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.     

Domains of Escherichia coli acyl carrier protein important for membrane-derived-oligosaccharide biosynthesis.

Acyl carrier protein participates in a number of biosynthetic pathways in Escherichia coli: fatty acid biosynthesis, phospholipid biosynthesis, lipopolysaccharide biosynthesis, activation of prohemolysin, and membrane-derived oligosaccharide biosynthesis. The first four pathways require the protein's prosthetic group, phosphopantetheine, to assemble an acyl chain or to transfer an acyl group from the thioester linkage to a specific substrate. By contrast, the phosphopantetheine prosthetic group is not required for membrane-derived oligosaccharide biosynthesis, and the function of acyl carrier protein in this biosynthetic scheme is currently unknown. We have combined biochemical and molecular biological approaches to investigate domains of acyl carrier protein that are important for membrane-derived oligosaccharide biosynthesis. Proteolytic removal of the first 6 amino acids from acyl carrier protein or chemical synthesis of a partial peptide encompassing residues 26 to 50 resulted in losses of secondary and tertiary structure and consequent loss of activity in the membrane glucosyltransferase reaction of membrane-derived oligosaccharide biosynthesis. These peptide fragments, however, inhibited the action of intact acyl carrier protein in the enzymatic reaction. This suggests a role for the loop regions of the E. coli acyl carrier protein and the need for at least two regions of the protein for participation in the glucosyltransferase reaction. We have purified acyl carrier protein from eight species of Proteobacteria (including representatives from all four subgroups) and characterized the proteins as active or inhibitory in the membrane glucosyltransferase reaction. The complete or partial amino acid sequences of these acyl carrier proteins were determined. The results of site-directed mutagenesis to change amino acids conserved in active, and altered in inactive, acyl carrier proteins suggest the importance of residues Glu-4, Gln-14, Glu-21, and Asp-51. The first 3 of these residues define a face of acyl carrier protein that includes the beginning of the loop region, residues 16 to 36. Additionally, screening for membrane glucosyltransferase activity in membranes from bacterial species that had acyl carrier proteins that were active with E. coli membranes revealed the presence of glucosyltransferase activity only in the species most closely related to E. coli. Thus, it seems likely that only bacteria from the Proteobacteria subgroup gamma-3 have periplasmic glucans synthesized by the mechanism found in E. coli.     

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.     

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.     

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.     

Amino acid residues of Escherichia coli acyl carrier protein involved in heterologous protein interactions

Acyl carrier protein (ACP) is a small, highly conserved protein with an essential role in a myriad of reactions throughout lipid metabolism in plants and bacteria where it interacts with a remarkable diversity of proteins. The nature of the proper recognition and precise alignment between the protein moieties of ACP and its many interactive proteins is not understood. Residues conserved among ACPs from numerous plants and bacteria were considered as possibly being crucial to ACP's function, including protein-protein interaction, and a method of identifying amino acid residue clusters of high hydrophobicity on ACP's surface was used to estimate residues possibly involved in specific ACP-protein interactions. On the basis of this information, single-site mutation analysis of multiple residues, one at a time, of ACP was used to probe the identities of potential contact residues of ACPSH or acyl-ACP involved in specific interactions with selected enzymes. The roles of particular ACP residues were more precisely defined by site-directed fluorescence analyses of various myristoyl-mutant-ACPs upon specific interaction with the Escherichia coli hemolysin-activating acyltransferase, HlyC. This was done by selectively labeling each mutated site, one at a time, with an environmentally sensitive fluoroprobe and observing its fluorescence behavior in the absence and presence of HlyC. Consequently, a picture of the portion of ACP involved in selected macromolecular interaction has emerged.     

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.     

Isolation and properties of acyl carrier protein phosphodiesterase of Escherichia coli.

