Intermediates in O2 Activation by Oxygenases at Non-heme Iron Centers

非血红素铁中心加氧酶激活 O2 的中间体

基本信息

批准号：
9895822
负责人：
JOHN D LIPSCOMB
金额：
$ 57.3万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-04-01 至 2021-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9895822
关键词：
Active Sites Area Atmosphere Bacteria Biological Catalysis Cell Death Chemistry Chloramphenicol Crystallization Detection Development Diffuse Dioxygen Dioxygenases Disease Energy Metabolism Environment Enzymes Freezing Genetic Materials Health Human Iron Kinetics Light Mammals Metals Methane Methane hydroxylase Methods Mixed Function Oxygenases Molecular Mononuclear Natural Products Nature Optics Organ Oxidases Oxidation-Reduction Oxides Oxygen Oxygenases Pathway interactions Pharmaceutical Preparations Process Production Reaction Reactive Oxygen Species Site-Directed Mutagenesis Source Spectrum Analysis Structure System Techniques Testing Time Work analog antineoplastic antibiotics biological systems carcinogenicity extradiol dioxygenase greenhouse gases human disease man novel public health relevance three dimensional structure

项目摘要

DESCRIPTION (provided by applicant): We propose to determine the molecular and regulatory mechanisms of a varied and versatile set of non-heme Fe- containing mono- and dioxygenases as a means to study the mechanisms of biological dioxygen activation. A major focus will be on the detection and characterization of reaction cycle intermediates. The most important O2 activation strategies used in nature are represented within the group of enzymes we have chosen, allowing the problem to be approached on a broad front. The activation of O2 by Fe-containing enzymes is highly regulated because escape of reactive oxygen species (ROS) causes damage to genetic material and other biomolecules, leading to cell death in bacteria and diseases in humans. Consequently, our study will delineate the stabilizing and destabilizing forces for the reactive intermediates in the O2 activation process. The mononuclear Fe-containing dioxygenase enzymes we study all use aromatic substrates and represent the major means by which natural and man-made aromatics are biodegraded. This provides the first line of defense against the toxic and carcinogenic effects of these compounds, and thus these enzymes have another substantial impact on human health. The dioxygenase enzymes proposed for study include: Fe(II)-containing extradiol dioxygenases, Fe(III)-containing intradiol dioxygenases, and redox cycling Rieske dioxygenases. The dinuclear Fe cluster-containing monooxygenases being studied are soluble methane monooxygenase (MMO) and two enzymes from the biosynthetic pathway for chloramphenicol, CmlA and CmlI. MMO is the principal barrier to release of the potent greenhouse gas methane into our atmosphere. CmlA and CmlI have numerous homologs in the natural product biosynthetic pathways for some of our most effective antibiotic and chemotherapy drugs, but remain mechanistically uncharacterized. We have developed hypotheses for the mechanisms by which the active site iron(s) in each type oxygenase we study is used to promote catalysis. These will now be tested by exploiting methods we have perfected to observe their reactions step by step as they occur. Past studies have led to the development of single turnover systems for each of the enzyme classes. Intermediates will be detected and trapped by coordinated use of stopped-flow transient kinetics, rapid mixing freeze quench (RFQ), site directed mutagenesis, and use of slow substrate analogs. The trapped intermediates will be characterized by a range of spectroscopies including optical, EPR, rRaman, NRVS, EXAFS, NIR CD, VTVH MCD, Mössbauer and a novel time resolved rRaman technique (TR3). Also, novel in crystallo techniques will be used in which the reactions are slowed (and often stopped at intermediates) by carrying them out in enzyme crystals. The 3D structures of intermediates trapped in crystallo will be solved crystallographically and through the use of newly developed time resolved techniques that employ the Linac Coherent Light Source. In ongoing studies, we have detected and trapped several widely postulated, but previously unobserved, intermediates in each of the enzyme classes. This work will yield fundamental information about the chemistry of oxygenases, oxygen, and metals in biological systems. The basic concepts that emerge will be useful in such areas as the mechanisms of similar enzymes in mammals and methods to interdict the production of deleterious diffusible ROS in humans.

