Evaluation of Bacillus anthracis thymidine kinase as a potential target for the development of antibacterial nucleoside analogs

Bacillus anthracis, which causes anthrax, has attracted attention because of its potential use as a biological weapon. The risk of multidrug resistance against B. anthracis increases the need for antibiotics with new molecular targets. Nucleoside analogs are well-known antiviral and anticancer prodrugs, and thymidine kinase catalyzes the rate-limiting step in the activation of pyrimidine nucleoside analogs used in chemotherapy. The thymidine kinase gene from B. anthracis Sterne strain (34F2) (Ba-TK) was cloned and expressed in E coli, and the product was purified and characterized regarding its substrate specificity. Ba-TK phosphorylated pyrimidine nucleosides and all natural nucleoside triphosphates served as phosphate donors. Size exclusion chromatography indicated a dimeric form of Ba-TK, regardless of the presence of ATP. Thymidine was the most efficient substrate with a low K-m value (0.6 mu m) and a V-max of 3.3 mu mol dTMP mg(-1) min(-1), but deoxyuridine (K-m = 4.2 mu M, V-max = 4.1 mu mol dUMP mg(-1) min(-1)) was also a good substrate. Several pyrimidine analogs were also tested and analogs with 5-position modifications showed higher activities compared to analogs with 3'- and N3-position modifications. Deoxyuridine analogs were the most potent inhibitors of B. anthracis growth in vitro. These results may be used to guide future development of nucleoside analogs against B. anthracis.