STRUCTURE, FUNCTION AND DYNAMICS OF TRANSHYDROGENASE

转氢酶的结构、功能和动力学

• 批准号: 6387189
• 负责人: YOUSSEF HATEFI
• 金额: $ 35.46万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至 2004-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6387189
• 关键词: Entamoeba histolytica; Escherichia coli; NAD(P) transhydrogenase; Rhodospirillum; X ray crystallography; bioenergetics; conformation; crystallization; enzyme mechanism; enzyme structure; hydrogen transport; site directed mutagenesis

This project is concerned with study of the structure and function of bovine mitochondrial, bacterial (Escherichia coli, Rhodospirillum rubrum) and Entamoeba histolytica transhydrogenases (TH), which catalyze hydride ion transfer between NAD(H) and NADP(H) in a reaction that is coupled to transmembrane proton translocation. As proton pumps TH are relatively simple structures, and excellent systems for study of proton translocation driven by substrate binding energy via protein conformation change. In mitochondria TH produces NADPH, which is utilized, among other things, by glutathione reductase and glutathione peroxidase to dissipate harmful H2O2 that results from dismutation of superoxide produced by the respiratory chain. SPECIFIC AIMS are (1) Structure Studies: (a) TH is composed of 3 domains: Extramembranous domains I (400-430 residues) and III(200 residues) that bind NAD(H) and NADP(H), respectively, and membrane-intercalated domain II (360-400 residues) that carries the proton channel. We have expressed in E. coli domains I and III of bovine and R. rubrum TH. Purified domains I and III reconstitute TH activity in the absence of domain II. We have solved the crystal structure of bovine domain III containing bound NADP at 1.2 Angstrom units resolution, and have a complete data set for native crystals of R. rubrum domain I at 2.1 Angstrom units resolution. It is proposed (a) to solve the crystal structures of R. rubrum and bovine domains I, R. rubrum domain III (q nucleotides), and E. histolytica contiguous domain III-I; (b) investigate by computer modeling of these crystal structures and those of appropriate mutants the docking of domains I and III; (c) solve the crystal structure of the entire E. coli TH. (2) Mechanism of Proton Translocation: In domain II, there are 3 clusters of high sequence conservation (60-80 percent). A His and an Asp in 2 of these clusters appear to be concerned (chemically or structurally) with proton translocation. This Asp is the only conserved negatively charged residue in domain II, and its mutation to Asn or Ile also alters the affinity (Km and Kd) of TH for NADPH. The above clusters have 10 conserved hydroxylated residues, which may be involved in proton translocation. In addition, domain III contains a conserved acidic patch which may interact with positively charged residues in the cytoplasmic-side loops of domain II. It is proposed to mutate these residues in the E. coli TH to investigate their role in proton translocation and in conformational energy transfer between domains II and III. Also, docked domains I-III models will be tested by mutation of complementary interface residues and activity assays.

该项目涉及研究牛线粒体，细菌（大肠杆菌，黄昏rubrum）和Entamoeba组织溶解性杂交酶（TH）的结构和功能，它们在反应中催化NAD（H）和NADP（H）之间的NAD（H）和NADP（h）之间的氢化物离子转移对转化蛋白转化的反应是转化的。 由于质子泵是相对简单的结构，也是通过蛋白质构象变化构成能量驱动的质子易位的出色系统。 在线粒体中，产生NADPH，除其他外，谷胱甘肽还原酶和谷胱甘肽过氧化物酶用于散发有害的H2O2，这是由于呼吸链产生的超氧化物的损失而导致的。具体目的是（1）结构研究：（a）TH由3个结构域组成：膜外域I（400-430个残基）和III（200个残基），分别结合NAD（H）和NADP（H），以及膜骨化结构域II（360-400残基），这些结构元（360-400残基）具有蛋白质通道。 我们已经在牛和R. rubrum th的大肠杆菌I和III中表达。 在没有域II的情况下，纯化的域I和III重新建立了TH活动。 我们已经解决了在1.2 Angstrom单位分辨率下包含绑定的NADP的牛域III的晶体结构，并为R。Rubrum域I i在2.1 Angstrom单元分辨率的天然晶体具有完整的数据集。 提出了（a）解决R. rubrum和牛域I的晶体结构I，R。rubrum结构域III（Q核苷酸）和E. astolosolytica连续域IIII-I； （b）通过对这些晶体结构的计算机建模和适当突变体的计算机建模进行调查，以扩展I和III的域； （c）解决整个大肠杆菌的晶体结构。 （2）质子易位的机制：在域II中，有3个高序列保护簇（60-80％）。 A中的两个簇中的A和ASP似乎（化学或结构上）与质子易位有关。该ASP是域II中唯一保守的负电荷残基，其突变对ASN或ILE的突变也会改变TH对NADPH的亲和力（KM和KD）。 上面的簇具有10个保守的羟基化残基，可能与质子易位有关。 此外，域III包含一个保守的酸性贴片，可以与域II的胞质侧环中的带正电的残基相互作用。 提议将这些残基突变在大肠杆菌中，以研究它们在质子易位和II和III结构域之间的构象能量转移中的作用。此外，对接域I-III模型将通过互补界面残基和活动分析的突变进行测试。