STRUCTURE, FUNCTION AND DYNAMICS OF TRANSHYDROGENASE

转氢酶的结构、功能和动力学

• 批准号: 6520280
• 负责人: Charles David Stout
• 金额: $ 37.04万
• 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/6520280
• 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.

该项目涉及牛线粒体、细菌（大肠杆菌、红色红螺菌）和溶组织内阿米巴转氢酶 (TH) 的结构和功能的研究，这些酶在以下反应中催化 NAD(H) 和 NADP(H) 之间的氢阴离子转移：与跨膜质子易位耦合。 由于质子泵 TH 的结构相对简单，并且是研究由底物结合能通过蛋白质构象变化驱动的质子易位的优秀系统。 TH 在线粒体中产生 NADPH，谷胱甘肽还原酶和谷胱甘肽过氧化物酶等利用 NADPH 来消除呼吸链产生的超氧化物歧化产生的有害 H2O2。具体目标是 (1) 结构研究：(a) TH 由 3 个结构域组成：分别结合 NAD(H) 和 NADP(H) 的膜外结构域 I（400-430 个残基）和 III（200 个残基）以及膜-携带质子通道的插入结构域 II（360-400 个残基）。 我们已在牛的大肠杆菌结构域 I 和 III 以及红红菌 TH 中表达。 纯化的结构域 I 和 III 在不存在结构域 II 的情况下重建 TH 活性。 我们已经在 1.2 埃单位分辨率下解析了含有结合 NADP 的牛结构域 III 的晶体结构，并在 2.1 埃单位分辨率下获得了 R. rubrum 结构域 I 天然晶体的完整数据集。 建议 (a) 解析 R. rubrum 和牛结构域 I、R. rubrum 结构域 III (q 个核苷酸) 和 E. histolytica 连续结构域 III-I 的晶体结构； (b) 通过计算机模拟这些晶体结构和适当突变体的结构域 I 和 III 的对接来研究； (c) 解析整个大肠杆菌 TH 的晶体结构。 (2)质子易位机制：在域II中，存在3个高度序列保守簇(60-80%)。 其中 2 个簇中的 His 和 Asp 似乎与质子易位有关（化学上或结构上）。该 Asp 是结构域 II 中唯一保守的带负电荷残基，其突变为 Asn 或 Ile 也会改变 TH 对 NADPH 的亲和力（Km 和 Kd）。 上述簇有10个保守的羟基化残基，可能参与质子易位。 此外，结构域 III 包含一个保守的酸性补丁，它可以与结构域 II 的细胞质侧环中带正电的残基相互作用。 建议对大肠杆菌 TH 中的这些残基进行突变，以研究它们在质子易位以及结构域 II 和 III 之间构象能量转移中的作用。此外，对接结构域 I-III 模型将通过互补界面残基的突变和活性测定进行测试。