NMR SOLUTION STRUCTURE OF THE MMOB COMPONENT

MMOB 组件的 NMR 解决方案结构

• 批准号: 2910033
• 负责人: George T. Gassner
• 金额: $ 1.35万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2910033
• 关键词: active sites; calorimetry; catalyst; chemical kinetics; enzyme complex; enzyme mechanism; enzyme structure; ionic bond; methane monooxygenase; nuclear magnetic resonance spectroscopy; oxygenases; protein structure; site directed mutagenesis

The soluble methane menooxygenase system isolated from M. capsulatus (Bath) consists of a reductase (MMOR), coupling protein (MMOB), and a hydroxylase (MMOH). The 16.5 kDa MMOB component has a profound influence on the type of chemistry catalyzed at the active site of MMOH. In a still unknown mechanism, MMOB directs the evolution of a series of oxygen intermediates that lead to the insertion of an atom of molecular oxygen into a CH bond of methane. In the absence of MMOB, MMOH still accepts electrons from substrate specificity of MMOH. This broad substrate specificity makes MMOH valuable system in both aqueous phase organic synthesis, and in the detoxification of contaminated environmental sites. High resolution homonuclear and heteronuclear NMR experiments will be used to solve the solution structure of MMOB. The three dimensional NMR structure will provide greater insight into the way in which MMOB tunes the reactivity potentials of the MMOH iron center. Searches based on the complementarity of the topologies and electrostatic surface potentials of MMOH and MMOB will be used in conjunction with chemical cross-linking data to identify probable MMOB binding sites on the surface of MMOH. Proposed binding sites will be confirmed through titration calorimetric studies involving synthetic peptides representative of secondary structural motifs in interface region. Individual amino acid side chains involved in the complex formation will be identified in high resolution NMR studies of complexes of MMOB with synthetic peptides. Amino acid side chains found to be involved in the binding interaction will be sequentially mutated to alanine. The relative contribution of individual amino acid side chains the macroscopic stability of the complexes formed between MMOB and MMOH will be measured based on calorimetric measurements.

从微囊藻中分离的可溶性甲烷加氧酶系统 (Bath)由一个还原酶(MMOR)、偶联蛋白(MMOB)和一个 羟基酶(MMOH)。16.5 kDa的MMOB组件具有深远的影响 关于在MMOH活性中心催化的化学类型。在静止状态下 未知机制，MMOB指导一系列氧的进化 导致分子氧原子插入的中间体 变成甲烷的CH键。在没有MMOB的情况下，MMOH仍然接受 电子来自底物的MMOH的专一性。这种宽广的衬底 特异性使MMOH在两个水相中都具有很高的价值 合成，以及在受污染的环境场所的解毒。 将使用高分辨率同核和异核核磁共振实验 解决了MMOB的溶液结构。三维核磁共振 结构将提供对MMOB曲调方式的更多洞察 MMOH铁中心的反应性势。基于搜索的搜索 拓扑结构与表面静电势的互补性 MMOH和MMOB将与化学交联数据一起使用 鉴定MMOH表面可能的MMOB结合位点。建议 结合部位将通过滴定量热法研究确定 涉及代表二级结构基序的合成肽 在界面区。单个氨基酸侧链参与 在高分辨率核磁共振研究中将识别复杂的地层 MMOB与合成肽的络合物。发现的氨基酸侧链 将参与结合相互作用的序列突变为 丙氨酸。单个氨基酸侧链的相对贡献 MMOB与MMOH形成络合物的宏观稳定性 将根据量热测量进行测量。