Structural and Functional Biochemistry of Nitrate Reductase and Its Catalytic Fragments

真核生物的生物化学，同化硝酸还原酶，现在已经研究了大约年，本实验室已经参与了这一追求近一半的时间。Ti项目是高等植物NADH：硝酸还原酶（NR）研究的继续。建议的重点是在大肠杆菌和毕赤酵母中表达的NR重组片段的结构和功能。利用重组的玉米NR细胞色素B还原酶（FAD）片段，在P.l.的实验室，他的瑞典合作者（Y。Lindqvist和Schneider）最近推导出了一个分辨率为2.5 A的全酶初始功能部分的三维模型。利用NR的第一个结构信息，第一个目标是进行定点诱变以确定哪些残基对于吡啶核苷酸特异性是重要的。除了对突变体进行基本的生化分析外，另一位合作者（俄亥俄州州立大学的Russ Hille博士）将进行物理生化研究。第二个目的是利用毕赤酵母表达系统生产NR的下一个片段，即由cyt B c cyt B还原酶片段组成的cyt c还原酶片段，为结晶和物理生化研究提供所需的定量蛋白。使用菠菜NADH：NR cDNA的一部分产生细胞色素c还原酶片段NR。这些研究有助于建立NR的更完整的结构模型，当与生理生化研究和关键突变体的产生相结合时，有助于鉴定NR功能的关键氨基酸残基。NR是一种独特的多中心氧化还原酶，是深入了解这类重要酶的核心。当然，NR在植物的光合代谢中也是中心的，因为植物获得的大部分氮都是由NR处理的。最近，NR作为硝酸盐检测的有用工具在商业上变得重要，并且可能在从污染水中去除硝酸盐方面变得重要。总之，NR生物化学的知识受到缺乏结构模型的限制，并且该项目代表了克服对关键酶的这种GaF理解的重大努力。 高等植物NADH：硝酸还原酶催化硝酸盐同化的第一步，这导致植物产生蛋白质。本实验室对硝酸还原酶的生物化学进行了近20年的研究。由于从植物来源获得足够量的纯硝酸还原酶是困难的，因此P.I.几年前开始使用重组DNA技术在细菌中产生它的片段。 这种方法导致了硝酸盐还原酶部分的第一个结构模型，该模型使用维生素B-2辅因子（黄素）催化硝酸盐还原的初始步骤。 硝酸还原酶的突变将用于增强对酶功能的理解。 除了通过这些研究获得的基本知识外，硝酸还原酶在从受污染的地下水中去除硝酸盐方面正变得具有商业重要性，该项目将有助于了解使这种酶用于环境清理所需的知识。

The biochemistry of eukaryoffc, assimilatory nitrate reductase has now been studied for about years and this laboratory has been involved in this pursuit for nearly half of this time period. Ti project is a continuation of studies of higher plant NADH:nitrate reductase (NR). The proposed foc is on the structure and function of the recombinant fragments of NR expressed in Eschenchia c and Pichia pastors. Using the recombinant corn NR cytochrome b reductase (FAD containir fragment produced in the P.l.'s laboratory, his Swedish collaborators (Drs. Y. Lindqvist and Schneider) recently deduced a three-dimensional model for this initial functional portion of holo-enzyme at 2.5 A resolution. W'th this first structural information on NR, the first objective i carry out site-directed mutagenesis to determine which residues are important for pyrid nucleotide specificity. In addition to basic biochemical analysis of the mutants, another collaborc (Dr. Russ Hille, Ohio State Univ.) will conduct physical biochemical studies. The second objectiv to produce the next fragment of NR, namely the cyt c reductase fragment composed of the cyt b c cyt b reductase fragments, using the Pichia expression system, which will provide the quantitie protein needed for crystallization and physical biochemical studies. The cyt c reductase fragmen NR is being produced using a portion of a spinach NADH:NR cDNA. These studies are cle' leading toward a more complete structural model for NR and, when combined with the phys biochemical studies and generation of key mutants, toward identification of the key amino c residues for NR functionality. NR is a unique multi-center redox enzyme and central to developin better understanding of this important class of enzymes. NR is, of course, also central in pl metabolism since most nitrogen obtained by plants is processed by NR. Recently, NR has becc commercially important as a useful tool for nitrate detection and may become important in nitr removal from contaminated water. In summary, knowledge of NR biochemistry is limited by a lac a structural model and this project represents a significant effort to overcome this gaF understanding of a key enzyme. %%% Higher plant NADH:nitrate reductase catalyzes the first step in nitrate assimilation, which leads to the production of proteins by plants. Biochemistry of nitrate reductase has been studied by this laboratory for nearly 20 years. Since it is diff'cult to obtain suffcient quantities of pure nitrate reductase from plant sources, the P.I. began several years ago to produce its fragments in bacteria using recombinant DNA technolgy. This approach led to the first structural model of the portion of the nitrate reductase which uses a vitamin b-2 cofactor(flavin)for catalyzing the intial step in nitatrate reduction. Mutations of the nitrate reductase will be used to enhance understanding of the enzyme functionality. In addition to basic knowledge gained via these studies, nitrate reductase is becoming commercially important in removal of nitrate from contaminated ground water and this project will contribute to the understanding needed to make this enzyme work for enviornmental cleanup.