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.     

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.     

Solution structure of B. subtilis acyl carrier protein

BACKGROUND: Acyl carrier protein (ACP) is a fundamental component of fatty acid biosynthesis in which the fatty acid chain is elongated by the fatty acid synthetase system while attached to the 4'-phosphopantetheine prosthetic group (4'-PP) of ACP. Activation of ACP is mediated by holo-acyl carrier protein synthase (ACPS) when ACPS transfers the 4'-PP moiety from coenzyme A (CoA) to Ser36 of apo-ACP. Both ACP and ACPS have been identified as essential for E. coli viability and potential targets for development of antibiotics. RESULTS: The solution structure of B. subtilis ACP (9 kDa) has been determined using two-dimensional and three-dimensional heteronuclear NMR spectroscopy. A total of 22 structures were calculated by means of hybrid distance geometry-simulated annealing using a total of 1,050 experimental NMR restraints. The atomic rmsd about the mean coordinate positions for the 22 structures is 0.45 +/- 0.08 A for the backbone atoms and 0.93 +/- 0.07 A for all atoms. The overall ACP structure consists of a four alpha-helical bundle in which 4'-PP is attached to the conserved Ser36 that is located in alpha helix II. CONCLUSIONS: Structural data were collected for both the apo and holo forms of ACP that suggest that the two forms of ACP are essentially identical. Comparison of the published structures for E. coli ACP and actinorhodin polyketide synthase acyl carrier protein (act apo-ACP) from Streptomyces coelicolor A3(2) with B. subtilis ACP indicates similar secondary structure elements but an extremely large rmsd between the three ACP structures (>4.3 A). The structural difference between B. subtilis ACP and both E. coli and act apo-ACP is not attributed to an inherent difference in the proteins, but is probably a result of a limitation in the methodology available for the analysis for E. coli and act apo-ACP. Comparison of the structure of free ACP with the bound form of ACP in the ACP-ACPS complex reveals a displacement of helix II in the vicinity of Ser36. The induced perturbation of ACP by ACPS positions Ser36 proximal to coenzyme A and aligns the dipole of helix II to initiate transfer of 4'-PP to ACP.     

Purification and characterization of recombinant spinach acyl carrier protein I expressed in Escherichia coli

Expression of plant acyl carrier protein (ACP) in Escherichia coli at levels above that of constitutive E. coli ACP does not appear to substantially alter bacterial growth or fatty acid metabolism. The plant ACP expressed in E. coli contains pantetheine and approximately 50% is present in vivo as acyl-ACP. We have purified and characterized the recombinant spinach ACP-I. NH2-terminal amino acid sequencing indicated identity to authentic spinach ACP-I, and there was no evidence for terminal methionine or formylmethionine. Recombinant ACP-I was found to completely cross-react immunologically with polyclonal antibody raised to spinach ACP-I. Recombinant ACP-I was a poor substrate for E. coli fatty acid synthesis. In contrast, Brassica napus fatty acid synthetase gave similar reaction rates with both recombinant and E. coli ACP. Similarly, malonyl-coenzyme A:acyl carrier protein transacylase isolated from E. coli was only poorly able to utilize the recombinant ACP-I while the same enzyme from B. napus reacted equally well with either E. coli ACP or recombinant ACP-I. E. coli acyl-ACP synthetase showed a higher reaction rate for recombinant ACP-I than for E. coli ACP. Expression of spinach ACP-I in E. coli provides, for the first time, plant ACP in large quantities and should aid in both structural analysis of this protein and in investigations of the many ACP-dependent reactions of plant lipid metabolism.     

Refinement of the NMR structures for acyl carrier protein with scalar coupling data

Structure determination of small proteins using NMR data is most commonly pursued by combining NOE derived distance constraints with inherent constraints based on chemical bonding. Ideally, one would make use of a variety of experimental observations, not just distance constraints. Here, coupling constant constraints have been added to molecular mechanics and molecular dynamics protocols for structure determination in the form of a psuedoenergy function that is minimized in a search for an optimum molecular conformation. Application is made to refinement of a structure for a 77 amino acid protein involved in fatty acid synthesis, Escherichia coli acyl carrier protein (ACP). 54 3JHN alpha coupling constants, 12 coupling constants for stereospecifically assigned side chain protons, and 450 NOE distance constraints were used to calculate the 3-D structure of ACP. A three-step protocol for a molecular dynamics calculation is described, in analogy to the protocol previously used in molecular mechanics calculations. The structures calculated with the molecular mechanics approach and the molecular dynamics approach using a rigid model for the protein show similar molecular energies and similar agreement with experimental distance and coupling constant constraints. The molecular dynamics approach shows some advantage in overcoming local minimum problems, but only when a two-state averaging model for the protein was used, did molecular energies drop significantly.     

