STRUCTURAL STUDIES OF MULTICOMPONENT BACTERIAL MONOOXYGENASES
多组分细菌单加氧酶的结构研究
基本信息
- 批准号:7955153
- 负责人:
- 金额:$ 0.21万
- 依托单位:
- 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:2009-04-01 至 2010-03-31
- 项目状态:已结题
- 来源:
- 关键词:CharacteristicsComplementComplexComputer Retrieval of Information on Scientific Projects DatabaseDataData SetEnzymesFamilyFundingGrantHydrocarbonsInstitutionMetalsMethane hydroxylaseMixed Function OxygenasesPhenol 2-monooxygenaseProtein BindingPseudomonasPublicationsReportingResearchResearch PersonnelResolutionResourcesRoleSiteSite-Directed MutagenesisSourceStructureUnited States National Institutes of HealthWorkcarboxylatedimerdivalent metalgenetic regulatory proteinimprovedmutantreconstitutionstructural biologytoluene 2-xylene monooxygenase
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.
Bacterial multicomponent monooxygenases are a family of enzymes that utilize a carboxylate-bridged diiron center to hydroxyalate a variety of hydrocarbon substrates. Essential for this activity is the formation of a complex between the hydroxylase and regulatory protein. The structure of the Pseudomonas sp. OX1 phenol hydroxylase, PHH-PHM complex was determined to 2.3 ¿ resolution. The regulatory protein binds on helices A, E, and F of the hydroxylase alpha subunit at the dimer interface 12 ¿ above the diiron center. Although the metal center resembled the structure of mixed-valent methane monooxygenase, significant structural changes were observed in helices E and F that were different than the configuration typically observed in the uncomplexed form of the hydroxylase. These changes have implications for substrate activation and substrate selectivity, the significance of which is currently being explored. Since determining this structure, structural work has focused on obtaining higher resolution data on the PHH-PHM complex with improved regulatory protein occupancy, and crystallographic characterization of various mutants of PHH and toluene/o-xylene monooxygenase hydroxylase from Pseudomonas sp. OX1. Higher resolution and structures of the complex with improved regulatory protein occupancy could confirm mechanistic conclusions and speculations about the structural effects of regulatory protein binding proposed upon analysis of the initial PHH-PHM data. Similarly, a structure of the hydroxylase in the absence of the regulatory protein may reveal critical, mechanistically relevant characteristics of the hydroxylase structure while also providing a platform to further investigate the structural effects of regulatory protein binding on the hydroxylase through comparison with the PHH-PHM structure. Diffraction data from these projects are only preliminary and require improvement and analysis before they may be reported. In effort to obtain a diferric or analogous structure of PHH or PHH-PHM, dithionite soaked divalent metal reconstituted and crystals of both species were pursued but not yet successfully obtained such to yield a quality diffraction data set for structure determination. Mechanistic data on mutant forms of ToMOH (T201X) and PHH (N204X) in which conserved residues near the diiron site were varied by site-directed-mutagenesis indicate roles for these residues, so the mutants were crystallized and their diffraction data collected to yield structural information that complements the mechanistic studies. This structural information is presently being analyzed and prepared for publication in conjuction with the relevant mechanistic studies and their results.
