A novel approach for over-expression, characterization, and isotopic enrichment of a homogeneous species of acyl carrier protein from Plasmodium falciparum

Abstract

Acyl carrier protein (ACP) plays a central role in fatty acid biosynthesis by transferring the acyl groups from one enzyme to another for the completion of the fatty acid synthesis cycle. Holo-ACP is the obligatory substrate for the synthesis of acyl-ACPs which act as the carrier and donor for various metabolic reactions. Despite its interactions with numerous proteins in the cell, its mode of interaction is poorly understood. Here, we report the over-expression of PfACP in minimal medium solely in its holo form and in high yield. Expression in minimal media provides a means to isotopically label PfACP for high resolution multi-nuclear and multi-dimensional NMR studies. Indeed, the proton-nitrogen correlated NMR spectrum exhibits very high chemical shift dispersion and resolution. We also show that holo-PfACP thus expressed is amenable to acylation reactions using Escherichia coli acyl-ACP synthetase as well as by standard chemical methods.

Chapter 17 Bacterial Fatty Acid Synthesis and its Relationships with Polyketide Synthetic Pathways

“…For preparation of unsaturated acyl-ACPs E. coli Aas may be required. It should also be noted that AasS is active on only a subset of bacterial ACPs (Jiang et al, 2006) whereas E. coli Aas has a broader specificity (Shanklin, 2000; Sharma et al, 2005). A second method for acyl-ACP synthesis is the use of a phosphopantetheinyl transferase such as the Sfp of Bacillus subtilis to transfer an acyl-phosphopantetheine group from acyl coenzyme A to apo ACP as discussed by Zhang & Tang in this volume, although the use of CoA thioesters makes this an expensive synthetic route and many acyl-CoAs are not commercially available.…”

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

“…Cloning, Expression, and Purification-Cloning, expression, and purification of P. falciparum holo-ACP in E. coli has already been described (23). E. coli holo-ACP synthase, apoPfACP and respective acyl-CoAs were used for the synthesis of butyryl-, octanoyl-, decanoyl-, dodecanoyl-, tetradecanoyl-, and hexadecanoyl-ACP using a modified protocol of Lambalot and Walsh (24) as described (23).…”

“…E. coli holo-ACP synthase, apoPfACP and respective acyl-CoAs were used for the synthesis of butyryl-, octanoyl-, decanoyl-, dodecanoyl-, tetradecanoyl-, and hexadecanoyl-ACP using a modified protocol of Lambalot and Walsh (24) as described (23). Uniformly labeled 13 …”

Mass Spectrometry-Based Systems Approach for Identification of Inhibitors ofPlasmodium falciparumFatty Acid Synthase

“…AcpS was cloned in pET22b and purified according to reference 17. Pfacp gene was cloned in pET28a(ϩ) (31), and the apo form was purified according to reference 27. Fifty micromolars apo-PfACP, 200 M acyl-CoA, 20 mM MgCl 2 , 3 to 5 g E. coli AcpS in 25 mM Tris-Cl, pH 7.5, and 0.5% Triton X-100 were used for synthesis reactions involving C 12 -C 16⌬1 -CoA.…”