title: Molecular cloning and characterization of Brugia malayi thymidylate kinase.
abstract: Thymidylate kinase (TMK) is a potential chemotherapeutic target because it is directly involved in the synthesis of deoxythymidine triphosphate, which is an essential component for DNA synthesis. The gene encoding thymidylate kinase of Brugia malayi was amplified by PCR and expressed in Escherichia coli. The native molecular weight of recombinant B. malayi thymidylate kinase (rBmTMK) was estimated to be ∼52kDa by gel filtration chromatography, suggesting a homodimeric structure. rBmTMK activity required divalent cation and Mg(2+) was found to be the most effective cation. The enzyme was sensitive to pH and temperature, it showed maximum activity at pH 7.4 and 37°C. The Km values for dTMP and ATP were 17 and 66μM, respectively. The turnover number kcat was found to be 38.09s(-1), a value indicating the higher catalytic efficiency of the filarial enzyme. The nucleoside analogues 5-bromo-2'-deoxyuridine (5-BrdU), 5-chloro-2'-deoxyuridine (5-CldU) and 3'-azido-3'-deoxythymidine (AZT) showed specific inhibitory effect on the enzyme activity and these effects were in good association with binding interactions and the scoring functions as compared to human TMK. Differences in kinetic properties and structural differences in the substrate binding site of BmTMK model with respect to human TMK can serve as basis for designing specific inhibitors against parasitic enzyme.
authors.affiliation: Division of Biochemistry, CSIR - Central Drug Research Institute, Lucknow 226001, Uttar Pradesh, India.
authors.forename: Pawan Kumar
authors.initials: PK
authors.lastname: Doharey
authors.affiliation: Division of Biochemistry, CSIR - Central Drug Research Institute, Lucknow 226001, Uttar Pradesh, India.
authors.forename: Manish Kumar
authors.initials: MK
authors.lastname: Suthar
authors.affiliation: Division of Biochemistry, CSIR - Central Drug Research Institute, Lucknow 226001, Uttar Pradesh, India.
authors.forename: Anita
authors.initials: A
authors.lastname: Verma
authors.affiliation: Division of Molecular and Structural Biology, CSIR - Central Drug Research Institute, Lucknow 226031, Uttar Pradesh, India.
authors.forename: Vikash
authors.initials: V
authors.lastname: Kumar
authors.affiliation: Division of Biochemistry, CSIR - Central Drug Research Institute, Lucknow 226001, Uttar Pradesh, India.
authors.forename: Sunita
authors.initials: S
authors.lastname: Yadav
authors.affiliation: Medicinal and Process Chemistry Division, CSIR - Central Drug Research Institute, Lucknow 226031, Uttar Pradesh, India.
authors.forename: Vishal M
authors.initials: VM
authors.lastname: Balaramnavar
authors.affiliation: Department of Biochemistry, Banaras Hindu University, Varanasi 221005, Uttar Pradesh, India.
authors.forename: Sushma
authors.initials: S
authors.lastname: Rathaur
authors.affiliation: Medicinal and Process Chemistry Division, CSIR - Central Drug Research Institute, Lucknow 226031, Uttar Pradesh, India.
authors.forename: Anil Kumar
authors.initials: AK
authors.lastname: Saxena
authors.affiliation: Division of Molecular and Structural Biology, CSIR - Central Drug Research Institute, Lucknow 226031, Uttar Pradesh, India.
authors.forename: Mohammad Imran
authors.initials: MI
authors.lastname: Siddiqi
authors.affiliation: Division of Biochemistry, CSIR - Central Drug Research Institute, Lucknow 226001, Uttar Pradesh, India. Electronic address: jkscdri@yahoo.com.
authors.forename: Jitendra Kumar
authors.initials: JK
authors.lastname: Saxena
mesh_terms: D000818:Animals; D017178:Brugia malayi / Q000201:enzymology* / Q000235:genetics; D020134:Catalytic Domain; D002413:Cations, Divalent / Q000378:metabolism; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D020536:Enzyme Activators / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000032:analysis; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000378:metabolism; D008958:Models, Molecular; D008970:Molecular Weight; D009703:Nucleoside-Phosphate Kinase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature
chemical_list: D002413:Cations, Divalent; D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D009703:Nucleoside-Phosphate Kinase; C020093:dTMP kinase; D008274:Magnesium
keywords: Brugia malayi; Drug target; Enzyme inhibition; Homology modelling and docking; Substrate specificity; Thymidylate kinase