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.     

Solution structures of the acyl carrier protein domain from the highly reducing type I iterative polyketide synthase CalE8

Biosynthesis of the enediyne natural product calicheamicins γ(1) (I) in Micromonospora echinospora ssp. calichensis is initiated by the iterative polyketide synthase (PKS) CalE8. Recent studies showed that CalE8 produces highly conjugated polyenes as potential biosynthetic intermediates and thus belongs to a family of highly-reducing (HR) type I iterative PKSs. We have determined the NMR structure of the ACP domain (meACP) of CalE8, which represents the first structure of a HR type I iterative PKS ACP domain. Featured by a distinct hydrophobic patch and a glutamate-residue rich acidic patch, meACP adopts a twisted three-helix bundle structure rather than the canonical four-helix bundle structure. The so-called 'recognition helix' (α2) of meACP is less negatively charged than the typical type II ACPs. Although loop-2 exhibits greater conformational mobility than other regions of the protein with a missing short helix that can be observed in most ACPs, two bulky non-polar residues (Met(992), Phe(996)) from loop-2 packed against the hydrophobic protein core seem to restrict large movement of the loop and impede the opening of the hydrophobic pocket for sequestering the acyl chains. NMR studies of the hydroxybutyryl- and octanoyl-meACP confirm that meACP is unable to sequester the hydrophobic chains in a well-defined central cavity. Instead, meACP seems to interact with the octanoyl tail through a distinct hydrophobic patch without involving large conformational change of loop-2. NMR titration study of the interaction between meACP and the cognate thioesterase partner CalE7 further suggests that their interaction is likely through the binding of CalE7 to the meACP-tethered polyene moiety rather than direct specific protein-protein interaction.     

Solution structure and dynamics of oxytetracycline polyketide synthase acyl carrier protein from Streptomyces rimosus

Type II polyketide synthases (PKSs) utilize a dedicated and essential acyl carrier protein (ACP) in the biosynthesis of a specific polyketide product. As part of our ongoing studies into the mechanisms and control of polyketide biosynthesis, we report the second structure of a polyketide synthase ACP. In this work, multidimensional, heteronuclear NMR was employed to investigate the structure and dynamics of the ACP involved in the biosynthesis of the commonly prescribed polyketide antibiotic, oxytetracycline (otc). An ensemble of 28 structures of the 95 amino acid otc ACP (9916Da) was computed by simulated annealing with the inclusion of 1132 experimental restraints. Atomic RMSDs about the mean structure for all 28 models is 0.66 A for backbone atoms, 1.15 A for all heavy atoms (both values calculated for the folded part of the protein (residues 3-80)), and 0.41 A for backbone atoms within secondary structure. Otc ACP adopts the typical right-handed, four-helix fold of currently known ACPs but with the addition of a 13-residue flexible C-terminus. A comparison of the global folds of all structurally characterized ACPs is described, illustrating that PKS ACPs show clear differences as well as similarities to FAS ACPs. (15)N relaxation experiments for the protein backbone also reveal that the long loop between helices I and II is flexible and helix II, a proposed site of protein-protein interactions, shows conformational exchange. The helices of the ACP form a rigid scaffold for the protein, but these are interspersed with an unusual proportion of flexible linker regions.     

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.     

Isolation of a cDNA clone for the acyl carrier protein-I of spinach

A 715 base pair cDNA clone coding for an acyl carrier protein (ACP) in spinach leaves has been isolated and characterized. The amino acid sequence indicated by the cDNA sequence closely matches the amino acid sequence of the ACP-I isoform. The presence of polyadenylation and DNA sequence coding for a precursor protein with a putative transit peptide, and the absence of hybridization between the cloned DNA and isolated spinach plastid DNA collectively show that the ACP-I gene is nuclear-encoded. The ACP-I cloned DNA did not cross-hybridize with mRNA from spinach tissues in which ACP-II has been found. Cross-hybridization with mRNA from tissues of Brassica campestris was either weak or undetectable. The cloning of an ACP-I gene represents an initial step in the molecular dissection of fatty acid synthetase in plants.     

