Deciphering the key residues in Plasmodium falciparum beta-ketoacyl acyl carrier protein reductase responsible for interactions with Plasmodium falciparum acyl carrier protein.
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 typeI pathway operating in humans. beta-Ketoacyl acyl carrier protein (ACP) reductase (FabG) performs the NADPH-dependent reduction of beta-ketoacyl-ACP to beta-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.
Karmodiya
Krishanpal
K
Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bangalore, India.
Modak
Rahul
R
Sahoo
Nirakar
N
Sajad
Syed
S
Surolia
Namita
N
D064431:3-Oxoacyl-(Acyl-Carrier-Protein) Reductase; D000213:Acyl Carrier Protein / Q000378:metabolism*; D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000378:metabolism; D001665:Binding Sites; D007700:Kinetics; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000378:metabolism; D016415:Sequence Alignment; D020349:Surface Plasmon Resonance
D000213:Acyl Carrier Protein; D009249:NADP; D001120:Arginine; D000429:Alcohol Oxidoreductases; D064431:3-Oxoacyl-(Acyl-Carrier-Protein) Reductase