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

基本信息

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
项目类别：
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

项目摘要

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.

该子项目是利用该技术的众多研究子项目之一资源由 NIH/NCRR 资助的中心拨款提供。子项目和研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金，因此可以在其他 CRISP 条目中表示。列出的机构是中心，不一定是研究者的机构。尿卟啉原 III 合酶 (URO-synthase) 是血红素生物合成途径中的第四种酶，催化线性四吡咯羟甲基联烷 (HMB) 的环化和 D 环异构化，形成尿卟啉原 (URO'gen) III，即环状四吡咯和生理学物质。血红素、叶绿素和钴胺素的前体。在缺乏 URO 合酶的情况下，HMB 快速、非酶促环化形成 URO'gen I，这是一种在先天性红细胞生成性卟啉症患者体内积聚的非生理性致病性异构体 [1]。我们的目标是表征该酶将 HMB 特异性转化为 URO III 的机制，避免 URO I 的形成，以及酶结构在提供立体特异性方面的作用。据报道，使用重组酶，人 URO 合酶的晶体结构分辨率为 1.85 [2]。但由于酶无法与底物类似物共结晶，绘制酶活性位点图谱和研究其反应机制的努力并未成功。因此，我们确定了人类 URO 合酶的 NMR 共振分配，并使用化学位移微扰方法和计算机对接实验 (Autodock v. 3.05) 来绘制酶的两个球状结构域之间的大裂隙中的活性位点残基图。 3]。我们现在已经通过 NMR 解析了该酶的 3D 溶液结构（未发表）。核磁共振和晶体结构非常相似，除了球状区域之间的裂缝大小不同。虽然晶体结构具有大的开裂，但在 NMR 溶液结构中，域更靠近。为了研究酶的结构和灵活性在 HMB 转化为 URO III 中的作用，我们对与底物、活化底物（氮杂富烯）、螺吡咯啉过渡态中间体和产品。在我们的工作假设中，晶体/NMR结构的开放/闭合构象代表了这些配体在反应机制期间可达到的不同状态，其中酶以允许氮杂富烯仅攻击吡咯的方式限制底物构象空间产生 III 异构体的碳，同时保护会导致非酶促形成的 I 异构体的碳攻击。为了估计 TeraGrid(tm) 开发帐户的 SU 要求，我们为 Dell PowerEdge 1950 上的溶剂化蛋白质配体系统确定了 24 小时/纳秒模拟时间的基准，该系统具有 8 个 2.66 GHz 内核，运行 NAMD 版本2.6。我们估计需要 10 ns 模拟实验，涉及野生型或定点突变酶形式的开放和封闭形式的复合物，具有酶底物的多个方向、预测的底物的活化氮杂富烯形式、建议的过渡态中间体和产品。将使用自由能微扰 (FEP) 方法对每个配体进行额外的模拟，以计算局部最小值中构象结合的自由能。这些实验将需要大约 30,000 SU 和大约 0.5 TB 的磁盘存储。参考文献 1. Anderson, K.E.，《塞西尔医学教科书》中的《卟啉症》，L. Goldman 和 C.J. Bennett，编辑。 2000 年，费城。 p。 1123-1132。 2. Mathews, M.A. 等人，人尿卟啉原 III 合酶的晶体结构。 Embo J，2001。20(21)：第 14 页。 5832-9。 3. Cunha, L.F. 等人，人尿卟啉原 III 合成酶：基于 NMR 的活性位点图谱。蛋白质：结构、功能和生物信息学，2007 年。出版中。