title: Biochemical and inhibition studies of glutamine synthetase from Leishmania donovani.
abstract: Leishmaniasis is a group of tropical diseases caused by protozoan parasites of the genus Leishmania. Leishmania donovani is a protozoan parasite that causes visceral leishmaniasis, a fatal disease if left untreated. Chemotherapy for leishmaniasis is problematic as the available drugs are toxic, costly and shows drug resistance, hence, there is a necessity to look out for the novel drug targets, chemical entities and vaccine. Glutamine synthetase (GS) catalyzes the synthesis of glutamine from glutamate and ammonia. In the present study, we have identified and characterized GS from L. donovani. The nucleotide sequence encoding putative glutamine synthetase like sequence from L. donovani (LdGS, LDBPK_060370) was cloned. A 43.5 kDa protein with 6X-His tag at the C-terminal end was obtained by overexpression of LdGS in Escherichia coli BL21 (DE3) strain. Expression of native LdGS in promastigotes and recombinant L. donovani glutamine synthetase (rLdGS) was confirmed by western blot analysis. An increase in expression of GS was observed at different phases of growth of the parasite. Expression of LdGS in promastigote and amastigote was confirmed by western blot analysis. Immunofluorescence studies of both the promastigote and amastigote stages of the parasite revealed the presence of LdGS in cytoplasm. GS exists as a single copy gene in parasite genome. Kinetic analysis of GS enzyme revealed Kmvalue of 26.3 ± 0.4 mM for l- glutamate and Vmaxvalue of 2.15 ± 0.07 U mg-1. Present study confirms the presence of glutamine synthetase in L. donovani and provides comprehensive overview of LdGS for further validating it as a potential drug target.
authors.affiliation: Department of Biotechnology, National Institute of Pharmaceutical Education and Research, SAS Nagar, Mohali 160062, Punjab, India.
authors.forename: Vinay
authors.initials: V
authors.lastname: Kumar
authors.affiliation: Department of Biotechnology, National Institute of Pharmaceutical Education and Research, SAS Nagar, Mohali 160062, Punjab, India.
authors.forename: Shailendra
authors.initials: S
authors.lastname: Yadav
authors.affiliation: Department of Biotechnology, National Institute of Pharmaceutical Education and Research, SAS Nagar, Mohali 160062, Punjab, India.
authors.forename: Neelagiri
authors.initials: N
authors.lastname: Soumya
authors.affiliation: Department of Biotechnology, National Institute of Pharmaceutical Education and Research, SAS Nagar, Mohali 160062, Punjab, India.
authors.forename: Rohit
authors.initials: R
authors.lastname: Kumar
authors.affiliation: Department of Biotechnology, National Institute of Pharmaceutical Education and Research, SAS Nagar, Mohali 160062, Punjab, India.
authors.forename: Neerupudi Kishore
authors.initials: NK
authors.lastname: Babu
authors.affiliation: Department of Biotechnology, National Institute of Pharmaceutical Education and Research, SAS Nagar, Mohali 160062, Punjab, India. Electronic address: sushmasingh@niper.ac.in.
authors.forename: Sushma
authors.initials: S
authors.lastname: Singh
mesh_terms: D000913:Antibodies, Protozoan; D001483:Base Sequence; D016054:DNA, Protozoan / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D017125:Genes, Protozoan / Q000235:genetics; D018503:Genome, Protozoan; D005974:Glutamate-Ammonia Ligase / Q000737:chemistry* / Q000187:drug effects* / Q000235:genetics / Q000276:immunology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007893:Leishmania donovani / Q000201:enzymology* / Q000254:growth & development; D007896:Leishmaniasis / Q000469:parasitology; D008670:Metals; D008970:Molecular Weight; D010767:Phosphorylcholine / Q000031:analogs & derivatives / Q000037:antagonists & inhibitors; D015800:Protozoan Proteins / Q000737:chemistry* / Q000187:drug effects* / Q000235:genetics / Q000276:immunology; D011994:Recombinant Proteins / Q000737:chemistry* / Q000187:drug effects* / Q000235:genetics / Q000276:immunology; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D013696:Temperature
chemical_list: D000913:Antibodies, Protozoan; D016054:DNA, Protozoan; D004791:Enzyme Inhibitors; D008670:Metals; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D010767:Phosphorylcholine; C039128:miltefosine; D005974:Glutamate-Ammonia Ligase
keywords: Glutamine synthetase; Kinetics; Leishmania; Localization