Isolation and characterization of Escherichia coli K-12 mutants lacking both 2-acyl-glycerophosphoethanolamine acyltransferase and acyl-acyl carrier protein synthetase activity.

2-Acyl-glycerophosphoethanolamine (2-acyl-GPE) acyltransferase and acyl-acyl carrier protein (acyl-ACP) synthetase are thought to be dual catalytic activities of a single inner membrane enzyme. A filter disc replica print method for the detection of acyl-ACP synthetase activity by colony fluorography was used to screen a mutagenized population of cells for acyl-ACP synthetase mutants (aas). All aas mutants lacked both acyl-ACP synthetase and 2-acyl-GPE acyltransferase activities in vitro. There was no detectable acyl-CoA-independent incorporation of exogenous fatty acids into phosphatidylethanolamine or the major outer membrane lipoprotein in aas mutants. Exogenous lysophospholipid uptake and acylation was also lacking in aas mutants. Lipoprotein acylation by phospholipids synthesized by the de novo biosynthetic pathway was not affected in aas mutants showing that this gene product was not directly involved in lipoprotein biogenesis. The aas mutants had an altered membrane phospholipid composition and accumulated both 2-acyl-GPE and acylphosphatidylglycerol. Acylphosphatidylglycerol accumulation was due to the transacylase activity of lysophospholipase L2 (the pldB gene product) since aas pldB double mutants accumulated 2-acyl-GPE, but not acylphosphatidylglycerol. The aas allele was mapped to 61 min of the Escherichia coli chromosome, and the deduced gene order in this region was thyA-aas-lysA. The biochemical, physiological, and genetic analyses of aas mutants support the conclusion that 2-acyl-GPE acyltransferase and acyl-ACP synthetase are two activities of the same protein and confirm that this enzyme system participates in membrane phospholipid turnover and governs the acyl-CoA independent incorporation of exogenous fatty acids and lysophospholipids into the membrane.     

Purification and characterization of fatty acyl-acyl carrier protein synthetase from Vibrio harveyi.

A Vibrio harveyi enzyme which catalyzes the ATP-dependent ligation of fatty acids to acyl carrier protein (ACP) has been purified 6,000-fold to apparent homogeneity by anion-exchange, gel filtration, and ACP-Sepharose affinity chromatography. Purified acyl-ACP synthetase migrated as a single 62-kDa band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and as an 80-kDa protein by gel filtration under reducing conditions. Activity of the purified enzyme was lost within hours in the absence of glycerol and low concentrations of Triton X-100. Acyl-ACP synthetase exhibited Kms for myristic acid, ACP, and ATP of 7 microM, 18 microM, and 0.3 mM, respectively. The enzyme was specific for adenine-containing nucleotides, and AMP was the product of the reaction. No covalent acyl-enzyme intermediate was observed. Enzyme activity was stimulated up to 50% by iodoacetamide but inhibited > 80% by N-ethylmaleimide: inhibition by the latter was prevented by ATP and ACP but not myristic acid. Dithiothreitol and sulfhydryl-directed reagents also influenced enzyme size, activity, and elution pattern on anion-exchange resins. The function of acyl-ACP synthetase has not been established, but it may be related to the capacity of V. harveyi to elongate exogenous fatty acids by an ACP-dependent mechanism.     

A soluble fatty acyl-acyl carrier protein synthetase from the bioluminescent bacterium Vibrio harveyi

An enzyme catalyzing the ligation of long chain fatty acids to bacterial acyl carrier protein (ACP) has been detected and partially characterized in cell extracts of the bioluminescent bacterium Vibrio harveyi. Acyl-ACP synthetase activity (optimal pH 7.5-8.0) required millimolar concentrations of ATP and Mg2+ and was slightly activated by Ca2+, but was inhibited at high ionic strength and by Triton X-100. ACP from either Escherichia coli (apparent Km = 20 microM) or V. harveyi was used as a substrate. Of the [14C]fatty acids tested as substrates (8-18 carbons), a preference for fatty acids less than or equal to 14 carbons in length was observed. Vibrio harveyi acyl-ACP synthetase appears to be a soluble hydrophilic enzyme on the basis of subcellular fractionation and Triton X-114 phase partition assay. The enzyme was not coinduced with luciferase activity or light emission in vivo during the late exponential growth phase in liquid culture. Acyl-ACP synthetase activity was also detected in extracts from the luminescent bacterium Vibrio fischeri, but not Photobacterium phosphoreum. The cytosolic nature and enzymatic properties of V. harveyi acyl-ACP synthetase indicate that it may have a different physiological role than the membrane-bound activity of E. coli, which has been implicated in phosphatidylethanolamine turnover. Acyl-ACP synthetase activity in V. harveyi could be involved in the intracellular activation and elongation of exogenous fatty acids that occurs in this species or in the reactivation of free myristic acid generated by luciferase.     

