Mutational Studies Reveal Lysine 352 on the Large Subunit is Indispensable for Catalytic Activity of bi-subunit Topoisomerase I from Leishmania Donovani
IR@IICB: CSIR-Indian Institute of Chemical Biology, Kolkata
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| Title |
Mutational Studies Reveal Lysine 352 on the Large Subunit is Indispensable for Catalytic Activity of bi-subunit Topoisomerase I from Leishmania Donovani
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| Creator |
Ganguly, Agneyo
Sengupta, Souvik Bose Dasgupta, Somdeb Roy, Amit Majumder, Hemanta K |
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| Subject |
Infectious Diseases and Immunology
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| Description |
From the vanadate complex crystal structure of Leishmania donovani topoisomerase I, several amino acid
residues have been implicated to be involved in the catalytic reaction. Although several predictions and
propositions have been made, the exact role of these amino acids has not yet been clearly demonstrated in
vitro. Among these residues, lysine 352 and arginine 314 stand as potential candidates for playing the role
of a general acid during the cleavage step. In this study,we have characterized the role of lysine 352 on the
large subunit, by site-directed mutagenesis and have tried to identify the general acid that can protonate
the 5�-O atom of the leaving strand. Studies with the mutant enzymes reveal that, relaxation activity was
severely affected when Lys352was mutated to arginine or alanine (K352R or K352A). Mutation of Arg314
to Lys (R314K) has very little effect on the relaxation activity.Detailed study reveals that, both cleavage and
religation steps are severely affected in case of K352R and K352A and the cleavage religation equilibrium
is shifted towards the cleavage. On the contrary, the R314K mutant exhibits only a slightly slower rate of
cleavage compared to wild-type enzyme. Cleavage assays with an oligonucleotide containing 5�-bridging
phosphorothiolate indicate that Lys352 acts as a general acid in the cleavage step. Altogether, this study
establishes the indispensable role of lysine 352 in the catalytic reaction of L. donovani topoisomerase I.
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| Publisher |
Elsevier
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| Date |
2009
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| Type |
Article
PeerReviewed |
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| Format |
application/pdf
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| Identifier |
http://www.eprints.iicb.res.in/1372/1/MOLECULAR_AND_BIOCHEMICAL_PARASITOLOGY___165(_1)_57%2D66_;2009[77].pdf
Ganguly, Agneyo and Sengupta, Souvik and Bose Dasgupta, Somdeb and Roy, Amit and Majumder, Hemanta K (2009) Mutational Studies Reveal Lysine 352 on the Large Subunit is Indispensable for Catalytic Activity of bi-subunit Topoisomerase I from Leishmania Donovani. Molecular and Biochemical Parasitology, 165 (1). pp. 57-66. |
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| Relation |
http://dx.doi.org/10.1016/j.molbiopara.2009.01.002
http://www.eprints.iicb.res.in/1372/ |
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