Molecular mechanisms of nitrate reduction, NO synthesis and post-translational regulation of plant nitrate reductases

植物硝酸还原酶（NR）是一种必需的同源二聚体酶，具有三个辅基（钼辅因子、细胞色素B、FAD）。硝酸还原酶（NR）的主要代谢功能是将硝酸盐还原为亚硝酸盐，这是植物氮素同化的第一步和限速步骤，NR还能将亚硝酸盐还原为信号分子一氧化氮（NO）。虽然NO的许多下游功能和过程是已知的，但NO如何由NR产生的酶促机制在很大程度上仍然未知。在催化过程中，NR经历了主要的构象变化，涉及有效的内部电子转移的结构域运动。在蛋白质水平上，14-3-3蛋白通过一个已知的高亲和力位点和一个最近发现的低亲和力位点与磷酸化NR结合，通过干扰内部电子转移来调节酶活性。尚未研究NR的NO合成活性是否以及如何也受到翻译后调节。我们的目标是了解植物硝酸还原酶还原亚硝酸盐的分子机制，以及硝酸盐和亚硝酸盐还原活性之间的相互作用。作为模型蛋白，我们将集中在表征的两个NR异构体从拟南芥，AtNIA 1和AtNIA 2，因为它通常被认为是AtNIA 1主要参与亚硝酸盐还原，而AtNIA 2主要发挥硝酸还原酶活性。我们将重组表达和纯化这两种酶的全长蛋白质，以及功能活性域片段在其野生型形式和突变体的功能重要的残基已被改变。我们将确定任何反应的纯酶的动力学特性，并确定任何反应的关键残基。此外，我们将通过活性和结合试验分析14-3-3s对AtNIA 1和AtNIA 2的功能和结合特性的影响，以揭示两种亚型之间的差异。由于已知酶在催化过程中经历构象变化，因此对NR的结构-功能关系的理解具有极大的兴趣。因此，我们将使植物NR在其活性和/或14-3-3-抑制状态下结晶。抑制复合物的稳定化将是结晶所需的，并且将通过相互作用配偶体之间的特异性共价交联来实现。由于14-3 -3中的相互作用位点尚不清楚，因此，我们将在此基础上对NR与14 - 3 - 3之间的低亲和力结合位点进行鉴定和表征。本项目旨在阐明NR还原亚硝酸盐的机理基础，揭示不同NR异构体的不同功能，并了解NR在其活性和14-3-3-抑制状态下的结构特性，以便了解双酶功能和一种生物体中双NR同种型的存在。

Plant nitrate reductase (NR) is an essential homodimeric enzyme with three prosthetic groups (molybdenum cofactor, cytochrome b, FAD). Besides its primary metabolic function to reduce nitrate to nitrite, which is considered as first and rate-limiting step in plant nitrogen assimilation, NR is also able to reduce nitrite to the signaling molecule nitric monoxide (NO). Although many downstream functions and processes of NO are known, the enzymatic mechanism how NO is produced by NR remains largely unknown. During catalysis NR undergoes major conformational changes involving domain movements for efficient internal electron transfer. On protein level, 14-3-3 protein binding to phosphorylated NR via one well-known high- and one recently discovered low-affinity site regulates the enzyme activity by disturbing the internal electron transfer. It has not yet been investigated whether and how the NO synthetic activity of NR is also subject to post-translational regulation. Our goal is to understand the molecular mechanism of nitrite reduction by plant nitrate reductase as well as the interplay between nitrate and nitrite reducing activities. As model proteins, we will focus on the characterization of the two NR isoforms from Arabidopsis thaliana, AtNIA1 and AtNIA2, because it is generally assumed that AtNIA1 is mainly involved in nitrite reduction while AtNIA2 exerts predominantly nitrate reductase activity. We will recombinantly express and purify both enzymes as full-length proteins as well as functionally active domain fragments in their wildtype form and as mutants where functionally important residues have been altered. We will determine the kinetic properties of the pure enzymes for either reaction and identify crucial residues for either reaction. Furthermore, we will analyze the impact of 14-3-3s on either activity for AtNIA1 and AtNIA2 with respect to function and binding properties via activity and binding assays, in order to reveal the differences between both isoforms. As the enzymes are known to undergo conformational changes during catalysis, understanding of the structure-function relationship of NR is of great interest. Therefore, we will crystallize plant NR in its active and/or in the 14-3-3-inhibited state. Stabilization of the inhibitory complex will be required for crystallization and will be achieved by specific covalent crosslinking between the interaction partners. As prerequisite, we will identify and characterize the recently described low-affinity binding site between NR and 14-3-3, as the interacting site in 14-3-3 is yet unknown.This project aims to elucidate the mechanistic basis of nitrite reduction by NR, to reveal the distinct functions of different NR isoforms, and to understand the structural properties of NR in its active and 14-3-3-inhibited state in order to understand the dual enzyme function and the presence of dual NR isoforms in one organism.