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.     

Acylation of plant acyl carrier proteins by acyl-acyl carrier protein synthetase from Escherichia coli

The acyl-acyl carrier protein synthetase from Escherichia coli has been examined for its ability to specifically acylate acyl carrier protein (ACP) from higher plants in order to develop an assay for plant ACP, and to prepare labeled acyl-ACP of plant origin. It was found that the E. coli enzyme was able to acylate ACP from spinach, soybean, avocado, corn, and several other plants. The acylation was very specific because, in crude extracts of spinach leaves where ACP represented approximately 0.1% of the total soluble protein, ACP was shown to be the only protein acylated. In contrast to other E. coli enzymes that display 2- to 10-fold lower rates with plant versus bacterial ACP, the kinetic constants (K_m and V_max) for acyl-ACP synthetase were found to be essentially identical for spinach and E. coli ACP when acylated with palmitic acid. Palmitic, myristic, lauric, stearic, and oleic acid could all be esterified to both spinach and E. coli ACP with similar specificity. Procedures are described that allow the assay of ACP in plant extracts at the nanogram level.     

Structural comparison of acyl carrier protein in acylated and sulfhydryl forms by two-dimensional 1H NMR spectroscopy

Sequence-specific assignments of 1H NMR resonances are obtained for the backbone protons of Escherichia coli acyl carrier protein, acylated with an eight-carbon chain covalently attached to the prosthetic group thiol (octanoyl-ACP). Comparison of 1H-1H sequential connectivities in the NOESY spectra of octanoyl-ACP and the unacylated protein (ACPSH) indicates that secondary structure is largely conserved on acylation. Changes in resonance positions observed for certain groups of residues are interpreted in terms of a model that describes the spatial reorientation of secondary structural elements in the protein resulting from introduction of the acyl chain.     

Localization of acyl carrier protein in Escherichia coli.

Acyl carrier protein was localized by immunoelectron microscopy in the cytoplasm of Escherichia coli. These data are inconsistent with the previous report of an association between acyl carrier protein and the inner membrane (H. Van den Bosch, J.R. Williamson, and P.R. Vagelos, Nature [London] 228:338-341, 1970). Moreover, bacterial membranes did not bind a significant amount of acyl carrier protein or its thioesters in vitro. A thioesterase activity specific for long-chain acyl-acyl carrier protein was associated with the inner membrane.     

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.     

Preliminary X-ray diffraction studies of acyl carrier protein from Escherichia coli

Crystals of the acyl carrier protein of Escherichia coli have been grown and analyzed by X-ray diffraction. The crystals grow in space group C2 with unit cell dimensions a = 46.8 A, b = 52.1 A, c = 47.3 A and beta = 93.2 degrees. An isomorphous derivative, HgCl2, has been identified and characterized.     

Chemical and posttranslational modification of Escherichia coli acyl carrier protein for preparation of dansyl-acyl carrier proteins

Escherichia coli acyl carrier protein (ACP) contains a single tyrosine residue at position 71. The combined o-nitration of apo-ACP Y71 by tetranitromethane and reduction to 3-aminotyrosyl-apo-ACP were performed to introduce a specific site for attachment of a dansyl fluorescent label. Conditions for purification and characterization of dansylaminotyrosyl-apo-ACP are reported. Dansylaminotyrosyl-apo-ACP was enzymatically phosphopantetheinylated and acylated in vitro with an overall approximately 30% yield of purified stearoyl-dansylaminotyrosyl-ACP starting from unmodified apo-ACP. The steady-state kinetic parameters k(cat) = 22 min(-1) and K(M) = 2.7 microM were determined for reaction of stearoyl-dansylaminotyrosyl-ACP with stearoyl-ACP Delta(9)-desaturase. These results show that dansylaminotyrosyl-ACP will function well for studying binding interactions with the Delta(9)-desaturase and suggest similar possibilities for other ACP-dependent enzymes. The efficient in vivo phosphopantetheinylation of E. coli apo-ACP by coexpression with holo-ACP synthase in E. coli BL21(DE3) using fructose as the carbon source is also reported.     

