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Electronic/molecular structure of enzyme heme pockets

酶血红素口袋的电子/分子结构

基本信息

批准号：
7585714
负责人：
GERD N LA MAR
金额：
$ 21.42万
依托单位：
UNIVERSITY OF CALIFORNIA AT DAVIS
依托单位国家：
美国
项目类别：
财政年份：
2001
资助国家：
美国
起止时间：
2001-05-01 至 2011-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7585714
关键词：
Active Sites Address Anisotropy Attention Bacteria Biliverdine Binding C-terminal Chemicals Cleaved cell Comparative Study Complement Complex Coupling Crystallography Cyanides Diphtheria Disease Distal Electronics Enzyme Inhibitor Drugs Enzyme Inhibitors Enzymes Exhibits Goals Heme Hemin Heterogeneity Human Hydrogen Bonding Individual Investigation Iron Ligands Modeling Molecular Molecular Structure Monitor Mutagenesis Nature Oxygenases Pattern Planet Mars Plants Porphyrins Procedures Process Property Protons Reaction Relative (related person)Research Research Personnel Resolution Rest Role Sequence Homology Series Site Solutions Solvents Structure Techniques Vertebrates Water alkalinity base cofactor cryogenics design electronic structure hemin-CN improved interest novel oxidation pathogenic bacteria polypeptide programs response role model tool

项目摘要

We propose the detailed study of functionally relevant molecular/electronic structural and dynamic properties of a series of heme oxygenase, HO, enzymes and their complexes with substrate/reaction intermediates in variable oxidation/spin/liagtion states, using high resolution solution 2D/3D NMR. HO, found in vertebrates, plants and bacteria, acts by a common mechanism and set of intermediates, using heme as both substrate and cofactor, to stereoselectively cleave heme into a-biliverdin, iron and CO. HO is unique in using the hydrpperoxy species as its activated form, and the structural properties of the active site that stabilize the species are not well understood except that ordered water molecules within a distal H-bond network are involved. We select three HOs, ispzyme #1 from human, hHO, and those from two pathogenic bacteria C. diphtheriae (CofHO) and N. meningitidis (NmHO), which share a common fold, but exhibit variable sequence homology for the residues involved in the H-bonding network. The target derivatives are substrate-free or app-HO, resting state HO-hemin-H/jO, HO-hemin-CN as a model for the unstable oxy complex, and HO-hemin-OH as a model for the reactive hydroperpxy species. Since all but one targeted HO derivative are paramagnetic, emphasis is placed on utilizing appropriately tailored 1D/2D/3D NMR to extract the wealth of unique information in hyperfine shifts. We will develop a new and highly sensitive NMR probe that directly reflect the degree of H-bonding between axial ligand to the hemin and the distal ordered-water/H-bond network, using the pair of complex HO-hemin-H2O/-OH, and to use this probe, as well as previously established procedures, to provide a detailed characterization of the solution structure. Our interests focus on comparison of local solution with cryogenic crystallographic molecular structure, with particular attention paid to extended H-bond networks with some remarkably robust H-bonds, and the ordered water molecules within these networks. We emphasize comparative studies among the various derivatives of one HO, and among the different HOs for a given derivative, to elucidate the relationship between variable strength H-bonds and axial ligand properties. In addition, we will characterize the influence of HO, substrate or intermediates and their axial ligands on dynamic properties related to entry and exit of substrate. Lastly, for NmHO, we will characterize the influence of heme substituents on its seating in the active site, determine the structure of the crystallographically disordered C-terminus found folded into the active site in solution, and illuminate the role of the C-terminus and the unique active site Cys113 in multiple, functionally relevant, microheterogeneities. The detailed description of the molecular structural and dynamic properties of heme oxygenase will improve our understanding of the varied roles of mammalian enzymes. The elucidation of the similarities and differences between bacterial and mammalian heme oxygenase will improve prospects for the design of selective inhibitors for the enzyme in pathogenic bacteria.

