Methane Monoxygenase Structure and Function

甲烷单加氧酶的结构和功能

• 批准号: 7147833
• 负责人: JOHN D LIPSCOMB
• 金额: $ 34.68万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7147833
• 关键词: Methanobacteriaceae; Mossbauer spectrometry; Raman spectrometry; active sites; bacterial proteins; circular dichroism; cofactor; crosslink; electron nuclear double resonance spectroscopy; electron spin resonance spectroscopy; enzyme complex; enzyme mechanism; enzyme reconstitution; enzyme structure; enzyme substrate; matrix assisted laser desorption ionization; metalloenzyme; methane monooxygenase; microorganism metabolism; nuclear magnetic resonance spectroscopy; oxidation reduction reaction; protein purification; protein structure function

DESCRIPTION (provided by applicant): We will investigate the 3D structure, catalytic mechanism, and regulation of soluble methane monooxygenase (MMO). MMO initiates the oxidation of CH4 to CO2 by methanotrophic bacteria. In this way, the atmospheric egress of nearly all of the enormous quantity of CH4 (greenhouse gas with 20 times the potency of CO2) generated by anaerobic bacteria is prevented. MMO also adventitiously catalyzes the oxidation of many other chemicals fostering applications in synthesis as well as biodegradation of abundant pollutants with human toxicity (e.g. trichloroethylene). MMO from Methylosinus trichosporium OB3b is composed of 3 proteins: hydroxylase (MMOH), reductase (MMOR), and "B" (MMOB). MMOH has a bis-u-hydroxo-bridged dinuclear Fe cluster needed for catalysis. Spectroscopic studies (optical, EPR, Mossbauer, EXAFS, ENDOR, rRaman, fluorescence, NMR, MCD, and CD), turnover of diagnostic substrates, and transient kinetics are being used to study the structure and mechanism. Transient kinetic studies have revealed 2 stable and 7 transient intermediates in the reaction cycle. 1 intermediate, compound Q, contains a bis-u-oxo-Fe(IV)-Fe(IV) cluster which reacts directly with CH4 to give CH3OH. Q is the first intermediate isolated in an oxygenase that can attack unactivated hydrocarbons. Ongoing studies suggest that MMOR and MMOB regulate catalysis by increasing the rate of Q formation and by controlling the rate of substrate entry into the active site of MMOH based on size. MMOB mutants have been purified that allow the rate of each step in the catalytic cycle to be individually regulated. Our recent studies have defined the interaction surfaces between the MMO components. The proposed studies will utilize fluorescence energy transfer, cross-linking, mass spec, and crystallography techniques to define the spatial orientation of the components as well as conformational changes that gate substrate binding. Reaction cycle intermediates will be trapped using new approaches based on steady state stabilization and surface freeze quenching. These will be spectroscopically characterized by newly developed X-ray absorption and cryoreduction techniques. This work should give us insight into: 1) novel O2 activation chemistry, 2) the nature of Q, 3) a new regulation strategy, and 4) design of small molecule catalysts for hydrocarbon oxidation. Finally, lessons learned from MMO should apply to the structurally and mechanistically similar human ribonucleotide reductase, which generates the building blocks for DNA.

描述（由申请人提供）：我们将研究3D结构，催化机理和可溶性甲烷单加氧酶（MMO）的调节。 MMO通过甲烷营养细菌启动CH4至CO2的氧化。这样，可以防止几乎所有大量的CH4（温室气体具有20倍CO2效力的温室气体）的大气出口。 MMO还出外催化了许多其他化学物质在合成中的氧化以及具有人类毒性的丰富污染物的生物降解（例如，三氯乙烯）。来自Trichosporium ob3b的MMO由3种蛋白质组成：羟化酶（MMOH），还原酶（MMOR）和“ B”（MMOB）。 MMOH具有催化所需的BIS-U-HYDROXO桥梁桥梁。光谱研究（光学，EPR，MOSSBAUER，EXAFS，ENDOR，RRAMAN，荧光，NMR，MCD和CD），诊断底物的周转率以及瞬时动力学用于研究结构和机制。瞬态动力学研究表明，在反应周期中，有2个稳定和7个瞬时中间体。 1个中级化合物Q包含一个Bis-U-oxo-Fe（IV）-FE（IV）群集，该簇直接与CH4反应以得到CH3OH。 Q是在氧合酶中孤立的第一个中间体，可以攻击未激活的烃。正在进行的研究表明，MMOR和MMOB通过增加Q形成速率并根据大小来控制底物进入MMOH活性位点的速率来调节催化。已经纯化MMOB突变体，以单独调节催化循环中每个步骤的速率。我们最近的研究定义了MMO组件之间的相互作用表面。拟议的研究将利用荧光能量转移，交联，质量规格和晶体学技术来定义组件的空间取向以及栅极底物结合的构象变化。反应循环中间体将使用基于稳态稳定和表面冻结的新方法捕获。这些将以新开发的X射线吸收和冷冻功能为特征。这项工作应该使我们深入了解：1）新型O2激活化学，2）Q的性质，3）一种新的调节策略，以及4）设计用于烃氧化的小分子催化剂。最后，从MMO中学到的经验教训应适用于结构和机械上相似的人核糖核苷酸还原酶，该核苷酸还原酶生成DNA的构件。