描述（由适用提供）：我们建议确定一种多种含有单聚糖和二氧酶的多种多样和多功能的分子和调节机制，以研究生物二氧化物激活的机制。重点将是反应周期中间体的检测和表征。自然界中使用的最重要的O2激活策略是在我们选择的酶组中表示的，允许在广泛的方面解决该问题。通过含有Fe的酶激活O2是高度调节的，因为活性氧（ROS）的逃生会损害遗传物质和其他生物分子，从而导致细菌的细胞死亡和人类疾病。因此，我们的研究将描述O2激活过程中反应性中间体的稳定和不稳定力。我们研究的单核Fe二氧酶酶使用芳香族底物，并代表了自然和人造芳香剂生物降解的主要手段。这提供了针对这些化合物的有毒和致癌作用的第一道防线，因此这些酶对人类健康产生了另一个实质性影响。提出的研究的二氧酶酶包括：含有Fe（II） - 含有二加氧酶的含有Fe（II），含有Fe（III）的含有二氧二加氧酶的Fe（III）和氧化还原循环rieske dioxyghyass。研究二氧酶的二氧酶是固体甲烷单加氧酶（MMO）和来自氯霉素，CMLA和CMLI的生物合成途径的两种酶。 MMO是将有效的温室气甲烷释放到我们大气中的主要障碍。 CMLA和CMLI在天然产物生物合成途径中具有许多同源物，用于我们一些最有效的抗生素和化学疗法药物，但在机械上仍然没有任何特征。我们已经为我们研究的每种类型的氧合酶中的活性位点铁（S）提出了假设，用于促进催化。现在将通过利用方法来测试这些方法，我们可以完美地观察它们的反应。过去的研究导致了每个酶类别的单个离职系统的发展。将通过协调使用停止流动瞬态动力学，快速混合冻干（RFQ），定向诱变以及使用缓慢的底物类似物来检测和捕获中间体。被困的中间体的特征将以各种光谱传感器为特征，包括光学，EPR，RRAMAN，NRVS，EXAFS，NIR CD，VTVH MCD，Mössbauer和新型时间解决RRAMAN TECHICE（TR3）。同样，将使用新颖的晶体技术中的新颖技术，其中反应缓慢（通常在中间体停止），通过在酶晶体中进行。将被困在Crystallo中的中间体的3D结构将通过晶体学求解，并通过使用新开发的时间解析技术，这些技术采用了Linac相干光源。在正在进行的研究中，我们检测到并将几个酶类中的几个中间体广泛张贴。这项工作将产生有关生物系统中氧酶，氧气和金属化学的基本信息。出现的基本概念将在哺乳动物中类似酶的机制和方法固定在人类中生产精致的扩散ROS的方法中很有用。

项目成果

期刊论文数量（7）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Contrasting Mechanisms of Aromatic and Aryl-Methyl Substituent Hydroxylation by the Rieske Monooxygenase Salicylate 5-Hydroxylase

DOI：
10.1021/acs.biochem.2c00610
发表时间：
2022-12-30
期刊：
BIOCHEMISTRY
影响因子：
2.9
作者：
Rogers，Melanie S.;Gordon，Adrian M.;Lipscomb，John D.
通讯作者：
Lipscomb，John D.

Determination of the iron(IV) local spin states of the Q intermediate of soluble methane monooxygenase by Kβ X-ray emission spectroscopy.

DOI：
10.1007/s00775-022-01953-4
发表时间：
2022-09
期刊：
Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry
影响因子：
0
作者：
通讯作者：

Corrigendum: Double-flow focused liquid injector for efficient serial femtosecond crystallography.

勘误表：用于高效串行飞秒晶体学的双流聚焦液体注射器。

DOI：
10.1038/srep46846
发表时间：
2017
期刊：
Scientific reports
影响因子：
4.6
作者：
Oberthuer,Dominik;Knoška,Juraj;Wiedorn,MaxO;Beyerlein,KennethR;Bushnell,DavidA;Kovaleva,ElenaG;Heymann,Michael;Gumprecht,Lars;Kirian,RichardA;Barty,Anton;Mariani,Valerio;Tolstikova,Aleksandra;Adriano,Luigi;Awel,Salah;Barth
通讯作者：
Barth

Enzyme Substrate Complex of the H200C Variant of Homoprotocatechuate 2,3-Dioxygenase: Mössbauer and Computational Studies.