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

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 尿卟啉原III合成酶(URO-Synthase)是血红素生物合成途径中的第四个酶，催化线性四氢吡咯羟甲基丁烷(HMB)的环化和D-环异构化，形成尿卟啉原(URO‘gen)III，它是环状四吡咯的产物，是血红素、叶绿素和钴胺的生理前体。在没有尿素合成酶的情况下，HMB迅速和非酶地环化形成URO‘gen I，这是一种非生理性和致病的异构体，在先天性红细胞生成性门静脉症患者中积累[1]。我们的目标是表征这种酶将HMB特异性地转化为Uro III，避免Uro I形成的机制，以及酶结构在提供立体特异性中的作用。利用重组酶[2]，以1.85的分辨率报道了人尿合酶的晶体结构。但是，由于不能将酶与底物类似物共结晶，绘制酶的活性部位并研究其反应机理的努力没有成功。因此，我们确定了人尿合酶的核磁共振共振归属，并使用化学位移微扰法和电子对接实验(Autodock v3.05)，绘制了该酶两个球状结构域之间的大裂缝中的活性位点残基[3]。我们现在已经通过核磁共振(未发表)解决了该酶的三维溶液结构。除了球状结构域之间的裂隙大小外，核磁共振和晶体结构非常相似。虽然晶体结构有一个大的开放裂隙，但在核磁共振溶液结构中，结构域更紧密。为了研究酶的结构和柔韧性在HMB转化为Uro III过程中的作用，我们对与底物络合的酶、活化的底物(氮杂富烯)、螺吡咯烷过渡态中间体和产物进行了分子动力学模拟。在我们的工作假设中，晶体/核磁共振结构的开放/封闭构象代表了这些配体在反应机理中可获得的不同状态，酶以这样的方式限制底物构象空间，允许氮杂富烯只对导致III异构体的吡咯碳进行攻击，同时保护会导致非酶形成I异构体的碳攻击。为了评估我们对TeraGrid(Tm)开发客户的SU要求，我们确定了在Dell PowerEdge 1950上运行NAMD版本2.6的8核2.66 GHz的溶剂化蛋白质-配体系统的基准时间为24小时/纳秒。我们估计需要10 ns的模拟实验，涉及野生型或定点突变酶形式的开放和封闭形式的复合体与酶底物的多个取向，底物的预测激活的氮富烯形式，建议的过渡态中间体，以及产品。另外还将使用自由能微扰(FEP)方法对每个配体进行额外的模拟，以计算局部极小值中构象结合的自由能。这些实验将需要大约30,000个SU和大约0.5 TB的磁盘存储。参考文献1.Anderson，K.E.，The Porporrias，收录于《塞西尔医学教科书》，L.Goldman和C.J.Bennett主编。2000年，费城。1123-1132页。2.Mathews，M.A.，等，人尿卟啉原III合成酶的晶体结构。EMBO J，2001。20(21)：第5832-9页。3.Cunha，L.F.等人，人尿卟啉原III合成：基于核磁共振的活性部位图谱。蛋白质：结构、功能和生物信息学，2007。在媒体上。

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.