CRYSTALLOGRAPHIC ANALYSIS OF METHANE MONOOXYGENASE SYS

这个项目是我们实验室和 斯蒂芬·J·利帕德在马萨诸塞州理工学院的研究。的 研究的主要目的是阐明三维 蛋白质的结构参与甲烷转化为 甲醇在嗜甲烷菌荚膜甲基球菌（Methylococcus capsulatus（Bath））中的作用。 这种可溶性甲烷单加氧酶系统（MMO）具有三个主要的生物活性。 组分，多亚基羟化酶，还原酶和电子 转移偶联蛋白，蛋白B。活性羟化酶是二聚体 每个原聚体由三条多肽链组成， 整个酶具有（α β γ）2构型， 分子量约为250 kDa。我们已经确定了 羟化酶的分辨率为2.2埃。的一个主要重点 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）。另外我们 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 决心。这些包括表征的减少形式的 羟化酶和铁耗尽的载脂蛋白形式的蛋白质。作为 对MMO羟化酶蛋白质的溶液机械工作的补充， 将研究含有结合碳氢化合物底物分子的晶体 以阐明双核铁核与底物的相互作用。 由于过量表达系统和纯化的可用性， 蛋白质B的方案，以及其在免疫系统中作用的有力证据。 调解的整体活化反应，我们计划下一步尝试共同- 并对该配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了解 与其他MMO蛋白的相互作用，还原酶和偶联 蛋白质B，并将使我们能够了解如何羟化酶蛋白质 环境调节二铁氧中心的反应化学。 MMO羟化酶与 核糖核苷酸还原酶（RR）的小亚基表明 关于前者的信息将有助于加深对RR的认识， 它本身也是抗病毒和抗肿瘤药物的靶点， 在DNA生物合成中起关键作用。

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.