title: Cloning, overexpression and characterization of Leishmania donovani triosephosphate isomerase.
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.
authors.affiliation: Division of Biochemistry, CSIR - Central Drug Research Institute, Lucknow 226001, UP, India.
authors.forename: Kishore
authors.initials: K
authors.lastname: Kumar
authors.forename: Prachi
authors.initials: P
authors.lastname: Bhargava
authors.forename: Uma
authors.initials: U
authors.lastname: Roy
mesh_terms: D000595:Amino Acid Sequence; D015153:Blotting, Western; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D006863:Hydrogen-Ion Concentration; D007893:Leishmania donovani / Q000201:enzymology* / Q000235:genetics; D008741:Methyl Methanesulfonate / Q000031:analogs & derivatives / Q000494:pharmacology; D008969:Molecular Sequence Data; D011487:Protein Conformation; D016415:Sequence Alignment; D014305:Triose-Phosphate Isomerase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; C014674:methyl methanethiosulfonate; D008741:Methyl Methanesulfonate; D014305:Triose-Phosphate Isomerase