Higher Plant Nitrate Reductase: Structure and Function of Its Domains

硝酸还原酶是植物代谢的中心酶。 的PI 将研究黄素腺嘌呤的生物化学 二核苷酸（FAD）结构域和组合的细胞色素B/FAD超- 玉米NADH的结构域：硝酸还原酶，将它们表达为 大肠杆菌中的重组蛋白。 FAD域具有 已经表达;它类似于全硝酸还原酶， 生物化学和免疫化学性质。 的晶体 重组FAD结构域，它可以抑制X射线，最近已经被 由瑞典的合作者获得，并分析其三维 结构正在进行中。 基于结构模型， 衍生，他将构建残基的定点突变体， 参与FAD和NADH的结合。 细胞色素B/FAD超 结构域，预期催化NADH依赖性细胞色素c 减少，将以E表示。大肠杆菌，纯化， 表征了 将提供纯化的重组超结构域 布兰登教授在瑞典的研究小组进行结晶。 研究中心- 定向诱变也将在该重组硝酸盐上进行 还原酶片段 这些研究将提供新的信息， 硝酸还原酶的功能和结构特性， 有助于深入了解细胞机制， 调节酶的活性。 %%% 玉米硝酸还原酶的结构与功能特性 将通过在大肠杆菌中表达部分酶来研究。 coli系统。 这种表达将允许产生足够的 酶的催化结构域的数量， 晶化 晶体的领域将用于X射线 衍射研究，以确定三维结构， 功能性蛋白质。 这些实验应该提供 重要的新见解的电子转移性质的 这些酶是其在硝酸盐还原中的功能所必需的。

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.