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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、HO、酶及其底物/反应络合物的性质中间体处于不同的氧化/自旋/凝聚状态，使用高分辨率的2D/3D核磁共振。何，发现于脊椎动物、植物和细菌中，通过一种共同的机制和一组中间体发挥作用，使用血红素同时作为底物和辅因子，立体选择性地将血红素裂解成α-胆绿素、铁和一氧化碳。HO 独特的是使用过氧化氢物种作为其活化形式，以及活性物质的结构性质稳定物种的位置并不是很清楚，除了在远端有秩序的水分子都涉及到氢键网络。我们选择了三个HO，ispzyme#1来自人类，HHO，以及来自两个致病细菌白喉杆菌(CofHO)和脑膜炎奈瑟菌(NmHO)，它们共享一个共同的折叠，但显示与氢键网络有关的残基的可变序列同源性。目标是衍生物是无底物或APP-HO，静止状态HO-氯化血红素-H/JO，HO-氯化血红素-CN作为模型不稳定的含氧络合物和HO-氯化高铁血红素作为反应性过氧化氢物种的模型。因为除了一种靶向HO衍生物是顺磁性的，重点是利用适当定制的 1D/2D/3D核磁共振在超精细位移中提取丰富的独特信息。我们将开发一种新的和直接反映轴向配体与氯化高铁血红素之间氢键程度的高灵敏核磁共振探针和远端有序水/氢键网络，使用络合物Ho-氯化血红素-H2O/-OH对，并使用这项调查以及以前建立的程序，以提供关于解决方案结构。我们的兴趣集中在局部溶液与低温结晶学的比较上。分子结构，特别关注扩展的氢键网络，其中一些强健的氢键，以及这些网络中有序的水分子。我们强调比较在同一个HO的各种衍生品之间，以及在给定衍生品的不同HOO之间进行研究，以阐明变强度氢键与轴向配体性质的关系。此外，我们将表征HO、底物或中间体及其轴向配体对动力学的影响与衬底进入和离开有关的性质。最后，对于NmHO，我们将表征血红素取代基在其活性中心的位置，决定了其结构的结晶学发现无序的C-末端折叠到溶液中的活性部位，并阐明了C-末端的作用和独特的活性位点Cys113在多个功能相关的微异质性中。详细的对血红素加氧酶的分子结构和动力学性质的描述将改善我们的了解哺乳动物酶的不同作用。阐明了它们之间的相似性和细菌和哺乳动物的血红素加氧酶的差异将改善设计的前景病原菌中该酶的选择性抑制物。

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.