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Molecular cloning and characterization of Brugia malayi thymidylate kinase

IR@CDRI: CSIR-Central Drug Research Institute, Lucknow

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Creator Doharey, P K
Sutha, M K
Verma, Anita
Kumar, Vikash
Yadav, Sunita
Balaramnavar, V M
Rathaur, Sushma
Saxena, A K
Siddiqi, M I
Saxena, J K
 
Date 2014-08-05T07:09:31Z
2014-08-05T07:09:31Z
2014
 
Identifier Acta Tropica, 2014, 133, 83-92
http://hdl.handle.net/123456789/1343
 
Description 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 Brugia malayi thymidylate kinase (rBmTMK) was estimated to be ~52 kDa 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 66 µM respectively. The turnover number kcat was found to be 38.09 s-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.
 
Format 752667 bytes
application/pdf
 
Language en
 
Relation CSIR-CDRI Communication No. 8625
 
Subject Thymidylate Kinase
Drug Target
Brugia Malayi
Substrate Specificity
Enzyme Inhibition
Homology Modelling And Docking
 
Title Molecular cloning and characterization of Brugia malayi thymidylate kinase
 
Type Article