Chakrabarty, Subhra Prakash and Saikumari, Yegnisettipalli Krishnaiah and Bopanna, Monnanda P and Balaram, Hemalatha (2008) Biochemical characterization of Plasmodium falciparum Sir2,a $NAD^+$-dependent deacetylase. In: Molecular and Biochemical Parasitology, 158 (2). pp. 139-151.
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In Plasmodium falciparum, the causative agent of cerebral malaria, silent information regulator 2 (Sir2) has been implicated in pathogenesis through its role in var gene silencing. P. falciparum Sir2 (PfSir2) in addition to the catalytic core, has a 13 residue N-terminal and 4 residue C-terminal extension over the shorter Archaeoglobus fulgidus Sir2. In this paper, we highlight our studies aimed at understanding the kinetic mechanism of PfSir2 and the role of N- and C-terminal extensions in protein function and oligomerization. Bisubstrate kinetic analysis showed that PfSir2 exhibits a rapid equilibrium ordered sequential mechanism, with peptide binding preceding $NAD^+$. This study also reports on surfactin as a novel Sir2 inhibitor exhibiting competitive inhibition with respect to $NAD^+$ and uncompetitive inhibition with acetylated peptide. This inhibition pattern with surfactin provides further support for ordered binding of substrates. Surfactinwas also found to be a potent inhibitor of intra-erythrocytic growth of P. falciparum with 50% inhibitory concentration in the low micromolar range. PfSir2, like the yeast homologs (yHst2 and Sir2p), is a trimer in solution. However, dissociation of trimer to monomers in the presence of $NAD^+$ is characteristic of the parasite enzyme. Oligomerization studies on N- and/or C-terminal deletion constructs of PfSir2 highlight the role of C-terminus of the protein in mediating homotrimerization. N-terminal deletion resulted in reduced catalytic efficiency although substrate affinity was not altered in the constructs. Interestingly, deletion of both the ends relaxed $NAD^+$ specificity.
|Item Type:||Journal Article|
|Additional Information:||Copyright of this article belongs to Elsevier.|
|Keywords:||Plasmodium falciparum Sir2;Kinetic parameters;Rapid equilibrium ordered kinetic mechanism;Surfactin inhibition;Subunit association.|
|Department/Centre:||Division of Biological Sciences > Molecular Biophysics Unit|
|Date Deposited:||10 Sep 2008 05:44|
|Last Modified:||19 Sep 2010 04:49|
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