Structural Insights into the Acyl Intermediates of the Plasmodium falciparum Fatty Acid Synthesis Pathway

Abstract

Acyl carrier protein (ACP) plays a central role in fatty acid biosynthesis. However, the molecular machinery that mediates its function is not yet fully understood. Therefore, structural studies were carried out on the acyl-ACP intermediates of Plasmodium falciparum using NMR as a spectroscopic probe. Chemical shift perturbation studies put forth a new picture of the interaction of ACP molecule with the acyl chain, namely, the hydrophobic core can protect up to 12 carbon units, and additional carbons protrude out from the top of the hydrophobic cavity. The latter hypothesis stems from chemical shift changes observed in C ␣ and C ␤ of Ser-37 in tetradecanoyl-ACP. 13 C, 15 N-Double-filtered nuclear Overhauser effect (NOE) spectroscopy experiments further substantiate the concept; in octanoyl (C 8 )-and dodecanoyl (C 12 )-ACP, a long range NOE is observed within the phosphopantetheine arm, suggesting an arch-like conformation. This NOE is nearly invisible in tetradecanoyl (C 14 )-ACP, indicating a change in conformation of the prosthetic group. Furthermore, the present study provides insights into the molecular mechanism of ACP expansion, as revealed from a unique side chain-to-backbone hydrogen bond between two fairly conserved residues, Ile-55 HN and Glu-48 O. The backbone amide of Ile-55 HN reports a pK a value for the carboxylate, ϳ1.9 pH units higher than model compound value, suggesting strong electrostatic repulsion between helix II and helix III. Charge-charge repulsion between the helices in combination with thrust from inside due to acyl chain would energetically favor the separation of the two helices. Helix III has fewer structural restraints and, hence, undergoes major conformational change without altering the overall-fold of P. falciparum ACP.In the malarial parasite Plasmodium falciparum, fatty acid biosynthesis occurs by a pathway distinct from the host. A number of enzymes involved in the process viz. ␤-ketoacyl acyl carrier protein (ACP) 5 synthase III, ␤-hydroxy acyl-ACP hydratase, and enoyl-ACP reductase are targets for drug design (1, 2). An indispensable component, crucial for each step of the pathway, is a small acidic protein, the ACP. ACP plays a pivotal role in a range of biochemical processes, like fatty acid biosynthesis (3), polyketide synthesis (4, 5), oligosaccharides (6), biotin, and nonribosomal peptide synthesis (7,8). Thus, the knowledge of structural features, which dictate ACP function, could offer new avenues for inhibitor design to disable several pathways of the parasite in parallel.Acyl carrier protein differs structurally in the host and the parasite. It exists as an independent protein in type II fatty acid synthesis pathway, observed in P. falciparum, Escherichia coli, spinach, and most prokaryotes. In the type II pathway, fatty acids are synthesized by multiple enzymes catalyzing different reactions. Conversely, mammalian ACP (malarial host) is an integral domain of one single multidomain, multifunctional fatty acid synthase (FAS) (type I pathway), each domain catalyzin...

NMR Solution Structure and Biophysical Characterization of Vibrio harveyi Acyl Carrier Protein A75H

“…Instead, the cavity observed in the VhACP A75H structure matches subpocket II of ACP that was recently described in molecular dynamics simulations (32). Evidence of subpocket II was also provided by NMR studies of P. falciparum ACP, and it appears likely that the acyl chains spend time in both subpockets in vivo (32,39). Further experiments are needed to determine how this influences acyl chain binding and whether potential inhibitors can be optimized based on the binding to both subcavities.…”

“…It is therefore not unexpected that the divalent cation binding affinity is different in apo-, holo-, and acyl-ACP (18,34). It has recently been suggested that the electrostatic repulsion between helices II and III of P. falciparum ACP is important for the prompt opening and extraction of the acyl chain (39). Therefore, divalent cation binding will likely affect this process by diminishing the repulsive forces between the two helices (Fig.…”

Fatty acid metabolism in the Plasmodium apicoplast: Drugs, doubts and knockouts

“…The function of PfACP has been demonstrated by complementation [129] and recombinant expression in E. coli [65,67], and also through its ability to support FASII enzymes in vitro [65,66]. There have been several structural studies of PfACP [130][131][132][133] and a considerable amount is known about the carrier's conformation, stability and redox state in bloodstage parasites. Intriguingly, PfACP has also been found to possess enzymatic activity, catalyzing both self-acylation and malonyl-transferase reactions in vitro [68,69].…”

Backbone chemical shift assignments of the acyl-acyl carrier protein intermediates of the fatty acid biosynthesis pathway of Plasmodium falciparum

“…Cloning, overexpression and purification of 15 N-labeled PfACP Proteins were expressed by cloning, overexpression and purification of P. falciparum holo and acyl-ACPs in E. coli as described (Upadhyay et al 2009 Inova 500, installed at the National Institute of Immunology, New Delhi, India, equipped with a triple resonance, Z pulsed field gradient probe. Experiments were performed at 300 K. NMR data was processed on a workstation running Red Hat Enterprize Linux 5.0, using NMRPipe/NMRDraw (Delaglio et al 1995) and analyzed using Sparky (Goddard and Kneller 2001).…”

“…In most of the acyl-ACP intermediates (with the exception of rat), the hydrophobic core increases in size to accommodate the growing acyl chain. The molecular basis of ACP expansion upon acyl chain insertion has recently been disclosed by our NMR studies on P. falciparum acyl-ACP intermediates (Upadhyay et al 2009), where the electrostatic repulsion in conjunction with thrust due to the growing acyl chain favors expansion of the hydrophobic core of the ACP , resulting in large chemical shift changes. In this paper, we report the backbone chemical shift assignments of the acyl-ACPs of P. falciparum viz.…”