Recombinant expression and biochemical characterization of the unique elongating β-ketoacyl-acyl carrier protein synthase involved in fatty acid biosynthesis of Plasmodium falciparum using natural and artificial substrates

Abstract

The human malaria parasite Plasmodium falciparum synthesizes fatty acids by using a type II synthase that is structurally different from the type I system found in eukaryotes. Because of this difference and the vital role of fatty acids, the enzymes involved in fatty acid biosynthesis of P. falciparum represent interesting targets for the development of new antimalarial drugs. β-Ketoacyl-acyl carrier protein (ACP) synthase (PfFabBF), being the only elongating β-ketoacyl-ACP synthase in P. falciparum, is a potential candidate for inhibition. In this study we present the cloning, expression, purification, and characterization of PfFabBF. Soluble protein was obtained when PfFabBF was expressed as a NusA fusion protein in Escherichia coli BL21(DE3)-CodonPlus-RIL cells under conditions of osmotic stress. The fusion protein was purified by affinity and ion exchange chromatography. Various acyl-P. falciparum acyl carrier protein (PfACP) substrates were tested for their specific activities, and their kinetic parameters were determined. Activity of PfFabBF was highest with C4:0- to C10:0-acyl-PfACPs and decreased with use of longer chain acyl-PfACPs. Consistent with the fatty acid synthesis profile found in the parasite cell, no activity could be detected with C16:0-PfACP, indicating that the enzyme is lacking the capability of elongating acyl chains that are longer than 14 carbon atoms. PfFabBF was found to be specific for acyl-PfACPs, and it displayed much lower activities with the corresponding acyl-CoAs. Furthermore, PfFabBF was shown to be sensitive to cerulenin and thiolactomycin, known inhibitors of β-ketoacyl-ACP synthases. These results represent an important step toward the evaluation of P. falciparum β-ketoacyl-ACP synthase as a novel antimalaria target. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.