Solution structure and backbone dynamics of the holo form of the frenolicin acyl carrier protein

During polyketide biosynthesis, acyl carrier proteins (ACPs) perform the central role of transferring polyketide intermediates between active sites of polyketide synthase. The 4'-phosphopantetheine prosthetic group of a holo-ACP is a long and flexible arm that can reach into different active sites and provide a terminal sulfhydryl group for the attachment of acyl groups through a thioester linkage. We have determined the solution structure and characterized backbone dynamics of the holo form of the frenolicin acyl carrier protein (fren holo-ACP) by nuclear magnetic resonance (NMR). Unambiguous assignments were made for 433 hydrogen atoms, 333 carbon atoms, and 84 nitrogen atoms, representing a total of 94.6% of the assignable atoms in this protein. From 879 meaningful NOEs and 45 angle constraints, a family of 24 structures has been calculated. The solution structure is composed of three major alpha-helices packed in a bundle with three additional short helices in intervening loops; one of the short helices slowly exchanges between two conformations. Superposition of the major helical regions on the mean structure yields average atomic rmsd values of 0.49 +/- 0.09 and 0.91 +/- 0.08 A for backbone and non-hydrogen atoms, respectively. Although the three-helix bundle fold is conserved among acyl carrier proteins involved in fatty acid synthases and polyketide synthases, a detailed comparison revealed that ACPs from polyketide biosynthetic pathways are more related to each other in tertiary fold than to their homologues from fatty acid biosynthetic pathways. Comparison of the free form of ACPs (NMR structures of fren ACP and the Bacillus subtilis ACP) with the substrate-bound form of ACP (crystal structure of butyryl-ACP from Escherichia coli) suggests that conformational exchange plays a role in substrate binding.     

Site-directed mutagenesis of the spinach acyl carrier protein-I prosthetic group attachment site

Site-directed mutagenesis was used to change the phosphopantetheine attachment site (Ser38) of spinach acyl carrier protein I (ACP-I) from a serine to a threonine or cysteine residue. 1. Although the native ACP-I is fully phosphopantethenylated when expressed in Escherichia coli, the TH-ACP-I and CY-ACP-I mutants were found to be completely devoid of the phosphopantetheine group. Therefore, the E. coli holoACP synthase requires serine for in vivo phosphopantetheine addition to spinach ACP-I. 2. Spinach holoACP synthase was completely inactive in vitro with either the TH-ACP-I or CY-ACP-I mutants. In addition, TH-ACP-I and CY-ACP-I were strong inhibitors of spinach holoACP synthase. 3. The mutant ACPs were weak or ineffective as inhibitors of spinach fatty acid synthesis and spinach oleoyl-ACP hydrolase. 4. Compared to holoACP-I, the mutant apoACP-I analogs had: (a) altered mobility in SDS and native gel electrophoresis, (b) altered binding to anti-(spinach ACP-I) antibodies and (c) altered isoelectric points. The combined physical, immunological and enzyme inhibition data indicate that attachment of the phosphopantheine prosthetic group alters ACP conformation.     

Expression of an active spinach acyl carrier protein-I/protein-A gene fusion

A synthetic gene encoding spinach acyl carrier protein I (ACP-I) was fused to a gene encoding the Fc-binding portion of staphylococcal protein A. This gene fusion, under the control of the P_R promoter, was expressed at high levels in Escherichia coli producing a 42 kDa fusion protein. This fusion protein was phosphopantethenylated in E. coli. In vitro the ACP portion of the fusion protein was able to participate in acyl ACP synthetase reactions, plant malonyl-CoA:ACP transacylase (MCT) reactions, and plant fatty acid synthetase (FAS) reactions. Inhibitory effects of high ACP concentrations on in vitro plant FAS were observed with the unfused ACP-1 but not with the fusion protein. As with unfused ACP-I, the fusion protein was a poor substrate for E. coli FAS reactions. When injected into rabbits, the fusion protein was also able to generate antiserum to spinach ACP-I.     

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.