tailieunhanh - Báo cáo khoa học: Deciphering the key residues in Plasmodium falciparum b-ketoacyl acyl carrier protein reductase responsible for interactions with Plasmodium falciparum acyl carrier protein

The type II fatty acid synthase (FAS) pathway ofPlasmodium falciparumis a validated unique target for developing novel antimalarials, due to its intrinsic differences from the type I pathway operating in humans. b-Ketoacyl acyl carrier protein (ACP) reductase (FabG) performs the NADPH-dependent reduction ofb-ketoacyl-ACP to b-hydroxyacyl-ACP, the first reductive step in the elongation cycle of fatty acid biosynthesis. | ễFEBS Journal Deciphering the key residues in Plasmodium falciparum b-ketoacyl acyl carrier protein reductase responsible for interactions with Plasmodium falciparum acyl carrier protein Krishanpal Karmodiya Rahul Modak Nirakar Sahoo Syed Sajad and Namita Surolia Molecular Biology and Genetics Unit JawaharlalNehru Centre for Advanced Scientific Research Jakkur Bangalore India Keywords fatty acid synthase fluorescence malaria protein-protein interactions surface plasmon resonance Correspondence N. Surolia Molecular Biology and Genetics Unit JawaharlalNehru Centre for Advanced Scientific Research Jakkur Bangalore 560064 India Fax 91 80 22082766 Tel 91 80 22082820-21 E-mail surolia@ Received 14 June 2008 revised 23 July 2008 accepted 25 July 2008 doi 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. p-Ketoacyl acyl carrier protein ACP reductase FabG performs the NADPH-dependent reduction of p-ketoacyl-ACP to b-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-indepen-dent 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 .