Methane Monoxygenase Structure and Function

甲烷单加氧酶的结构和功能

• 批准号: 7450847
• 负责人: JOHN D LIPSCOMB
• 金额: $ 33.68万
• 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/7450847
• 关键词: Active Sites; Acyl Carrier Protein; Aerobic; Affect; Alcohols; Alkanes; Alkenes; Alkynes; Ally; Anaerobic Bacteria; Area; Bacteria; Binding; Biodegradation; Biological Models; Biology; Carbon; Carbon Dioxide; Catalysis; Chemicals; Chemistry; Class; Collaborations; Complex; Compound Q; Crystallography; Cytochrome P450; DNA; Diagnostic; Electron Nuclear Double Resonance; Electron Transport; Energy Transfer; Enzymes; Ethers; Ethyl Ether; Exhibits; Family; Ferritin; Fluorescence; Fostering; Freezing; Goals; Grant; Hemerythrin; Human; Hydrocarbons; Kinetics; Learning; Lytic; Maps; Mediating; Metals; Methane; Methane hydroxylase; Methods; Methylosinus trichosporium; Mixed Function Oxygenases; Molecular; Mono-S; Mutation; NADH; Nature; Nicotinamide adenine dinucleotide; Object Attachment; One-Step dentin bonding system; Optics; Oxidoreductase; Oxygen; Oxygenases; Process; Proteins; Protons; Range; Rate; Reaction; Reactive Oxygen Species; Regulation; Relative (related person); Research; Research Personnel; Resolution; Ribonucleotide Reductase; Roentgen Rays; Solutions; Source; Space Perception; Specificity; Structure; Study models; Surface; System; Techniques; Thinking; Time; Toluene; Toxic effect; Trichloroethylene; Vision; Work; absorption; abstracting; alkene monooxygenase; base; catalyst; chemical property; chemical reaction; cofactor; crosslink; desaturase; design; greenhouse gases; insight; mutant; new growth; novel; novel diagnostics; novel strategies; oxidation; pollutant; prevent; programs; ribonucleotide reductase M2; size; small molecule; three dimensional structure; tool; uteroferrin

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）：我们将研究可溶性甲烷单加氧酶（MMO）的三维结构、催化机制和调控。MMO启动甲烷氧化细菌将CH 4氧化为CO2。通过这种方式，几乎所有由厌氧细菌产生的大量CH 4（温室气体，其效力是CO2的20倍）的大气排放都被阻止。MMO还可以催化许多其他化学品的氧化，促进对人类毒性的大量污染物（如三氯乙烯）的合成和生物降解。来自发孢甲基弯菌OB 3 B的MMO由3种蛋白质组成：羟化酶（MMOH）、还原酶（MMOR）和“B”（MMO B）。MMOH具有催化所需的双-U-羟基桥接的双核Fe簇。光谱研究（光学，EPR，穆斯堡尔，EXAFS，ENDOR，rRaman，荧光，NMR，MCD和CD），诊断底物的周转率和瞬态动力学正在被用来研究结构和机制。瞬态动力学研究揭示了2个稳定和7个瞬态中间体的反应周期。1的中间体化合物Q含有双-u-氧代-Fe（IV）-Fe（IV）簇合物，其直接与CH 4反应生成CH 3OH。Q是在加氧酶中分离的第一个中间体，其可以攻击未活化的烃。正在进行的研究表明，MMOR和MMOB通过增加Q形成的速率和通过基于大小控制底物进入MMOH活性位点的速率来调节催化。MMOB突变体已被纯化，允许催化循环中的每个步骤的速率被单独调节。我们最近的研究已经确定了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、质谱和晶体学技术来定义组分的空间取向以及门底物结合的构象变化。反应循环中间体将被捕获使用基于稳态稳定化和表面冷冻淬灭的新方法。这些将通过新开发的X射线吸收和低温还原技术进行光谱表征。这项工作应该让我们深入了解：1）新的O2活化化学，2）Q的性质，3）新的调节策略，以及4）用于烃氧化的小分子催化剂的设计。最后，从MMO中吸取的教训应该适用于结构和机制相似的人类核糖核苷酸还原酶，它产生DNA的构建模块。