Biochemical characterization of Plasmodium falciparum Sir2, a NAD+-dependent deacetylase.
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.
Chakrabarty
Subhra Prakash
SP
Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bangalore 560064, India.
Saikumari
Yegnisettipalli Krishnaiah
YK
Bopanna
Monnanda P
MP
Balaram
Hemalatha
H
D000595:Amino Acid Sequence; D000818:Animals; D019281:Dimerization; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007700:Kinetics; D055666:Lipopeptides; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism*; D010456:Peptides, Cyclic / Q000494:pharmacology; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D021122:Protein Subunits / Q000378:metabolism; D015800:Protozoan Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D037761:Sirtuins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D004791:Enzyme Inhibitors; D055666:Lipopeptides; D010456:Peptides, Cyclic; D021122:Protein Subunits; D015800:Protozoan Proteins; D009243:NAD; C009365:surfactin peptide; D037761:Sirtuins