Acyl carrier protein interacts with melittin

Acyl carrier protein (ACP) from Escherichia coli has been shown to form complexes with melittin, a cationic peptide from bee venom. ACP is a small (Mr 8847), acidic, Ca2(+)-binding protein, which possesses some characteristics resembling those of regulatory Ca2(+)-binding proteins including interaction with melittin. Complexing between melittin and ACP which occurred both in the presence and absence of Ca2+ was evident by chemical cross-linking the two peptides, fluorescence changes (including anisotropy measurements), and inhibition by melittin of the activity of a nonaggregated fatty acid synthetase from Euglena. Also, anti-Apis mellifera antibodies which contained antibodies against melittin specifically inhibited the same enzyme system activity relative to non-immune IgG.     

Novel E. coli β-ketoacyl-acyl carrier protein synthase III inhibitors as targeted antibiotics

β-Ketoacyl-acyl carrier protein synthase (KAS) III is a condensing enzyme that initiates fatty acid biosynthesis in most bacteria. We determined three pharmacophore maps from receptor-oriented pharmacophore-based in silico screening of the X-ray structure of Escherichia coli KAS III (ecKAS III) and choose 16 compounds as candidate ecKAS III inhibitors. Binding inhibitors were characterized using saturation-transfer difference NMR spectroscopy (STD-NMR), and binding constants were determined with fluorescence quenching experiments. Based on the results, we propose that the antimicrobial compound, 4-cyclohexyliminomethyl-benzene-1,3-diol (YKAs3003), is a potent inhibitor of pathogenic KAS III, displaying minimal inhibitory concentration (MIC) values in the range 128–256 μg/mL against various bacteria.     

Biochemical characterization of 3-ketoacyl-acyl carrier protein synthase II from leek epidermis

In order to define its possible involvement in production of stearic acid for wax biosynthesis, the presence of 3-ketoacyl acyl synthase II (KAS II) activity was investigated in different tissues of leek (Allium porrum L.) leaves. KAS II activity was identified in sheath and lamina epidermis, as well as in underlying parenchyma. In all three tissues, activity was inhibited by 50% on addition of 100 microM cerulenin, and showed an absolute requirement for acyl-ACP substrates. More interestingly, the different tissues did not display similar KAS II substrate specificities. Parenchyma and lamina epidermis tissues presented typical KAS II activities, since C(18:0)-ACP was the exclusive product. In contrast, in sheath epidermis, KAS II activity resulted in the synthesis of acyl-chains up to 22 carbons in length, suggesting the existence in this tissue of an unusual KAS II. This activity was sufficient to elongate all of the palmitoyl-ACP produced by the fatty acid synthase, suggesting that C(18:0) is the substrate of the microsomal elongases involved in wax biosynthesis.     

Acyl carrier protein: structure-function relationships in a conserved multifunctional protein family.

Acyl carrier protein (ACP) is a universal and highly conserved carrier of acyl intermediates during fatty acid synthesis. In yeast and mammals, ACP exists as a separate domain within a large multifunctional fatty acid synthase polyprotein (type I FAS), whereas it is a small monomeric protein in bacteria and plastids (type II FAS). Bacterial ACPs are also acyl donors for synthesis of a variety of products, including endotoxin and acylated homoserine lactones involved in quorum sensing; the distinct and essential nature of these processes in growth and pathogenesis make ACP-dependent enzymes attractive antimicrobial drug targets. Additionally, ACP homologues are key components in the production of secondary metabolites such as polyketides and nonribosomal peptides. Many ACPs exhibit characteristic structural features of natively unfolded proteins in vitro, with a dynamic and flexible conformation dominated by 3 parallel alpha helices that enclose the thioester-linked acyl group attached to a phosphopantetheine prosthetic group. ACP conformation may also be influenced by divalent cations and interaction with partner enzymes through its "recognition" helix II, properties that are key to its ability to alternately sequester acyl groups and deliver them to the active sites of ACP-dependent enzymes. This review highlights recent progress in defining how the structural features of ACP are related to its multiple carrier roles in fatty acid metabolism.     

Acyl carrier protein structural classification and normal mode analysis

All acyl carrier protein primary and tertiary structures were gathered into the ThYme database. They are classified into 16 families by amino acid sequence similarity, with members of the different families having sequences with statistically highly significant differences. These classifications are supported by tertiary structure superposition analysis. Tertiary structures from a number of families are very similar, suggesting that these families may come from a single distant ancestor. Normal vibrational mode analysis was conducted on experimentally determined freestanding structures, showing greater fluctuations at chain termini and loops than in most helices. Their modes overlap more so within families than between different families. The tertiary structures of three acyl carrier protein families that lacked any known structures were predicted as well.     

Beta-Ketoacyl-acyl carrier protein synthetase. Characterization of the acyl-enzyme intermediate.

Beta-Ketoacyl-acyl carrier protein (ACP) synthetase catalyzes the condensation reaction of fatty acid synthesis in Escherichia coli. The homogeneous enzyme reacts with hexanoyl-CoA to form hexanoyl-enzyme which was isolated and characterized. Hexanoyl-enzyme contains 2 mol of hexanoate/mol of enzyme (molecular weight 66,000); it is liable at alkaline pH, and it reacts with neutral hydroxylamine to form hexanoyl hydroxamic acid. Hexanoate was cleaved from the enzyme when hexanoyl-enzyme was subjected to performic acid oxidation. These properties indicate that hexanoyl-enzyme is a thioester. Studies of the circular dichroism spectra of fully acylated and nonacylated forms of the enzyme indicated that the secondary structure of the enzyme is relatively unperturbed by the presence of the hexanoyl groups. An alpha helical content of 65% was estimated for the enzyme from the circular dichroism spectrum. Hexanoyl-enzyme is active in both partial reactions that comprise the beta-ketoacyl-ACP synthetase reaction; it reacts with ACP to form hexanoyl-ACP and with malonyl-ACP to form beta-ketooctanoyl-ACP. Although the hexanoate of hexanoyl-enzyme is transferred very rapidly to ACP, the physiological acceptor in this reaction, it is also transferred very slowly to CoA, dithiothreitol, and 2-mercaptoethanol, indicating that the enzyme can react nonspecifically with a number of unrelated mercaptans.