Improved purification of acyl carrier protein

An improved method for the purification of acyl carrier protein from Escherichia coli is described. The method consists of four steps: a 2-propanol extraction, batch adsorption to DEAE-cellulose, ammonium sulfate fractionation, and acid precipitation. The purification can be carried out in a few days and yields between 120 and 150 mg of pure acyl carrier protein per kilogram wet weight of cells.     

Structural homology of spinach acyl carrier protein and Escherichia coli acyl carrier protein based on NMR data

Acyl carrier proteins (ACPs) from spinach and from Escherichia coli have been used to demonstrate the utility of proton NMR for comparison of homologous structures. The structure of E. coli ACP had been previously determined and modeled as a rapid equilibrium among multiple conformational forms (Kim and Prestegard, Biochemistry 28:8792–8797, 1989). Spinach ACP showed two slowly exchanging forms and could be manipulated into one form for structural study. Here we compare this single form to postulated multiple forms of E. coli ACP using the limited amount of NOE data available for the spinach protein. A number of long-range NOE contacts were present between homologous residues in both spinach and E. coli ACP, suggesting tertiary structural homology. To allow a more definitive structural comparison, a method was developed to use spinach ACP NOE constraints to search for regions of structural divergence from two postulated forms of E. coli ACP. The homologous regions of the two protein sequences were aligned, additional distance constraints were extracted from the E. coli structure, and these were mapped onto the spinach sequence. These distance constraints were combined with experimental NOE constraints and a distance geometry simulated annealing protocol was used to test for compatibility of the constraints. All of the experimental spinach NOE constraints could be successfully combined with the E. coli data, confirming the general hypothesis of structural homology. A better fit was obtained with one form, suggesting a preferential stabilization of that form in the spinach case. Proteins 27:131–143 © 1997 Wiley-Liss, Inc.     

Analyses of co-operative transitions in Plasmodium falciparum beta-ketoacyl acyl carrier protein reductase upon co-factor and acyl carrier protein binding

The type II fatty acid synthase pathway of Plasmodium falciparum is a validated unique target for developing novel antimalarials because of its intrinsic differences from the type I pathway operating in humans. beta-Ketoacyl-acyl carrier protein reductase is the only enzyme of this pathway that has no isoforms and thus selective inhibitors can be developed for this player of the pathway. We report here intensive studies on the direct interactions of Plasmodiumbeta-ketoacyl-acyl carrier protein reductase with its cofactor, NADPH, acyl carrier protein, acetoacetyl-coenzyme A and other ligands in solution, by monitoring the intrinsic fluorescence (lambdamax 334 nM) of the protein as a result of its lone tryptophan, as well as the fluorescence of NADPH (lambdamax 450 nM) upon binding to the enzyme. Binding of the reduced cofactor makes the enzyme catalytically efficient, as it increases the binding affinity of the substrate, acetoacetyl-coenzyme A, by 16-fold. The binding affinity of acyl carrier protein to the enzyme also increases by approximately threefold upon NADPH binding. Plasmodiumbeta-ketoacyl-acyl carrier protein reductase exhibits negative, homotropic co-operative binding for NADPH, which is enhanced in the presence of acyl carrier protein. Acyl carrier protein increases the accessibility of NADPH to beta-ketoacyl-acyl carrier protein reductase, as evident from the increase in the accessibility of the tryptophan of beta-ketoacyl-acyl carrier protein reductase to acrylamide, from 81 to 98%. In the presence of NADP+, the reaction proceeds in the reverse direction (Ka=23.17 microM-1). These findings provide impetus for exploring the influence of ligands on the structure-activity relationship of Plasmodiumbeta-ketoacyl-acyl carrier protein reductase.     

