Functional elucidation of YbiB - the defining member of the novel TrpD2 family of prokaryotic nucleoside triphosphatases

该项目的目的是功能性阐明从大肠杆菌中提取的酶YbiB作为迄今尚未表征的TrpD2蛋白家族的定义成员。TrpD2蛋白在结构上与色氨酸生物合成酶——苯甲酸磷酸核糖基转移酶（TrpD）和在核苷酸回收途径中起重要作用的II类核苷磷酸化酶（NP-II）具有高度的相似性。然而，我们既没有检测到YbiB的TrpD活性，也没有检测到NP-II活性。YbiB的晶体结构显示二聚体蛋白表面有两个突出的带正电的凹槽，这是负责核酸高亲和力结合的。有趣的是，ybiB基因位于lexa调控的操纵子中。LexA抑制因子控制细菌对DNA损伤的SOS反应。基于这些结果，我们假设YbiB和TrpD2蛋白代表了SOS系统中迄今未知的组成部分。根据这一假设，我们最近可以证明YbiB具有8-oxo- dgtp特异性三磷酸酶活性。这种活性的相关性是从核苷酸库中去除氧化损伤的dGTP。尽管在许多生物体中存在同功能的Nudix水解酶MutT，但像YbiB这样的第二种酶的存在在生物学上是有意义的：MutT也能在一定程度上水解四种标准核苷酸，而YbiB则没有这种副反应。因此，在SOS病例中，YbiB的表达可以明显增加到更高的水平，而不会对核苷酸库产生负面影响。我们的目的是详细描述YbiB和其他TrpD2代表在DNA修复和核苷酸池卫生背景下的功能。首先，我们将研究8-oxo-dGTP是否是这些酶的主要底物，或者其他受损的核苷酸是否以类似甚至更高的效率转化。此外，我们将测试核酸或蛋白质的结合是否增强了相对较低的YbiB活性。此外，我们计划将YbiB与底物类似物和核酸共结晶。所得到的结构将有助于我们理解TrpD2蛋白的功能模式。最后，我们将研究磷酸核糖基转移酶III类超家族及其成员TrpD2， TrpD和NP-II的结构-功能关系。因此，计划中的实验不仅将阐明迄今为止未知的SOS反应成员的功能，而且还将揭示一个有趣的蛋白质超家族的进化。

The aim of this project is the functional elucidation of the enzyme YbiB from Escherichia coli as a defining member of the hitherto uncharacterized TrpD2 protein family. The TrpD2 proteins show high structural similarity both to anthranilate phosphoribosyltransferase (TrpD), an enzyme from tryptophan biosynthesis, and to class II nucleoside phosphorylases (NP-II), which play an important role in the nucleotide salvage pathway. However, we could neither detect TrpD activity nor NP-II activity for YbiB. The crystal structure of YbiB shows two prominent positively charged grooves on the surface of the dimeric protein, which are responsible for the high-affinity binding of nucleic acids. Interestingly, the ybiB gene is located in a LexA-regulated operon. The LexA repressor controls the bacterial SOS response to DNA damage. Based on these results we hypothesized that YbiB and thereby the TrpD2 proteins represent a hitherto unknown component of the SOS system.In accordance with this hypothesis, we could recently show that YbiB has an 8-oxo-dGTP-specific triphosphatase activity. The relevance of this activity is the removal of oxidatively damaged dGTP from the nucleotide pool. In spite of the presence of the iso-functional Nudix hydrolase MutT in many organisms, the existence of a second enzyme like YbiB is biologically meaningful: MutT also hydrolyzes the four standard nucleotides to a certain extent, whereas YbiB does not show this side reaction. Therefore, the expression of YbiB could be increased in the SOS case to a significantly higher level without negative effects for the nucleotide pool.Our aim is to characterize in detail the function of YbiB and other TrpD2 representatives in the context of DNA repair and nucleotide pool sanitation. First, we will investigate whether 8-oxo-dGTP is the main substrate of these enzymes, or whether other damaged nucleotides are converted with similar or even higher efficiency. Furthermore, we will test whether the binding of nucleic acids or proteins enhances the relatively low activity of YbiB. Additionally, we plan to co-crystallize YbiB with substrate analogues and nucleic acids. The resulting structures will contribute to our understanding of the mode of function of the TrpD2 proteins. Finally, we will examine the structure-function relationships within the phosphoribosyltransferase class III superfamily with its members TrpD2, TrpD and NP-II. Thus, the planned experiments will not only elucidate the function of a hitherto unknown member of the SOS response, but also shed light on the evolution of an interesting protein superfamily.