Leptospira interrogans triosephosphate isomerase: exploring the structural determinants of stability, high reaction rate and specificity.

Leptospires are zoonotic pathogens that cause significant socio-economic burden in developing countries, world-wide. The pathogenic species Leptospira interrogans (Li) is an important and interesting target for investigating the enzymes essential to its metabolic needs and adaptations. We cloned and expressed triosephosphate isomerase (LiTIM), a central metabolic flux regulator of Li, in AA200, E. coli TIM null strain. LiTIM was obtained as an active dimer (D-GAP[->]DHAP kcat = 1740 s-1 and Km (D-GAP) = 0.21 mM, at 25 {degrees}C) with mid-transition concentrations, Cm, 0.8 mM and 2.6 mM, respectively, for guanidine hydrochloride and urea induced equilibrium unfolding. We report the high resolution X-ray structures of LiTIM in apo and substrate (DHAP) bound forms. Our analysis highlights key features of TIM that regulate the mode of substrate binding and transition state stabilization and thus play a decisive role in attainment of high proficiency for the isomerisation reaction while avoiding the elimination reaction. Unexpected differences in the effect of temperature on stability and activity were observed for the three mesophilic pathogenic TIMs viz. from Li, Plasmodium falciparum (Pf) and Trypanosoma brucei (Tb). LiTIM and TbTIM (Tm = 46.5 {degrees}C) were more susceptible to unfolding and precipitation compared to PfTIM (Tm = 67 {degrees}C). In contrast, the initial (or zero point) activity of PfTIM rises till 50 {degrees}C and saturates unlike LiTIM and TbTIM which show a rise till 55 {degrees}C and 60 {degrees}C, respectively. These observations could be rationalized by sequence comparison and examination of the structures of the three TIMs.