Malonyl-CoA: acyl carrier protein transacylase from Helicobacter pylori: Crystal structure and its interaction with acyl carrier protein

Malonyl-CoA: acyl carrier protein transacylase (MCAT) is a critical enzyme responsible for the transfer of the malonyl moiety to holo-acyl carrier protein (ACP) forming the malonyl-ACP intermediates in the initiation step of type II fatty acid synthesis (FAS II) in bacteria. MCAT has been considered as an attractive drug target in the discovery of antibacterial agents. In this study, the crystal structure of MCAT from Helicobacter pylori (Hp) at 2.5 angstroms resolution is reported, and the interaction of HpMCAT with HpACP is extensively investigated by using computational docking, GST-pull-down, and surface plasmon resonance (SPR) technology-based assays. The crystal structure results reveal that HpMCAT has a compact folding composed of a large subdomain with a similar core as in alpha/beta hydrolases, and a similar ferredoxin-like small subdomain as in acylphosphatases. The docking result suggests two positively charged areas near the entrance of the active site of HpMCAT as the ACP-binding region. Binding assay research shows that HpMCAT demonstrates a moderately binding ability against HpACP. The solved 3D structure of HpMCAT is expected to supply useful information for the structure-based discovery of novel inhibitors against MCAT, and the quantitative study of HpMCAT interaction with HpACP is hoped to give helpful hints in the understanding of the detailed catalytic mechanisms for HpMCAT.     

Purification and NMR characterization of acyl carrier protein

The acyl carrier protein preparation obtained using the 2-propanol method of Rock and Cronan (Rock, C. O., and Cronan, J. E., Jr. (1981) Methods Enzymol. 71, 341-351) can be further purified with reversed-phase high-performance liquid chromatography. A homogeneous sample of acyl carrier protein is obtained as determined by NMR and reversed-phase high-performance liquid chromatography.     

The primary structure of spinach acyl carrier protein

Acyl carrier protein (ACP) from spinach leaves has been purified to homogeneity by high-performance liquid chromatography with an anion-exchange column. The amino acid sequence of one major ACP in spinach leaves, ACP-I, has been determined by automated Edman degradation. It consists of the following 82 amino acids: (sequence in text). Sequencing of the intact polypeptide provided data for the first 57 residues. Cleavage of the succinylated ACP with CNBr at Met-46, followed by sequencing of the fragment mixture, provided data for the final 36 residues. The C-terminal alanine was confirmed by carboxypeptidase Y digestion. The spinach ACP has 40, 70, and 25% homology with Escherichia coli, barley, and rabbit ACPs, respectively. The results not only provide the first complete sequence of a plant ACP, but also provide insight into the structural and evolutionary relationships among plant, animal, and bacterial ACPs.     

Mammalian mitochondria contain a soluble acyl carrier protein

Plant and fungal mitochondria contain type II fatty acid synthesis systems closely related to those of bacteria in which the individual reactions are catalyzed by separate soluble proteins acting on intermediates bound to acyl carrier protein (ACP). Mammalian mitochondria are thought to synthesize fatty acids, but evidence for the key ACP component was lacking since the only reported ACP was the SDAP subunit of the membrane-bound NADH:ubiquinone oxidoreductase, We report that most of the SDAP is found in the soluble (matrix) fraction of bovine heart mitochondria and is therefore available to carry the intermediates of type II fatty acid synthesis.     

A mechanism based protein crosslinker for acyl carrier protein dehydratases

Recent advances in the structural study of fatty acid synthase (FAS) and polyketide synthase (PKS) biosynthetic enzymes have illuminated our understanding of modular enzymes of the acetate pathway. However, one significant and persistent challenge in such analyses is resolution of the acyl carrier protein (ACP), a small (～9 kDa) protein to which biosynthetic intermediates are tethered throughout the biosynthetic cycle. Here we report a chemoenzymatic crosslinking strategy in which the installation of a historical suicide substrate scaffold upon the 4′-phosphopantetheine (PPant) arm of the ACP is used to capture the active site of acyl carrier protein dehydratase (DH) domains in FAS. Through the synthesis of a small panel of related probes we identify structural features essential for ACP–DH crosslinking, and apply gel-based assays to demonstrate the stability as well as purification strategies for isolation of the chemoenzymatically modified ACP. Applying these carrier protein crosslinking techniques to the structural analysis of FAS and PKS complexes has the potential to provide snapshots of these biosynthetic assembly lines at work.     

