Structural Studies of NAGS

NAGS的结构研究

• 批准号: 7113157
• 负责人: DASHUANG SHI
• 金额: $ 12.31万
• 依托单位: CHILDREN'S RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-01 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7113157
• 关键词: Escherichia coli; Mammalia; X ray crystallography; active sites; acyltransferase; aminoacid biosynthesis; ammonia; arginine; blood chemistry; chemical aggregate; chemical stability; chimeric proteins; crystallization; enzyme mechanism; enzyme structure; genetic disorder; genetic manipulation; genetic screening; green fluorescent proteins; maltose; method development; mutant; postdoctoral investigator; protein structure function; solubility; structural biology; urea cycle

DESCRIPTION (provided by applicant): This research training application is aimed to provide both didactic and research training experience to allow the candidate to become an independent investigator. The purpose of training is to expand the expertise of the candidate in molecular biology and molecular microbiology which will complement his knowledge and experience in crystallography. Thus, three of the mentors have expertise in molecular biology and the other two will provide expertise in various aspects of structural analysis of proteins. The research projects aim to understand the correlations between three-dimensional structure and function of N-acetylglutamate synthase (NAGS) encoded by the most recently cloned gene of this unique urea cycle enzyme, thought to be involved in the regulation of ureagenesis. Very little is known on the structure of any NAGS protein. The NAGS genes of bacteria, fungi and mammals are much more diverse than other arginine-biosynthesis and urea-cycle genes. It is very interesting, therefore, to explore at the protein level whether the three-dimensional structures are also very different and how the enzymes respond differently to L-arginine. A strategy to overcome the insolubility and aggregation of mammalian and E. coil NAGS is proposed. This includes using random mutagenesis of the NAGS gene followed by GFP screening and functional assay to identify the highly soluble and stable mutants. The second methodology uses the fusion of NAGS to a soluble protein such as green fluorescent protein (GFP) or maltose binding protein (MBP) to increase protein solubility and stability. Different length of amino acid linkers with different properties will be produced to examine how they affect the crystallization behavior. The ultimate goal is to study the three-dimensional structures of NAGS enzymes from both lower and higher organisms and to understand their catalytic and regulatory mechanisms, and relationship to inherited defects causing hyperammonaemia.

描述（由申请人提供）： 该研究培训申请旨在提供教学和研究培训经验，以使候选人成为独立的研究者。培训的目的是扩大分子生物学和分子微生物学中候选者的专业知识，这将补充他在晶体学方面的知识和经验。因此，三个导师在分子生物学方面具有专业知识，另外两个将在蛋白质结构分析的各个方面提供专业知识。该研究项目旨在了解N-乙酰谷氨酸合酶（NAGS）的三维结构与功能之间的相关性，并由该独特的尿素周期酶的最新克隆基因编码，被认为与尿素发生调节有关。在任何NAGS蛋白的结构上，知之甚少。细菌，真菌和哺乳动物的NAG基因比其他精氨酸生物合成和尿素周期基因多样化。因此，在蛋白质水平上探索三维结构是否也非常不同，以及酶对L-精氨酸的反应方式非常有趣。提出了一种克服哺乳动物和E.卷。这包括使用NAGS基因的随机诱变，然后进行GFP筛选和功能测定，以鉴定高度可溶和稳定的突变体。第二种方法使用NAG融合到可溶性蛋白（例如绿色荧光蛋白（GFP）或麦芽糖结合蛋白（MBP））以提高蛋白质溶解度和稳定性。具有不同特性的氨基酸接头长度将产生不同的特性，以检查它们如何影响结晶行为。最终目标是研究来自较低生物体和高等生物体的NAG酶的三维结构，并了解它们的催化和调节机制，以及与引起多余血症血症的遗传缺陷的关系。

项目成果

Structure and catalytic mechanism of a novel N-succinyl-L-ornithine transcarbamylase in arginine biosynthesis of Bacteroides fragilis.

脆弱拟杆菌精氨酸生物合成中新型N-琥珀酰-L-鸟氨酸转氨甲酰酶的结构和催化机制。

• DOI: 10.1074/jbc.m601229200
• 发表时间: 2006
• 期刊: The Journal of biological chemistry
• 作者: Shi,Dashuang;Morizono,Hiroki;Cabrera-Luque,Juan;Yu,Xiaolin;Roth,Lauren;Malamy,MichaelH;Allewell,NormaM;Tuchman,Mendel
• 通讯作者: Tuchman,Mendel