Molecular cloning and characterization of Brugia malayi thymidylate kinase

Abstract

Thymidylate kinase (TMK) is a potential chemotherapeutic target because it is directly involved in the synthesis of deoxythymidine triphosphate, which is an essential component for DNA synthesis. The gene encoding thymidylate kinase of Brugia malayi was amplified by PCR and expressed in Escherichia coli. The native molecular weight of recombinant B. malayi thymidylate kinase (rBmTMK) was estimated to be ∼52kDa by gel filtration chromatography, suggesting a homodimeric structure. rBmTMK activity required divalent cation and Mg2+ was found to be the most effective cation. The enzyme was sensitive to pH and temperature, it showed maximum activity at pH 7.4 and 37°C. The Km values for dTMP and ATP were 17 and 66μM, respectively. The turnover number kcat was found to be 38.09s−1, a value indicating the higher catalytic efficiency of the filarial enzyme. The nucleoside analogues 5-bromo-2′-deoxyuridine (5-BrdU), 5-chloro-2′-deoxyuridine (5-CldU) and 3′-azido-3′-deoxythymidine (AZT) showed specific inhibitory effect on the enzyme activity and these effects were in good association with binding interactions and the scoring functions as compared to human TMK. Differences in kinetic properties and structural differences in the substrate binding site of BmTMK model with respect to human TMK can serve as basis for designing specific inhibitors against parasitic enzyme.