2-Acylglycerolphosphoethanolamine acyltransferase/acyl-acyl carrier protein synthetase is a membrane-associated acyl carrier protein binding protein

Two enzymatic activities, 2-acylglycerolphosphoethanolamine (2-acyl-GPE) acyltransferase and acyl-acyl carrier protein (acyl-ACP) synthetase, were solubilized and purified from Escherichia coli membranes. Electrophoretic analysis of the final product of the purification procedure revealed a single protein species with an apparent molecular mass of 27 kilodaltons. The ratio of acyltransferase to synthetase activities remained the same throughout the purification scheme indicating that both activities were catalyzed by the same enzyme. 2-Acyl-GPE acyltransferase exhibited an apparent ACP Km of 64 nM under standard assay conditions that increased to 10 microM when the assay was conducted in the presence of 0.4 M LiCl. Acyl-ACP synthetase activity was not detected in the absence of 0.4 M LiCl, and the apparent ACP Km for this reaction was 16 microM. Direct evidence that ACP was a subunit of the acyltransferase/synthetase was obtained by the adsorption of both catalytic activities to an ACP-Sepharose affinity column and by the binding of [3H]ACP to the purified enzyme preparation. The apparent Km for acyl-ACP was 13 microM, and the rate of acyl transfer from this acyl donor was enhanced by the addition of 0.4 M LiCl indicating that the exchange of enzyme-bound ACP for acyl-ACP was a determinant factor in the rate of phosphatidylethanolamine formation from acyl-ACP. These data indicate that the 2-acyl-GPE acyltransferase and acyl-ACP synthetase reactions are catalyzed by the same membrane protein that possesses a high affinity binding site for soluble ACP.     

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.     

大肠杆菌holo-ACP的过表达、分离纯化及长链脂酰ACP的合成

[目的]获得高纯度大肠杆菌holo-ACP和多种长链脂酰ACP,为研究细菌脂肪酸、类脂A和N-酯酰高丝氨酸内脂等物质的合成提供底物.[方法和结果]采用PCR方法扩增得到大肠杆菌酰基载体蛋白基因(acpP)和holo-ACP合成酶基因(acpS).使用载体pBAD24、pBAD34和pET28b分别克隆了acpP和acpS,得到pBAD-ACP、pET-ACP和pET-ACP-ACPS 3个ACP表达质粒和一个AcpS表达质粒pBAD-ACPS.分别用3个ACP表达质粒转化大肠杆菌DH5a和BL21(DE3),构建了DH5αpBAD-ACP、BL21(DE3)/pET-ACP和BL21(DE3)/pET-ACP-ACPS 3种ACP生产菌株.与holo-ACP纯化常用菌株DK574相比,虽然三菌株在诱导时均能过量表达ACP,但是holo-ACP所占比例偏低.为了提高ACP生产菌株holo-ACP的产量,用质粒pBAD-ACPS分别转化上述3种ACP生产菌株,获得了3种携带双质粒的ACP生产菌株.表达结果显示携带pBAD-ACP和pBAD-ACPS双质粒的DH5a菌株比DK574菌株能产生更多的holo-ACP,且纯度也得到提高(纯度达99%).同时使用UNOsphere Q阴离子交换层析从这一菌株培养物中分离纯化到了高纯度的holo-ACP,并以纯化到的holo-ACP和多种长链脂肪酸为底物在哈氏弧菌脂酰ACP合成酶的催化下,合成了多种长链脂酰ACP.[结论]通过研究获得一株holo-ACP高产菌株,并证明在大肠杆菌菌株中,同时表达acpP基因和acpS基因,有利于holo-ACP的产生.