Biochemical characterization of Plasmodium falciparum Sir2, a NAD+-dependent deacetylase
- 作者：Subhra Prakash Chakrabarty ; Yegnisettipalli Krishnaiah Saikumari ; Monn ; a P. Bopanna ; Hemalatha Balaram
- 关键词：Plasmodium falciparum Sir2 ; Kinetic parameters ; Rapid equilibrium ordered kinetic mechanism ; Surfactin inhibition ; Subunit association
- 刊名：Molecular and Biochemical Parasitology
- 出版年：2008
- 期刊代码：108_01666851
- 类别：bio
- 出版时间：April 2008
- 卷：158
- 期：2
- 页码：139-151
- 文件大小：571 K
摘要
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. Surfactin was 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.