THE ROLE OF UROPORPHYRINOGEN III SYNTHASE STRUCTURE AND FLEXIBILITY IN THE FORM

尿卟啉原 III 合酶结构的作用和形式的灵活性

• 批准号: 7956247
• 负责人: DAVID T BISHOP
• 金额: $ 0.08万
• 依托单位: CARNEGIE-MELLON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7956247
• 关键词: Accounting; Active Sites; Benchmarking; Binding; Bioinformatics; Biomedical Research; Carbon; Chemicals; Chlorophyll; Cleaved cell; Cobalamin; Complex; Computer Retrieval of Information on Scientific Projects Database; Computer Simulation; Cyclization; Development; Docking; Enzymes; Erythropoietic Porphyria; Free Energy; Funding; Grant; Heme; High Performance Computing; Hour; Human; Hydroxymethylbilane Synthase; Institution; Isomerism; Lead; Ligands; Maps; Medicine; Methods; Molecular Conformation; Multienzyme Complexes; Pathway interactions; Patients; Philadelphia; Physiological; Porphyrias; Proteins; Pyrroles; Reaction; Recombinants; Reporting; Research; Research Personnel; Resolution; Resources; Role; Running; Site; Solutions; Source; Structure; System; Tetrapyrroles; Textbooks; Time; United States National Institutes of Health; Uroporphyrinogen III; Uroporphyrinogen III Synthetase; Uroporphyrinogens; Work; analog; base; enzyme structure; enzyme substrate; flexibility; hydroxymethylbilane; molecular dynamics; mutant; nanosecond; protein structure function; research study; simulation

