STRUCTURE AND DYNAMICS OF METAL-CONTAINING PROTEINS

这个项目代表了一个持续的努力，以确定结构和动力学的作用，在确定金属蛋白质的功能。特别是，从恶臭假单胞菌樟脑羟化酶途径的分子识别和蛋白质间电子转移的机制是调查的目标。该系统由三种蛋白质组成，含血红素的细胞色素P-450/cam（CYP 101）、Fe 2S 2铁氧还蛋白putidaredoxin（Pdx）和NADH依赖性黄素蛋白putidaredoxin还原酶（PdR）。Cypo 101催化樟脑通过分子氧的5-外羟基化反应，该反应需要由Pdx依次向细胞色素提供两个电子。还需要Pdx作为用于衬底翻转的效应器。PdR通过氧化PdR结合的NADH将电子提供给Pdx。樟脑羟化酶系统是参与类固醇激素生物合成和外源性物质加工、药物代谢和致癌作用的人P450酶的良好模型。寻求改进的方法来确定顺磁性蛋白质，如Pdx的结构和动力学，使用选择性同位素标记和多核NMR方法。已经开发了Pdx CYP 101相互作用的模型，并将使用蛋白质工程（诱变）和溶液复合物的NMR表征进行检测。NMR方法学的进步，沿着氘代介质中表达技术的改进，使得首次呈现均匀2 H（13 C），15 N-标记的CYP 101的高分辨率多维NMR谱。一种新的金属酶E2的结构-活性关系也正在研究中。E2参与甲硫氨酸补救途径，并根据E2活性位点中结合的金属从相同底物产生不同的产物。我们已经确定了全球折叠E2的NMR和正在调查的差异，底物结合不同的含金属的异构体的酶。蛋氨酸补救途径在反馈抑制多胺生物合成中起重要作用，而多胺生物合成与哺乳动物的组织病理学密切相关。

This project represents a continuing effort to determine the role of structure and dynamics in determining function of metalloproteins. In particular, mechanisms of molecular recognition and interprotein electron transfer in the camphor hydroxylase pathway from Pseudomonas putida are the targets of investigation. This system consists of three proteins, the heme-containing cytochrome P-450/cam (CYP101), the Fe2S2 ferredoxin putidaredoxin (Pdx) and the NADH-dependent flavoprotein putidaredoxin reductase(PdR). Cypo101 catalyzes the 5-exo-hydroxylation of camphor by molecular oxygen, a reaction that requires two electrons supplied to the cytochrome sequentially by Pdx. Pdx is also required as an effector for substrate turnover. The electrons are supplied to Pdx by PdR by oxidation of PdR-bound NADH. The camphor hydroxylase system is a good model for human P450 enzymes involved in steroid hormone biosynthesis and processing of xenobiotics, drug metabolism and carcinogenesis. Improved methods are sought for determining structure and dynamics of paramagnetic proteins such as Pdx using selective isotope labeling and multinuclear NMR methods. A model for Pdx CYP101 interactions has been developed and will be tested using protein engineering (mutagenesis) and NMR characterization of the solution complexes. Advances in NMR methodology, along with improved expression techniques in deuterated media, have allowed the first high resolution multi-dimensional NMR spectra of a uniformly 2H(13C), 15N- labeled CYP101 to be presented. Structure-activity relationships of a novel metalloenzyme, E2, are also under investigation. E2 is involved in the methionine salvage pathway, and produces different products from the same substrate depending on the metal bound in the E2 active site. We have determined the global fold of E2 by NMR and are investigating the differences in substrate binding between the different metal-containing isoforms of the enzyme. The methionine salvage pathway is important for feedback inhibition of polyamine biosynthesis, which are associated with tissue pathology in mammals.