RESEARCH ARTICLE Structural and Kinetic Characterization of

Leishmania spp. is a protozoan parasite and the causative agent of leishmaniasis. Thymi-dine kinase (TK) catalyses the transfer of the γ-phosphate of ATP to 2’-deoxythymidine (dThd) forming thymidine monophosphate (dTMP). L.major Type II TK (LmTK) has been previously shown to be important for infectivity of the parasite and therefore has potential as a drug target for anti-leishmanial therapy. In this study, we determined the enzymatic prop-erties and the 3D structures of holo forms of the enzyme. LmTK efficiently phosphorylates dThd and dUrd and has high structural homology to TKs from other species. However, it sig-nificantly differs in its kinetic properties from Trypanosoma brucei TK since purines are not substrates of the enzyme and dNTPs such as dUTP inhibit LmTK. The enzyme had Km and kcat values for dThd of 1.1 μM and 2.62 s-1 and exhibits cooperative binding for ATP. Addi-tionally, we show that the anti-retroviral prodrug zidovudine (3-azido-3-deoxythymidine, AZT) and 5’-modified dUrd can be readily phosphorylated by LmTK. The production of re-combinant enzyme at a level suitable for structural studies was achieved by the construction of C-terminal truncated versions of the enzyme and the use of a baculoviral expression sys