CRYSTALLOGRAPHIC ANALYSIS OF METHANE MONOOXYGENASE SYS

这个项目是我们实验室和那个实验室合作完成的。 麻省理工学院的史蒂芬·J·利帕德。这个 这项研究的主要目的是阐明三维 甲烷转化为甲烷的蛋白质的结构 甲烷营养细菌中的甲醇胶囊甲氧球菌属。 这种可溶的甲烷单加氧酶系统(MMO)主要有三种 组分、多亚单位羟基酶、还原酶和电子 转移偶联蛋白，蛋白B。活性羟基酶是一种二聚体。 每个原基由三条多肽链组成，使得 整个酶具有(α-β-伽马)2构型和总的 分子量约为250 kDa。我们已经确定了 羟基酶的分辨率为2.2埃。这次会议的一个主要焦点是 目前的建议是将这种蛋白质的结构扩展到极限 天然晶体的分辨率(1.8埃)。此外，我们 最初计划利用这些结构信息来调查 几种密切相关结构的氧活化机理 决定。其中包括对简化形式的 羟基酶和缺铁蛋白的载脂蛋白形式。作为一名 补充了对MMO羟基酶蛋白的溶解机制工作， 含有结合的碳氢化合物底物分子的晶体将被研究 目的：阐明底物与双核铁核的相互作用。 由于可获得过表达系统和纯化 蛋白质B的方案，以及它在 整个激活反应的调解，我们计划下一步尝试联合- 并对该络合物进行了结晶和结构测定。 这些详细的结构研究将有助于理解 与其他MMO蛋白的相互作用、还原酶和偶联 蛋白B，并将使我们能够了解羟基酶蛋白是如何 环境对二铁氧中心的反应化学性质有调节作用。 MMO羟基酶和MMO羟基酶的结构和功能相似性 核糖核苷酸还原酶(RR)的小亚基提示 关于前者的信息将有助于加深对RR的洞察， 本身就是抗病毒和抗肿瘤药物的靶子，因为它 DNA生物合成中的关键作用。

This project is a collaborative effort between our laboratory and that of Stephen J. Lippard at the Massachusetts Institute of Technology. The major objective of the research is to elucidate the three dimensional structure of the proteins involved in the conversion of methane to methanol in methanotrophic bacterium, Methylococcus capsulatus (Bath). This soluble methane monooxygenase system (MMO) has three major components, a multi-subunit hydroxylase, a reductase, and an electron transfer coupling protein, protein B. The active hydroxylase is a dimer with each protomer composed of three polypeptide chains such that the entire enzyme has an (alpha beta gamma) 2 configuration and a total molecular weight of roughly 250kDa. We have determined the structure of the hydroxylase to 2.2 Angstroms resolution. One major focus of the current proposal is to extend the structure of this protein to the limit of resolution of the native crystals (1.8 Angstroms). In addition, we plan initially to utilize this structural information to investigate the mechanism of oxygen activation by several closely related structure determinations. These include characterization of the reduced form of the hydroxylase and the iron depleted apo form of the protein. As a complement to solution mechanistic work on the MMO hydroxylase protein, crystals containing bound hydrocarbon substrate molecules will be studied to elucidate the interaction of substrate with the dinuclear iron core. Due to the availability of an overexpression system and purification scheme for protein B, and the strong evidence of its role in the mediation of the overall activation reaction, we plan next to attempt co- crystallization and structure determination of this complex as well. These detailed structural studies will facilitate an understanding of interactions with the other MMO proteins, the reductase and the coupling protein B, and will enable us to learn how the hydroxylase protein environment modulates the reaction chemistry of the diiron oxo center. The structural and functional similarity between the MMO hydroxylase and the small subunit of the enzyme ribonucleotide reductase (RR) suggest that information about the former will help to sharpen insights about RR, itself a target of antiviral and antitumor pharmaceuticals because of its pivotal function in the biosynthesis of DNA.