Cloning, overexpression and characterization of Leishmania donovani triosephosphate isomerase

Experimental Parasitology
Kishore KumarUma Roy

Abstract

Triosephosphate isomerase (TIM) is a major enzyme in the glycolytic pathway, which catalyzes the interconversion of glyceraldehyde 3-phosphate to dihydroxyacetone phosphate. Here, we report cloning, expression and purification of a catalytically active recombinant TIM of Leishmania donovani (LdTIM). The recombinant LdTIM had a pH optimum in the range of 7.2-9.0, found stable at 25°C for 30 min and K(m) and V(max) for the substrate glyceraldehyde 3-phosphate was 0.328±0.02mM and 10.05mM/min/mg, respectively. The cysteine-reactive agent methylmethane thiosulphonate (MMTS) was used as probe, in order to test its effect on enzyme activity. The MMTS induced 75% enzyme inactivation within 15 min at 250 μM concentration. The biochemical characterization of LdTIM described in this work is the essential step towards deeper understanding of its role in parasite survival. The purification of LdTIM in bioactive form provides important tools for further functional and structural studies.

References

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Citations

Jul 5, 2013·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Ifedayo Victor Ogungbe, William N Setzer
Oct 21, 2016·Biochimica Et Biophysica Acta·Alonso A Lopez-ZavalaLuis G Brieba
Jan 28, 2017·Developmental and Comparative Immunology·Fei LiuFuhua Li
Nov 5, 2014·Chemical Reviews·Advait S NagleValentina Molteni

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