Deoxyribonucletides (dNTPs) are synthesized via the de novo and salvage pathways. The de novo synthesis involves initial synthesis of ribonucleotides whereas the salvage pathway uses deoxyribonucleosides. Deoxynucleoside kinases (dNKs) initiate the salvage pathway and this step is often considered as rate limiting. The two pathways eventually coincide to one where nucleoside monophosphate kinases are the shared enzymes. In this thesis two thymidine kinases (TKs) and a thymidylate kinase (TMPK) are cloned, expressed and characterized and they originate from Ureaplasma urealyticum (Uu) and Bacillus anthracis (Ba). Uu causes urethritis and is associated with complications in pregnancy, e.g. premature births, infertility, spontaneous abortions and chronic lung disease in the infants. Ba causes anthrax; cutaneous, inhalational and gastrointestinal, where the two latter have a high mortality rate. Ba is also considered as a potent bioweapon because of its spore forming ability. TK from Uu (Uu-TK) was strictly pyrimidine specific and used all nucleoside triphosphates as phosphate donors, except dTTP that was a feedback inhibitor. Further studies discovered that analogs with halogen substitutions at the 5-position gave the highest activity. Analogs with modifications at the N3- or 3’-position showed good to moderate activities while 2’-substituents were not substrates. A fluorine substitution was tolerated in the 2’-arabinosyl position. These results correlated well with the active site structure of Uu-TK. The Uu-TK structure contained a unique domain, the lasso domain with a structural zinc ion, and belonged structurally to another enzyme family than the other dNKs. TK and TMPK from Ba (Ba-TK and Ba-TMPK) were strictly pyrimidine and thymidylate specific, respectively. Ba-TK used all nucleoside triphosphates as phosphate donors, except dTTP, and phosphorylated several nucleoside analogs. The analog activities of Ba-TK were similar to that of Uu-TK. Ba-TMPK used ATP and dATP as phosphate donors and a number of analogs as substrates. FMAUMP (1-(2-deoxy-2-fluoro-D-arabinofuranosyl)-5-methyluracil-5’-monophosphate) was the best substrate and its nucleoside form was a potent inhibitor. Enzymes in thymidylate synthesis are potential targets for antibacterial therapy and the studies conducted in this thesis have discovered several potential leads, which will contribute to future design of antibiotics
