STRUCTURE AND DYNAMICS OF METAL-CONTAINING PROTEINS

这个项目代表了确定结构和动力学在确定金属蛋白功能中的作用的持续努力。特别是恶臭假单胞菌樟脑羟化酶途径中分子识别和蛋白间电子传递的机制是研究的对象。该系统由含血红素的细胞色素P-450/CaM(CyP101)、Fe2S2铁氧还蛋白putidaredosin(PDX)和依赖NADH的黄素蛋白putidaredosin还原酶(PDR)三种蛋白质组成。Cypo101催化分子氧催化樟脑的5-外羟基化反应，该反应需要由PDX顺序提供给细胞色素的两个电子。PDX还需要作为底物周转的效应器。电子由PDR通过氧化PDR结合的NADH提供给PDX。樟脑羟化酶系统是人类P450酶的良好模型，参与类固醇激素的生物合成和外源物质的加工、药物代谢和致癌。寻求利用选择性同位素标记和多核核磁共振方法来确定顺磁性蛋白质如PDX的结构和动力学的改进方法。已经开发了PDX CYP101相互作用的模型，并将使用蛋白质工程(诱变)和溶液络合物的核磁共振表征进行测试。随着核磁共振方法的进步，以及在氚介质中表达技术的改进，首次出现了均匀的2H(13C)，15N标记的CYP101的高分辨率多维核磁共振谱。一种新的金属酶E2的构效关系也在研究中。E2参与蛋氨酸回收途径，根据E2活性部位结合的金属的不同，在同一底物上产生不同的产物。我们已经通过核磁共振确定了E2的全局折叠，并正在研究该酶不同金属异构体之间底物结合的差异。甲硫氨酸挽救途径对于反馈抑制多胺生物合成是重要的，多胺生物合成与哺乳动物的组织病理学有关。

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.