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A knowledge of the structures of acyl chain loaded species of the acyl carrier protein (ACP) as used in fatty acid biosynthesis and a range of other metabolic events, is essential for a full understanding of the molecular recognition at the heart of these processes. To date the only crystal structure of an acylated species of ACP is that of a butyryl derivative of Escherichia coli ACP. We have now determined the structures of a family of acylated E. coli ACPs of varying acyl chain length. The acyl moiety is attached via a thioester bond to a phosphopantetheine linker that is in turn bound to a serine residue in ACP. The growing acyl chain can be accommodated within a central cavity in the ACP for transport during the elongation stages of lipid synthesis through changes in the conformation of a four alpha-helix bundle. The results not only clarify the means by which a substrate of varying size and complexity is transported in the cell but also suggest a mechanism by which interacting enzymes can recognize the loaded ACP through recognition of surface features including the conformation of the phosphopantetheine linker.

Acyl carrier protein (ACP) is an essential cofactor in biosynthesis of fatty acids and many other reactions that require acyl transfer steps. We have determined the first crystal structures of an acylated form of ACP from E. coli, that of butyryl-ACP. Our analysis of the molecular surface of ACP reveals a plastic hydrophobic cavity in the vicinity of the phosphopantethylated Ser36 residue that is expanded and occupied by the butyryl and beta-mercaptoethylamine moieties of the acylated 4'-phosphopantetheine group in one of our crystal forms. In the other form, the cavity is contracted, and we propose that the protein has adopted the conformation after delivery of substrate into the active site of a partner enzyme.

Acyl carrier proteins carry the lipid substrate to the enzymes of the fatty acid synthase system. Crystals of Escherichia coli acyl carrier protein to which a butyryl group has been attached via a thioester link to the phosphopantetheine prosthetic arm have been obtained by the hanging-drop vapour-diffusion method. These crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 27.6, b = 41.6, c = 63.7 A. The asymmetric unit appears to contain one subunit, corresponding to a packing density of 2.1 A(3) Da(-1). Crystals of the selenomethionine-substituted (SeMet) protein were obtained using different conditions and belong to space group P6(3), with unit-cell parameters a = b = 63.4, c = 37.0 A, alpha = beta = 90, gamma = 120 degrees and with a monomer in the asymmetric unit (V(M) = 2.5 A(3) Da(-1)). Crystals of a SeMet butyryl-ACP I62M variant were obtained using the conditions for the native protein. Like the native crystals, these belong to space group P2(1)2(1)2(1) and have unit-cell parameters a = 27.3, b = 41.9, c = 64.5 A. A data set suitable for MAD phasing was collected from the crystals of the I62M variant to 1.8 A resolution on the ESRF beamline ID14-4.

Abstract: The predicted mature portion of a putative 3-hydroxyacyl-ACP dehydratase (DH) from Arabidopsis was linked to an N-terminal poly-histidine-tag and the fusion protein expressed in Escherichia coli. Soluble dehydratase was present on induction at 25°C and pure dehydratase eluted from a nickel-affinity column in 0.2–0.5M imidazole. High concentrations of imidazole were necessary to retain enzyme solubility. The dehydratase reaction is reversible and 3-hydroxybutyryl- and 2-butenoyl-ACP substrates were prepared from E. coli apo-ACP. Analysis of these suggested contamination of apo-ACP with dehydratase and an additional reverse-phase chromatographic step was required during acyl carrier protein (ACP) preparation. Activity of purified dehydratase was demonstrated by mass spectrometry using 2-butenoyl-ACP, providing the first functional experimental evidence for plant DH gene sequences. [Copyright &y& Elsevier]