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 ( Li TIM), a central metabolic flux regulator of Li , in AA200, E. coli TIM null strain. Li TIM was obtained as an active dimer (D-GAP→DHAP k cat = 1740 s−1 and K m (D-GAP) = 0.21 mM, at 25 °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 Li TIM 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 ). Li TIM and Tb TIM (Tm = 46.5 °C) were more susceptible to unfolding and precipitation compared to Pf TIM (Tm = 67 °C). In contrast, the initial (or zero point) activity of Pf TIM rises till 50 °C and saturates unlike Li TIM and Tb TIM which show a rise till 55 °C and 60 °C, respectively. These observations could be rationalized by sequence comparison and examination of the structures of the three TIMs. ### Competing Interest Statement The authors have declared no competing interest. * WHO : World Health Organization TIM : Triosephosphate isomerase Li : Leptospira interrogans Pf : Plasmodium falciparum Tb : Trypanosoma brucei DHAP : Dihydroxyacetone phosphate D-GAP : D-glyceraledhyde-3-phosphate MG : Methylglyoxal CD : Circular dichroism SDS-PAGE : Sodium dodecyl sulfate polyacrylamide gel electrophoresis LC-ESI/MS : Liquid chromatography coupled electrospray ionisation mass spectrometry GdmCl : Guanidine hydrochloride