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.
