title: Biochemical characterization of dihydroorotase of Leishmania donovani: Understanding pyrimidine metabolism through its inhibition.
abstract: De novo pyrimidine biosynthesis pathway is well developed and functional in protozoan parasite Leishmania donovani. The dihydroorotase (LdDHOase) is third enzyme of the pathway. The enzyme was cloned, expressed in E. coli BL21 (DE3), purified to homogeneity and biochemically characterized. The estimated kcat for the forward reaction and reverse reactions were 2.1 ± 0.1 s-1and 1.1 ± 0.15 s-1, respectively. Homology modeling and docking studies were done to find out potential inhibitors for LdDHOase. Biotin sulfone and Kaempferol were found to be potential inhibitors of LdDHOase based on docking studies. These inhibitors were verified using recombinant LdDHOase and their anti-leishmanial effect was evaluated. Moreover, alterations in expressions of de novo as well as salvage pathways enzymes, after treatment of L. donovani with dihydroorotase inhibitor(s) were evaluated and discussed as survival mechanism of the pathogen. Further, effect of inhibition of cytidine deaminase, a key enzyme of salvage pathway of pyrimidine biosynthesis, was also evaluated on parasitic survival and alteration in gene expression of enzymes of both pathways. Further, effect of both pathways inhibition was also evaluated. The data suggests that the inhibition of single pathway can be overcome by increased expression of enzyme(s) of alternate pathway and both pathways seem to be equally important in the pathogen. When both pathways are simultaneously inhibited, parasite shows significant DNA damage and parasitic death.
authors.affiliation: Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Assam 781039, India.
authors.forename: Kartikeya
authors.initials: K
authors.lastname: Tiwari
authors.affiliation: Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Assam 781039, India.
authors.forename: Ritesh
authors.initials: R
authors.lastname: Kumar
authors.affiliation: Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Assam 781039, India. Electronic address: vdubey@iitg.ernet.in.
authors.forename: Vikash Kumar
authors.initials: VK
authors.lastname: Dubey
mesh_terms: D001710:Biotin / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology; D003564:Cytidine Deaminase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D004080:Dihydroorotase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D044949:Kaempferols / Q000737:chemistry / Q000494:pharmacology; D007700:Kinetics; D007893:Leishmania donovani / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D062105:Molecular Docking Simulation; D015394:Molecular Structure; D009963:Orotic Acid / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D000072417:Protein Domains; D015800:Protozoan Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011743:Pyrimidines / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013379:Substrate Specificity; D013450:Sulfones / Q000737:chemistry / Q000494:pharmacology
chemical_list: D044949:Kaempferols; D015800:Protozoan Proteins; D011743:Pyrimidines; D011994:Recombinant Proteins; D013450:Sulfones; C004768:4,5-dihydroorotic acid; C104349:biotin sulfone; D009963:Orotic Acid; D001710:Biotin; C006552:kaempferol; D004080:Dihydroorotase; D003564:Cytidine Deaminase; C030986:pyrimidine
keywords: Anti-leishmanial; LdDHOase; Leishmania donovani; Pyrimidine biosynthesis