Deciphering the key residues in Plasmodium falciparum β-ketoacyl acyl carrier protein reductase responsible for interactions with Plasmodium falciparum acyl carrier protein

Karmodiya, Krishanpal ; Modak, Rahul ; Sahoo, Nirakar ; Sajad, Syed ; Surolia, Namita (2008) Deciphering the key residues in Plasmodium falciparum β-ketoacyl acyl carrier protein reductase responsible for interactions with Plasmodium falciparum acyl carrier protein FEBS Journal, 275 (19). pp. 4756-4766. ISSN 1742-464X

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Official URL: http://onlinelibrary.wiley.com/doi/10.1111/j.1742-...

Related URL: http://dx.doi.org/10.1111/j.1742-4658.2008.06608.x

Abstract

The type II fatty acid synthase (FAS) pathway of Plasmodium falciparum is a validated unique target for developing novel antimalarials, due to its intrinsic differences from the type I pathway operating in humans. β-Ketoacyl acyl carrier protein (ACP) reductase (FabG) performs the NADPH-dependent reduction of β-ketoacyl-ACP to β-hydroxyacyl-ACP, the first reductive step in the elongation cycle of fatty acid biosynthesis. In this article, we report intensive studies on the direct interactions of Plasmodium FabG and Plasmodium ACP in solution, in the presence and absence of its cofactor, NADPH, by monitoring the change in intrinsic fluorescence of P. falciparum FabG (PfFabG) and by surface plasmon resonance. To address the issue of the importance of the residues involved in strong, specific and stoichiometric binding of PfFabG to P. falciparum ACP (PfACP), we mutated Arg187, Arg190 and Arg230 of PfFabG. The activities of the mutants were assessed using both an ACP-dependent and an ACP-independent assay. The affinities of all the PfFabG mutants for acetoacetyl-ACP (the physiological substrate) were reduced to different extents as compared to wild-type PfFabG, but were equally active in biochemical assays with the substrate analog acetoacetyl-CoA. Kinetic analysis and studies of direct binding between PfFabG and PfACP confirmed the identification of Arg187 and Arg230 as critical residues for the PfFabG-PfACP interactions. Our studies thus reveal the significance of the positively charged/hydrophobic patch located adjacent to the active site cavities of PfFabG for interactions with PfACP.

Item Type:Article
Source:Copyright of this article belongs to John Wiley and Sons.
Keywords:Fatty Acid Synthase; Fluorescence; Malaria; Protein-Protein Interactions; Surface Plasmon Resonance
ID Code:61607
Deposited On:15 Sep 2011 12:46
Last Modified:15 Sep 2011 12:46

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