Structure - function analysis of Arabidopsis thaliana CC-type glutaredoxins ROXY1 and ROXY9

电子转移反应在所有生物体中起着重要作用。例如，呼吸过程中氧气的减少是塑造我们星球的复杂生命进化的基础。虽然氧气对于有氧代谢是必不可少的，但它也会导致生物分子的不必要的氧化。这些可以通过抗氧化系统的组分来减少。作为这种保护系统的重要组成部分，谷氧还蛋白（GRX）催化二硫键的还原。另外或可替代地，它们中的一些配位铁-硫（FeS）簇，从而促进FeS簇的合成、转移到靶蛋白以及氧化还原和铁传感。在活性位点含有CP（Y/F）C或CGFS基序变体的GRX存在于所有类型的生物体中。有趣的是，第三种类型的GRX-以CC（M/L）（C/S）基序为特征-仅在陆地植物的基因组序列中发现。与CPYC和CGFS型GRX相反，这些陆地植物特异性CC型GRX的生物化学和生物物理特征描述得很少。一个标志是它们能够与转录因子的TGACG结合蛋白（TGA）家族成员相互作用并对其进行调节。这个提议提出了一个问题，即CC型谷氧还蛋白是否仍然需要进化上较老的经典GRX的功能来调节TGA活性。我们专注于两个21拟南芥谷氧还蛋白（ROXY 1和ROXY 9），因为他们最有可能调节其同源的TGA通过不同的机制。ROXY 1负调节TGA因子PAN，以确定花发育过程中花瓣的正确数量。ROXY 9可以抑制调节生长和防御反应的冗余因子TGA 1和TGA 4的活性。关键氨基酸突变的蛋白质的特征在于其氧化还原状态，氧化还原酶活性，FeS簇结合能力和在植物中的功能。这些分析将显示ROXY 1和ROXY 9的体内活性需要哪些体外性质。

Electron transfer reactions play essential roles in all living organisms. The reduction of oxygen during respiration, for instance, is the basis for the evolution of the complex life that is shaping our planet. Although being essential for aerobic metabolism, oxygen also leads to unwanted oxidation of biomolecules. These can be reduced by components of the anti-oxidative system. Serving as essential components of this protective system, glutaredoxins (GRXs) catalyze the reduction of disulfide bonds. In addition or alternatively, some of them coordinate iron-sulfur (FeS) clusters and thus facilitate FeS cluster synthesis, transfer to target proteins and redox and iron sensing. GRXs containing variants of a CP(Y/F)C or a CGFS motif in the active site occur in all types of organisms. Intriguingly, a third type of GRXs - being characterized by a CC(M/L)(C/S) motif - is only found in the genome sequences of land plants. In contrast to CPYC- and CGFS-type GRXs, the biochemical and biophysical features of these land plant-specific CC-type GRXs are poorly described. A hallmark is their ability to interact with and regulate members of the TGACG-binding protein (TGA) family of transcription factors. This proposal asks the question whether CC-type glutaredoxins still require the functions of the evolutionary older canonical GRXs to regulate TGA activity. We focus on two of the 21 Arabidopsis thaliana glutaredoxins (ROXY1 and ROXY9), since they most likely regulate their cognate TGAs by different mechanisms. ROXY1 negatively regulates TGA factor PAN to determine the correct number of petals during flower development. ROXY9 can repress the activities of the redundant factors TGA1 and TGA4, which regulate growth and defense responses. Proteins with mutations in critical amino acids will be characterized with respect to their redox states, oxidoreductase activities, FeS cluster binding capacities and in planta functions. These analyses will show, which in vitro properties are required for the in vivo activities of ROXY1 and ROXY9.