The acyl carrier protein (ACP) phosphodiesterase of Escherichia coli catalyzes the hydrolytic cleavage of the 4'-phosphopantetheine residue from ACP, with the generation of apo-ACP (P. R. Vagelos and A. R. Larrabee, J. Biol. Chem. 242:1776-1781, 1967). Although it has been postulated to play a role in the regulation of fatty acid synthesis, presently available evidence makes this unlikely, and its physiological function requires further investigation. We have now purified the enzyme from E. coli more than 3,000-fold and have identified it as a protein of Mr 25,000, as judged from its migration during electrophoresis in gels containing sodium dodecyl sulfate. The enzyme has remarkable thermostability, being protected against irreversible inactivation at 90 degrees C by the presence of sodium dodecyl sulfate. A partial sequence of the amino terminus of the enzyme is as follows: H2N-Ser-Lys-Val-Leu-Val-Leu-Lys-Ser-?-Ile-Leu-Ala-Gly-Tyr-Ser-. Other properties of the enzyme are also described.     

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.     

The gene encoding Escherichia coli acyl carrier protein lies within a cluster of fatty acid biosynthetic genes.

The gene encoding Escherichia coli acyl carrier protein (ACP) has been isolated and sequenced. The ACP gene (called acpP) was located on the genetic map between fabF and fabD which encode two fatty acid biosynthetic enzymes, 3-ketoacyl-ACP synthase II and malonyl CoA-ACP transacylase, respectively. An open reading frame between acpP and fabD encodes a 26.5-kDa protein that has significant sequence identity (greater than 40%) with two acetoacetyl-CoA reductases and thus is believed to encode a 3-ketoacyl-ACP reductase. This gene (called fabG) is cotranscribed with acpP. Thus, the gene encoding ACP, the key carrier protein of fatty acid synthesis, is located within a cluster of fatty acid biosynthetic genes.     

Resin-based investigation of acyl carrier protein interaction networks in Escherichia coli

Protein-protein interactions play an integral role in metabolic regulation. Elucidation of these networks is complicated by the changing identity of the proteins themselves. Here we demonstrate a resin-based technique that leverages the unique tools for acyl carrier protein (ACP) modification with non-hydrolyzable linkages. ACPs from Escherichia coli and Shewanella oneidensis MR-1 are bound to Affigel-15 with varying acyl groups attached and introduced to proteomic samples. Isolation of these binding partners is followed by MudPIT analysis to identify each interactome with the variable of ACP-tethered substrates. These techniques allow for investigation of protein interaction networks with the changing identity of a given protein target.     

Beta-methylthio-aspartic acid: identification of a novel posttranslational modification in ribosomal protein S12 from Escherichia coli.

Utilizing microscale chemical derivatization reactions and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, we have identified a novel posttranslational modification of aspartic acid, beta-methylthio-aspartic acid. The modified residue is located at position 88 in ribosomal protein S12 from Escherichia coli, a phylogenetically conserved protein that has been implicated in maintaining translational accuracy of the ribosome.     

The Escherichia coli alkylation response protein AidB is a redox partner of flavodoxin and binds RNA and acyl carrier protein

Microorganisms are exposed to a wide variety of exogenous and endogenous chemical agents that alkylate DNA. Escherichia coli cells exhibit an adaptive response that recognizes and repairs alkylated DNA lesions using Ada, AlkA, and AlkB enzymes. Another alkylation response protein, the DNA-binding flavoprotein AidB, was proposed to repair DNA or protect it from chemical alkylating agents, but direct evidence for its role is lacking. Here, AidB was shown to form tight complexes with both flavodoxin and acyl carrier protein. In addition, electron transfer between 1-electron and 2-electron reduced flavodoxin to oxidized AidB was observed, although with very small rate constants. AidB was found to bind to RNA, raising the prospect that the protein may have a role in protection of RNA from chemical alkylation. Finally, the reagent N-methyl-N′-nitro-N-nitrosoguanidine was eliminated as a direct substrate of the enzyme.