Epsilon Open Archive

Abstract

There are two routes to produce deoxyribonucleoside triphosphates (dNTPs) precursors for DNA synthesis, the de novo and the salvage pathways. Deoxyribonucleoside kinases (dNKs) perform the initial phosphorylation of deoxyribonucleosides (dNs). Furthermore, they can act as activators for several medically important nucleoside analogs (NAs) for treatment against cancer or viral infections. Several disorders are characterized by mutations in enzymes involved in the nucleotide biosynthesis, such as Lesch-Nyhan disease that is linked to hypoxanthine guanine phosphoribosyltransferase (HPRT). In this thesis, the structures of human thymidine kinase 1 (TK1), a mycoplasmic deoxyadenosine kinase (Mm-dAK), and phosphoribosyltransferase domain containing 1 (PRTFDC1) are presented. Furthermore, a structural investigation of Drosophila melanogaster dNK (Dm-dNK) N64D mutant was carried out. The obtained structural information reveals the basis for substrate specificity for TKs and the bacterial dAKs. The TK1 revealed a structure different from other known dNK structures, containing an α/β domain similar to the RecA-F1ATPase family, and a lasso-like domain stabilized by a structural zinc. The Mm-dAK structure was similar to its human counterparts, but with some alterations in the proximity of the active site. Furthermore several residues important for substrate specificity were identified. The crystal structure of PRTFDC1 was structurally very similar to its homolog HPRT. PRTFDC1 was classified as having an unknown function and with structural and biochemical data we showed that PRTFDC1 has some phosphoribosyltransferase activity. A changed behavior of the Dm-dNK N64D mutant was previously observed. This mutant displayed an increased sensitivity towards NAs and a decreased feedback inhibition. Complexes with substrate and feedback inhibitor provided an explanation for the changed behavior of the mutant. The structural data presented here, provide a foundation for substrate specificity for dNKs. The information of differences between human and bacterial enzymes will be of importance for the design of new anti-bacterial agents. Mutational studies to improve desired properties of an enzyme are an important issue in suicide gene/chemotherapy. Although we have found a potential function of PRTFDC1 there are still a lot of questions concerning its biological role to answer.