Photoaffinity cross-linking of acyl carrier protein to Escherichia coli membranes.

Acyl Carrier Protein (ACP) is a small acidic protein which interacts with the various enzymes implicated in the biosynthesis of fatty acids in E. coli. It also interacts with the inner membrane proteins implicated in the biosynthesis of phospholipids. Samples of radioactive ACP were prepared with high specific activities and bearing photoactivable aryl azide derivatives. Two photoactivable reagents were used: para azido phenacyl bromide (pAPA) which reacts with the SH of the ACP prosthetic group and the N-hydroxysuccinimide ester of 4-azido salicylic acid (NHS-ASA) which reacts with the amino groups of the protein. Various methods were used to demonstrate that ACP could be cross-linked specifically to an inner membrane protein of E. coli, most probably to the glycerol-3-phosphate acyl transferase (GPAT). This covalent link should provide a powerful tool for further analysis of the structure of GPAT and its role in phospholipid biosynthesis. These photoactivable aryl azide derivatives of ACP could also be very useful for studying the interaction of ACP with the soluble enzymes implicated in fatty acid biosynthesis.     

Membrane-binding sites for acyl carrier protein in Escherichia coli

We report that membrane vesicles of Escherichia coli contain protein-binding sites for acyl carrier protein. Scatchard analysis of the binding indicates a dissociation constant around 0.35 micrometers and a maximum number of protein-binding sites around 50 pmol per mg of membrane protein. Binding is on the inner membrane while the outer membrane is devoid of binding sites. These results are consistent with the fact that some acyl carrier protein-dependent enzymes implicated in phospholipid- and membrane-derived oligosaccharide biosynthesis are localized in the cytoplasmic membrane.     

The enigmatic acyl carrier protein phosphodiesterase of Escherichia coli: genetic and enzymological characterization

The acyl carrier proteins (ACPs) of fatty acid synthesis are functional only when modified by attachment of the prosthetic group, 4'-phosphopantetheine (4'-PP), which is transferred from CoA to the hydroxyl group of a specific serine residue. Almost 40 years ago Vagelos and Larrabee reported an enzyme from Escherichia coli that removed the prosthetic group. We report that this enzyme, called ACP hydrolyase or ACP phosphodiesterase, is encoded by a gene (yajB) of previously unknown function that we have renamed acpH. A mutant E. coli strain having a total deletion of the acpH gene has been constructed that grows normally, showing that phosphodiesterase activity is not essential for growth, although it is required for turnover of the ACP prosthetic group in vivo. ACP phosphodiesterase (AcpH) has been purified to homogeneity for the first time and is a soluble protein that very readily aggregates upon overexpression in vivo or concentration in vitro. The purified enzyme has been shown to cleave acyl-ACP species with acyl chains of 6-16 carbon atoms and is active on some, but not all, non-native ACP species tested. Possible physiological roles for AcpH are discussed.     

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.     

On the structure-function relationship of acyl carrier protein of Escherichia coli.

The conformations of Escherichia coli acyl carrier protein (ACP) and acetylated ACP have been studied as a function of pH and salt concentration by circular dichroism measurements. The results show that the amino groups of ACP in their protonated form are important for maintaining the native conformation of the protein at physiological pH. However, externally added cations (divalent more effectively than monovalent ones) can substitute for the ammonium groups in maintaining the ordered structure pf ACP. It is suggested that both the ammonium groups of ACP and externally added cations reduce the repulsion between carboxylate groups of ACP and thereby prevent the unfolding of the protein. A reduction of the number of negatively charged carboxylate groups by either protonation or chemical modification abolished the requirement for either ammonium groups or other cations. A qualitative agreement between the effect of salt on the conformation and on the biological activity of acetylated ACP has been observed. The single arginine residue of acetylated ACP has been modified by treatment with a trimer of 2,3-butanedione with the resulting derivative of ACP retaining most of its biological activity.