Higher Plant Nitrate Reductase: Structure and Function of Its Domains

点击翻译按钮获取中文摘要

Nitrate reductase is a central enzyme in plant metabolism. The PI will investigate the biochemistry of the flavin adenine dinucleotide (FAD) domain and the combined cytochrome b/FAD super- domain of corn NADH:nitrate reductase by expressing them as recombinant proteins in Escherichia coli. The FAD domain has already been expressed; it is similar to holo-nitrate reductase in biochemical and immunochemical properties. Crystals of the recombinant FAD domain, which diffract x-rays have recently been obtained by collaborators in Sweden and analysis of its 3-D structure is underways. Based on the structural model being derived, he will construct site-directed mutants of residues involved in binding FAD and NADH. The cytochrome b/FAD super- domain, which is expected to catalyze NADH-dependent cytochrome c reduction, will be expressed in E. coli, purified and characterized. Purified recombinant super-domain will be supplied to Professor Branden's group in Sweden for crystallization. Site- directed mutagenesis will also be done on this recombinant nitrate reductase fragment. These studies will provide new information on functional and structural properties of nitrate reductase and may be useful for gaining insight into cellular mechanisms involved in regulation of the enzyme's activity. %%% The structural and functional properties of corn nitrate reductase will be investigated by expressing portions of the enzyme in an E. coli system. Such expression will allow production of sufficient quantities of the enzyme's catalytic domains to allow crystallization. Crystals of the domains will be used in X-ray diffraction studies to determine the three dimensional structure of the functional protein. These experiments should provide significant new insights into electron transfer properties of the enzyme which are essential to its function in nitrate reduction.