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Uroporphyrinogen III synthase (URO-synthase), the fourth enzyme in the heme biosynthetic pathway, catalyzes the cyclization and D-ring isomerization of the linear tetrapyrrole hydroxymethylbilane (HMB), to form uroporphyrinogen (URO'gen) III, the cyclic tetrapyrrole and physiologic precursor of heme, chlorophyll, and cobalamin. In the absence of URO-synthase, HMB rapidly and non-enzymatically cyclizes to form URO'gen I, the non-physiologic and pathogenic isomer that accumulates in patients with congenital erythropoietic porphyria [1]. Our objective is to characterize the mechanism by which this enzyme converts HMB specifically to URO III, avoiding URO I formation, and the role of enzyme structure in providing stereospecificity. The crystal structure of human URO-synthase has been reported at 1.85 resolution using a recombinant enzyme [2]. But efforts to map the enzyme's active site and to investigate its reaction mechanism were not successful due to the inability to co-crystallize the enzyme with a substrate analogue. Therefore, we have determined the NMR resonance assignments for human URO-synthase and used the chemical shift perturbation method and in silico docking experiments (Autodock v. 3.05), to map the active site residues in the large cleft between the enzyme's two globular domains [3]. We have now solved the 3D solution structure of this enzyme by NMR (unpublished). The NMR and crystal structures are very similar, except in the size of the cleft between the globular domains. While the crystal structure has a large open cleft, in the NMR solution structure the domains are closer together. To investigate the role of the enzyme's structure and flexibility in the conversion of HMB to URO III, we are performing molecular dynamics simulations on the enzyme complexed with the substrate, the activated substrate (an azafulvene), the spiro-pyrrolenine transition state intermediate, and the product. In our working hypothesis, the open/closed conformations of the crystal/NMR structures represent different states accessible to these ligands during the reaction mechanism, with the enzyme constraining the substrate conformational space in such a way to allow attack of the azafulvene only on the pyrrole carbon that results in the III isomer while protecting the carbon attack that would lead to the non-enzymatically formed I isomer. In order to estimate our SU requirements for a TeraGrid(tm) development account, we have determined a benchmark of 24 hours/nanosecond simulation time for our solvated protein-ligand system on a Dell PowerEdge 1950, with 8 cores at 2.66 GHz running NAMD version 2.6. We estimate requiring 10 ns simulation experiments involving complexes of open and closed forms of wild-type or site-directed mutant enzyme forms with multiple orientations of the enzyme's substrate, the predicted activated azafulvene form of the substrate, the proposed transition state intermediate, and the product. Additional simulations to calculate the free energy of binding of conformations in local minima will be performed for each ligand using free energy perturbation (FEP) methods. These experiments will require about 30,000 SU, and about 0.5 terabytes of disk storage. References 1. Anderson, K.E., The Porphyrias, in Cecil Textbook of Medicine, L. Goldman and C.J. Bennett, Editors. 2000, Philadelphia. p. 1123-1132. 2. Mathews, M.A., et al., Crystal structure of human uroporphyrinogen III synthase. Embo J, 2001. 20(21): p. 5832-9. 3. Cunha, L.F., et al., Human uroporphyrinogen III syntahse: NMR-Based mapping of the active site. Proteins: Structure, Function, and Bioinformatics, 2007. in press.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 尿中肌原子III合酶（URO-合成酶），血红素生物合成途径中的第四个酶，催化线性四吡喃甲甲基甲基甲烷（HMB）的环化和D环异构化，形成尿中的III II IIIIIIIIIIIIIIIIIIIIII'GROMOLICORINIC II II，URO''''''''U'''''''血红素，叶绿素和钴胺素的前体。在缺乏URO合并酶的情况下，HMB迅速和非酶环化形成Uro'gen I，这是一种非生理和致病性异构体，可在先天性红细胞生成性斑岩的患者中累积[1]。我们的目标是表征该酶将HMB专门转换为URO III的机制，避免了Uro I的形成以及酶结构在提供立体特定性中的作用。使用重组酶以1.85的分辨率报道了人URO合酶的晶体结构[2]。但是，由于无法与底物类似物共结合酶的共结晶，因此绘制酶的活性位点并研究其反应机制的努力并没有成功。因此，我们已经确定了人URO-合成酶的NMR共振分配，并使用了化学位移扰动方法和计算机对接实验（Autodockv。3.05），以在酶的两个球状域之间绘制较大裂缝中的活性位点残基[3]。现在，我们通过NMR（未发表）解决了该酶的3D溶液结构。 NMR和晶体结构非常相似，除了球状结构域之间的裂缝大小。虽然晶体结构具有较大的开放裂缝，但在NMR溶液结构中，域更加紧密。为了研究该酶的结构和柔韧性在HMB转换为URO III中的作用，我们正在与与底物复合的酶进行分子动力学模拟，激活的底物（AZAFULEVENE），SIPERO-吡咯烷氨酸过渡状态中间体和产物。 In our working hypothesis, the open/closed conformations of the crystal/NMR structures represent different states accessible to these ligands during the reaction mechanism, with the enzyme constraining the substrate conformational space in such a way to allow attack of the azafulvene only on the pyrrole carbon that results in the III isomer while protecting the carbon attack that would lead to the non-enzymatically formed I isomer.为了估计我们对TeraGrid（TM）开发帐户的SU要求，我们确定了我们在Dell PowerEdge上的溶剂化蛋白质配体系统的24小时/纳秒模拟时间的基准，其中8个核心为2.66 GHz，运行NAMD版本2.6。我们估计需要10个NS仿真实验，涉及野生型或位置定向的突变酶的开放和封闭形式的复合物，并具有多种取向酶的底物，预测的已激活的植物形式，拟议的过渡状态中间体和产品。将使用自由能扰动（FEP）方法为每个配体进行局部最小值的构象结合的自由能的其他模拟。这些实验将需要大约30,000个SU，约0.5吨的磁盘存储。参考文献1。Anderson，K.E。，Porphyrias，在Cecil医学教科书中，L。Goldman和C.J. Bennett，编辑。 2000年，费城。 p。 1123-1132。 2。Mathews，M.A。等，人类尿素原III合酶的晶体结构。 Embo J，2001。20（21）：p。 5832-9。 3。Cunha，L.F。等人，人类肾纤维蛋白原III Sanntahse：基于NMR的活性位点的映射。蛋白质：结构，功能和生物信息学，2007年。