我们建议对功能相关的分子/电子结构和动力学进行详细研究一系列血红素加氧酶、H2O、酶及其与底物/反应的复合物的特性使用高分辨率溶液 2D/3D NMR 检测可变氧化/自旋/连接状态的中间体。何，存在于脊椎动物、植物和细菌中，通过共同机制和一组中间体发挥作用，使用血红素作为底物和辅因子，将血红素立体选择性地裂解成α-胆绿素、铁和CO。其独特之处在于使用氢过氧物质作为其活化形式，并且活性物质的结构特性除了远端的有序水分子外，稳定物种的位点尚不清楚。涉及氢键网络。我们选择三种 HO，来自人类的 ispzyme #1、hHO 以及来自两个致病菌白喉杆菌 (CofHO) 和脑膜炎奈瑟菌 (NmHO) 具有共同的折叠，但氢键网络中涉及的残基表现出可变的序列同源性。目标衍生物是无底物或 app-H2O、静息态 HO-血红素-H/jO、HO-血红素-CN 作为模型不稳定的氧复合物，以及 HO-hemin-OH 作为反应性 Hydroperpxy 物种的模型。因为除了一种目标 H2O 衍生物是顺磁性的，重点是利用适当定制的 1D/2D/3D NMR 可提取超精细位移中丰富的独特信息。我们将开发一个新的和高灵敏度核磁共振探针，直接反映轴向配体与氯化血红素之间的氢键结合程度和远端有序水/氢键网络，使用一对复杂的HO-血红素-H2O/-OH，并使用该探针以及先前建立的程序，以提供详细的特征解决方案结构。我们的兴趣集中在局部解决方案与低温晶体学的比较分子结构，特别关注扩展的氢键网络，其中一些显着强大的氢键，以及这些网络中有序的水分子。我们强调比较研究一种 HO 的各种衍生物之间以及给定衍生物的不同 HO 之间的研究，以阐明不同强度的氢键和轴向配体性质之间的关系。此外，我们将表征 H2O、底物或中间体及其轴向配体对动态的影响与基材的进入和退出相关的特性。最后，对于 NmHO，我们将描述以下因素的影响：血红素取代基在其活性位点的位置上，决定了晶体学的结构发现无序的 C 末端折叠到溶液中的活性位点，并阐明了 C 末端的作用以及多个功能相关的微观异质性中独特的活性位点 Cys113。详细的血红素加氧酶的分子结构和动态特性的描述将提高我们的了解哺乳动物酶的不同作用。相似性和相似性的阐明细菌和哺乳动物血红素加氧酶之间的差异将改善设计的前景病原菌酶的选择性抑制剂。

项目成果

期刊论文数量（20）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

1H NMR study of the influence of mutation on the interaction of the C-terminus with the active site in heme oxygenase from Neisseria meningitidis: implications for product release.

1H NMR 研究突变对脑膜炎奈瑟菌血红素加氧酶 C 末端与活性位点相互作用的影响：对产品释放的影响。

DOI：
10.1021/bi1000867
发表时间：
2010
期刊：
Biochemistry
影响因子：
2.9
作者：
Peng,Dungeng;Ma,Li-Hua;Ogura,Hiroshi;Yang,En-Che;Zhang,Xuhong;Yoshida,Tadashi;LaMar,GerdN
通讯作者：
LaMar,GerdN

Influence of substrate modification and C-terminal truncation on the active site structure of substrate-bound heme oxygenase from Neisseriae meningitidis. A 1H NMR study.

DOI：
10.1021/bi200978g
发表时间：
2011-10-18
期刊：
BIOCHEMISTRY
影响因子：
2.9
作者：
Peng, Dungeng;Satterlee, James D.;Ma, Li-Hua;Dallas, Jerry L.;Smith, Kevin M.;Zhang, Xuhong;Sato, Michihiko;La Mar, Gerd N.
通讯作者：
La Mar, Gerd N.

1H NMR detection of immobilized water molecules within a strong distal hydrogen-bonding network of substrate-bound human heme oxygenase-1.