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。
细菌多组分单加氧酶是一类利用羧酸桥联的双铁中心对多种碳氢化合物底物进行羟基化的酶家族。这种活性的关键是在羟基酶和调节蛋白之间形成一个复合体。对假单胞菌的结构进行了研究。OX1苯酚羟基酶,PHH-PHM络合物的分辨率为2.3°。调节蛋白结合在双铁中心上方二聚体界面上的羟基酶α亚基的螺旋A、E和F上。尽管金属中心类似于混合价甲烷单加氧酶的结构,但在螺旋E和F中观察到了显著的结构变化,这与典型的非络合形式的羟基酶的构型不同。这些变化对底物活化和底物选择性有影响,其意义目前正在探索中。自确定这种结构以来,结构工作主要集中在获得更高分辨率的PHH-PHM复合体的数据,以及假单胞菌PHH和甲苯/邻二甲苯单加氧酶羟基酶的各种突变体的结晶学特征。OX1.更高的分辨率和结构以及调节蛋白占有率的提高可以证实通过分析初始PHH-PHM数据而得出的关于调节蛋白结合结构效应的机械性结论和推测。同样,在缺乏调节蛋白的情况下,羟基酶的结构可能揭示羟基酶结构的关键的、机械相关的特征,同时也提供了一个平台,通过与PHH-PHM结构的比较,进一步研究调节蛋白结合对羟基酶的结构影响。这些项目的衍射数据只是初步数据,需要改进和分析,然后才能报告。为了获得PHH或PHH-PHM的二亚铁或类似结构,重新构造了二亚硫酸盐浸泡的二价金属,并尝试了这两种物种的晶体,但尚未成功获得,从而产生了用于结构测定的质量衍射数据集。ToMOH(T201X)和PHH(N204X)突变形式的机制数据通过定点突变改变了双铁位点附近的保守残基,表明了这些残基的作用,因此突变体被结晶并收集它们的衍射数据,以产生补充机制研究的结构信息。目前正在结合相关的机理研究及其结果对这些结构信息进行分析并准备出版。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Stephen J. Lippard其他文献
X-ray structure of a dodecamer duplex containing the major cisplatin d(GpG) intrastrand cross-link
- DOI:
10.1016/0162-0134(95)97300-f - 发表时间:
1995-08-01 - 期刊:
- 影响因子:
- 作者:
Patricia M. Takahara;Amy C. Rosenzweig;Christin A. Frederick;Stephen J. Lippard - 通讯作者:
Stephen J. Lippard
Frank Albert Cotton (1930–2007)
弗兰克·艾伯特·科顿(1930 年至 2007 年)
- DOI:
10.1038/446626a - 发表时间:
2007-04-04 - 期刊:
- 影响因子:48.500
- 作者:
Stephen J. Lippard - 通讯作者:
Stephen J. Lippard
High resolution crystal structures of the hydroxylase protein of methane monooxygenase
- DOI:
10.1016/0162-0134(95)97479-a - 发表时间:
1995-08-01 - 期刊:
- 影响因子:
- 作者:
Amy C. Rosenzweig;Pär Nordlund;Stephen J. Lippard;Christin A. Frederick - 通讯作者:
Christin A. Frederick
Conjugués de nanoparticule de polynucléotide polyvalente en tant que véhicules de distribution pour un agent chimiothérapique
多核苷酸多价纳米粒子结合物与化学药物分配载体
- DOI:
- 发表时间:
2010 - 期刊:
- 影响因子:0
- 作者:
Chad A. Mirkin;David A. Giljohann;W. Daniel;Stephen J. Lippard;Shanta Dhar - 通讯作者:
Shanta Dhar
Structural, mechanistic, and model studies for methane monooxygenase
- DOI:
10.1016/0162-0134(95)97192-s - 发表时间:
1995-08-01 - 期刊:
- 影响因子:
- 作者:
Stephen J. Lippard - 通讯作者:
Stephen J. Lippard
Stephen J. Lippard的其他文献
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{{ truncateString('Stephen J. Lippard', 18)}}的其他基金
STRUCTURAL STUDIES OF BACTERIAL MULTICOMPONENT MONOOXYGENASES
细菌多组分单加氧酶的结构研究
- 批准号:
8362193 - 财政年份:2011
- 资助金额:
$ 0.21万 - 项目类别:
INVESTIGATIONS OF CISPLATIN-DNA CROSS-LINKS ON NUCLEOSOME CORE PARTICLES
核小体核心颗粒上顺铂-DNA 交联的研究
- 批准号:
8169250 - 财政年份:2010
- 资助金额:
$ 0.21万 - 项目类别:
STRUCTURAL STUDIES OF BACTERIAL MULTICOMPONENT MONOOXYGENASES
细菌多组分单加氧酶的结构研究
- 批准号:
8170154 - 财政年份:2010
- 资助金额:
$ 0.21万 - 项目类别:
STRUCTURAL STUDIES OF MULTICOMPONENT BACTERIAL MONOOXYGENASES
多组分细菌单加氧酶的结构研究
- 批准号:
8169251 - 财政年份:2010
- 资助金额:
$ 0.21万 - 项目类别:
STRUCTURAL STUDIES OF BACTERIAL MULTICOMPONENT MONOOXYGENASES
细菌多组分单加氧酶的结构研究
- 批准号:
7954158 - 财政年份:2009
- 资助金额:
$ 0.21万 - 项目类别:
CHEMISTRY AND BIOLOGY OF PLATINUM ANTICANCER DRUGS
铂类抗癌药物的化学和生物学
- 批准号:
7955152 - 财政年份:2009
- 资助金额:
$ 0.21万 - 项目类别:
Nonheme Diiron Centers and the Biological Oxidation of Hydrocarbons
非血红素二铁中心和碳氢化合物的生物氧化
- 批准号:
7923548 - 财政年份:2009
- 资助金额:
$ 0.21万 - 项目类别:
STRUCTURAL STUDIES OF BACTERIAL MULTICOMPONENT MONOOXYGENASES
细菌多组分单加氧酶的结构研究
- 批准号:
7954496 - 财政年份:2009
- 资助金额:
$ 0.21万 - 项目类别:
STRUCTURAL STUDIES OF BACTERIAL MULTICOMPONENT MONOOXYGENASES
细菌多组分单加氧酶的结构研究
- 批准号:
7721732 - 财政年份:2008
- 资助金额:
$ 0.21万 - 项目类别:
STRUCTURAL STUDIES OF BACTERIAL MULTICOMPONENT MONOOXYGENASES
细菌多组分单加氧酶的结构研究
- 批准号:
7597911 - 财政年份:2007
- 资助金额:
$ 0.21万 - 项目类别:
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