Backbone chemical shift assignments of the acyl-acyl carrier protein intermediates of the fatty acid biosynthesis pathway of Plasmodium falciparum
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- Upadhyay, S.K., Misra, A., Surolia, N. et al. Biomol NMR Assign (2010) 4: 83. doi:10.1007/s12104-010-9212-2
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We report the backbone chemical shift assignments of the acyl-acyl carrier protein (ACP) intermediates of the fatty acid biosynthesis pathway of Plasmodium falciparum. The acyl-ACP intermediates butyryl (C4), -octanoyl (C8), -decanoyl (C10), -dodecanoyl (C12) and -tetradecanoyl (C14)-ACPs display marked changes in backbone HN, Cα and Cβ chemical shifts as a result of acyl chain insertion into the hydrophobic core. Chemical shift changes cast light on the mechanism of expansion of the acyl carrier protein core.
KeywordsBackbone assignmentsAcyl-ACPsPlasmodium falciparumFatty acid biosynthesis
Malaria is one of the major health concerns in tropical countries, liable for high rates of mortality. Fatty acid biosynthesis pathway of Plasmodium sp. has been considered as a drug target against this disease, owing to the differences in fatty acid biosynthesis pathway proteins between the host and the parasite (Lu et al. 2005; Lu and Tonge 2008). In Plasmodium, fatty acids are synthesized by the type II pathway, multiple enzymes catalyzing various functions, while in the host, fatty acid biosynthesis is brought about by one single multidomain, fatty acid synthase, performing multiple functions (type I pathway). The structural differences between the two fatty acid pathways offer unique opportunities for drug design, targeting the protozoal pathway exclusively. A common cofactor, indispensable to both the pathways is the acyl carrier protein, which serves to shuttle the lengthening acyl chains to the catalytic site of FAS enzymes.
Acyl carrier protein is a domain of fatty acid synthase (FAS) in type I pathway, while in type II pathway, it is an independent protein. Acyl carrier protein is expressed as an apo protein (inactive), and converted to holo-ACP (active) by the transfer of a 4′-PP moiety from coenzyme A to a conserved serine residue, with ACP synthase acting as a catalyst. The acyl chains gets covalently tethered to the terminal sulfhydryl group of 4-phosphopentetheine (4′-PP) moiety of ACP, which in turn transfers the acyl chain to the active site of FAS enzymes. The mechanism by which the ACP molecule carries the acyl chain and transfers to the active site of FAS enzymes remains unknown. Numerous structural studies have been carried out by different groups to understand the mechanism of formation of acyl-ACP intermediates (Wu et al. 2009; Roujeinikova et al. 2002, 2007; Zornetzer et al. 2006; Sharma et al. 2006). 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 molecule, resulting in large chemical shift changes. In this paper, we report the backbone chemical shift assignments of the acyl-ACPs of P. falciparum viz. butyryl, octanoyl, decanoyl, dodecanoyl, and tetradecanoyl-ACP.
Materials and methods
Cloning, overexpression and purification of 15N-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).
NMR samples comprised of uniformly labeled [1H, 15N, and 13C] protein, 50 mM sodium phosphate buffer, pH 6.5, 100 mM NaCl, 2 mM DTT, 0.5% Sodium azide (NaN3), 90% H2O in 10% D2O. Concentration of protein was kept at 1 mM during all the NMR experiments. Two and three dimensional NMR experiments, viz. 1H15N HSQC, 1H15N TOCSY, HNCACB, CBCAcoNH, CCcoNH, HNcoCA, 13C15N-filtered experiments were acquired on a Varian 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). The data was multiplied by a phase shifted sinebell apodization function in all dimensions.
1H15N HSQC spectra were acquired using 1,024 data points in t2 dimension and 512 data points in t1 dimension. 1H15N HSQC-TOCSY experiments were collected with 1,024 (t3) × 72 (t1) × 48 (t2) data points and a mixing time of 150 ms. CBCAcoNH, HNCACB, CCcoNH experiments were collected with 1,024 (t3) × 36 (t1) × 24 (t2). Data was linear predicted in the forward direction for up to half the number of experimental points in the indirect dimensions. The temperature was maintained at 300 K during all experiments. 1 mM Sodium 4,4-dimethyl-4-silapentanesulfonate (DSS) was used as a chemical shift standard.