Higher Plant Nitrate Reductase: Structure and Function of Its Domains

硝酸还原酶是植物代谢的中心酶。PI将通过在大肠杆菌中以重组蛋白的形式表达玉米NADH：硝酸还原酶的黄素腺嘌呤二核苷酸(FAD)结构域和结合的细胞色素b/FAD超级结构域来研究它们的生物化学。FAD结构域已经被表达，它在生化和免疫化学性质上与全硝酸还原酶相似。瑞典的合作者最近获得了能够衍射X射线的重组FAD结构域的晶体，对其三维结构的分析正在进行中。基于所推导的结构模型，他将构建与FAD和NADH结合的残基的定点突变体。细胞色素b/Fad超结构域有望催化NADH依赖的细胞色素c还原，将在大肠杆菌中表达、纯化和鉴定。纯化的重组超结构域将提供给瑞典的布兰登教授的团队进行结晶。还将对该重组硝酸还原酶片段进行定点突变。这些研究将提供关于硝酸还原酶功能和结构特性的新信息，并可能有助于深入了解参与该酶活性调节的细胞机制。玉米硝酸还原酶的结构和功能特性将通过在大肠杆菌系统中表达该酶的部分片段来研究。这样的表达将允许产生足够数量的酶的催化域，以允许结晶。这些结构域的晶体将用于X射线衍射研究，以确定功能蛋白质的三维结构。这些实验应该为该酶的电子转移性质提供重要的新见解，这些性质对于其在硝酸盐还原中的功能是必不可少的。

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.