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

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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.