了蛋白质结晶。最近，我们获得了一个高质量的密度图，这将导致确定NAG的第一个三维结构(来自淋病奈瑟菌)。由于NAGS可能是尿失禁的调节因子，其功能受精氨酸的变构影响，因此现在有可能了解精氨酸效应的机制(S)，并比较NAGS在肝脏和肠道组织中的调节。遗传性NAG缺乏症、有机酸血症和丙戊酸治疗中的NAG缺乏会导致高氨血症，从而导致脑损伤、发育障碍和死亡。更好地了解NAG/NAGS系统将改进对这些疾病的诊断和治疗。该项目的具体目标是1)解决NAG的连接和非连接结构，并表征催化机制和精氨酸的作用；2)表征NAGS蛋白的生化性质，重点是精氨酸对结构和功能的影响；3)通过表征和比较NAGS在肝脏和肠道中的表达调控，区分肝尿失禁特有的调控机制；4)确定导致遗传性NAGS缺陷的自然发生突变的功能影响。在这一系统进化发展的背景下，将利用生化、结晶学和分子方法深入了解NAGS基因和蛋白质的结构生物学、生化、病理生理学、基因/表型相关性。在解决了NAGS的第一个三维结构后，难治性NAGS蛋白的其他结构将变得可用。这将导致构建哺乳动物NAG的结构模型，从催化机制出发，确定精氨酸效应的机制，以及突变导致NAGS功能障碍和高氨血症的影响。