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）是一种酶，可产生同源辅因子N-乙酰谷氨酸（NAG），这是哺乳动物的第一种和速率限制酶（CPS I）的必不可少的变构激活剂，在哺乳动物中，是第一个承诺的蛋白酶依克氨酸的蛋白酶酶，以培养基化蛋白质构酸化。我们的克隆和表达小鼠和人类nags基因以及来自各种物种的许多其他NAG基因，现在可以将结构/功能洞察到这种有趣的蛋白质中。我们在许多蛋白质细菌（X. Campestris，M。Maris，O。Alexandrii，X。Axonopodis和X. fastidiosa dixon）中找到了一种基因，用于双功能的NAGS/NAGK，类似于哺乳动物NAGS，我们已经获得了哺乳动物的NAGS和我们获得的蛋白质水晶。最近，我们获得了一个高质量密度图，这将导致确定NAG的第一个三维结构（来自N. Gonorrhoeae）。由于NAGS可能是尿素发生的调节剂，其功能会受到精氨酸的变构影响，因此现在有可能了解精氨酸效应的机制，并比较肝与肠道，组织中NAG的调节。 NAG缺乏遗传的NAG缺乏，有机酸血症和丙戊酸治疗会导致高症血症，经常导致脑损伤，发育障碍和死亡。更好地了解NAG/NAG系统将改善这些疾病的诊断和治疗。该项目的具体目的是1）解决NAG的配体和无配合的结构，并表征了催化和精氨酸的作用的机制； 2）表征跨门的NAGS蛋白的生化特性，重点是精氨酸对结构和功能的影响； 3）通过表征和比较肝脏和肠中NAGS表达的调节来区分特定于肝尿素发生的调节机制； 4）确定导致遗传性na不足的天然突变突变的功能效应。将采用生化，晶体学和分子方法，以深入了解NAGS基因和蛋白质的结构生物学，生化，病理生理学，基因型/表型相关性。在解决了符号的第一个三维结构之后，将可用难治性nags蛋白的其他结构。这将导致构建哺乳动物na的结构模型，以催化机制衍生，确定精氨酸效应的机制以及引起NAGS功能障碍和高症血症的突变的作用。