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.     

Preparative enzymatic synthesis and hydrophobic chromatography of acyl-acyl carrier protein.

We have used purified preparations of acyl-acyl carrier protein synthetase to prepare pure, native acyl-acyl carrier proteins (acyl-ACP) ranging in chain lengths from C10:0 to C delta 9 18:1. Factors affecting yield are explored and reaction conditions are presented that yield 0.8 to 0.9 mg of C16:0-ACP/ml of reaction mix. Ohter acyl groups, such as C10:0 and C delta 9 18:1 are poorer substrates and gave correspondingly lower yields. Acyl-Acp synthetase may be recovered from the reaction mixture using blue-Sepharose CL-6B and recycled. ACP and acyl-ACP are separated by hydrophobic chromatography on octyl-Sepharose CL-4B. Mixtures of acyl-ACPs could be resolved according to acyl chain length using octyl-Sepharose CL-4B columns eluted with a 2-propanol gradient. The high resolution obtained using 2-propanol gradients to separate acyl-ACP species suggests that similar techniques would be applicable to the chromatography of protein mixtures on hydrophobic supports.     

Purification and characterization of beta-ketoacyl-ACP synthetase I from Spinacia oleracea leaves

beta-Ketoacyl-acyl carrier protein (ACP) synthetase I was purified 180-fold from crude extracts of spinach leaves. The purified preparation was completely free from other component enzymes of the de novo fatty acid synthetase (FAS) system. Its molecular weight was estimated to be 56,000 by gel filtration. The apparent Km value for malonyl-CoA in the presence of ACP and malonyl-CoA:ACP transacylase was 4 microM. Purified synthetase I was highly active with acyl-ACP having chain lengths from C2 to C14, with hexanoyl-ACP being the most effective substrate, but palmitoyl-ACP was far less effective and stearoyl-ACP almost inactive. The antibiotic, cerulenin, strongly inhibited synthetase I activity. The inhibition by cerulenin was protected by prior incubation with hexanoyl-ACP, decanoyl-ACP, and myristoyl-ACP. The synthetase was inhibited by 1 mM p-CMB and 5 mM NEM, but not by 1 mM arsenite.     

Lysophospholipid flipping across the Escherichia coli inner membrane catalyzed by a transporter (LplT) belonging to the major facilitator superfamily

The transfer of phospholipids across membrane bilayers is protein-mediated, and most of the established transporters catalyze the energy-dependent efflux of phospholipids from cells. This work identifies and characterizes a lysophospholipid transporter gene (lplT, formally ygeD) in Escherichia coli that is an integral component in the 2-acylglycerophosphoethanolamine (2-acyl-GPE) metabolic cycle for membrane protein acylation. The lplT gene is adjacent to and in the same operon as the aas gene, which encodes the bifunctional enzyme 2-acyl-GPE acyltransferase/acyl-acyl carrier protein synthetase. In some bacteria, acyltransferase/acyl-ACP synthetase (Aas) and LplT homologues are fused in a single polypeptide chain. 2-Acyl-GPE transport to the inside of the cell was assessed by measuring the Aas-dependent formation of phosphatidylethanolamine. The Aas-dependent incorporation of [3H]palmitate into phosphatidylethanolamine was significantly diminished in deltalplT mutants, and the LplT-Aas transport/acylation activity was independent of the proton motive force. The deltalplT mutants accumulated acyl-GPE in vivo and had a diminished capacity to transport exogenous 2-acylglycerophosphocholine into the cell. Spheroplasts prepared from wild-type E. coli transported and acylated fluorescent 2-acyl-GPE with an apparent K(d) of 7.5 microM, whereas this high-affinity process was absent in deltalplT mutants. Thus, LplT catalyzes the transbilayer movement of lysophospholipids and is the first example of a phospholipid flippase that belongs to the major facilitator superfamily.     

Preparation of fatty-acylated derivatives of acyl carrier protein using Vibrio harveyi acyl-ACP synthetase.

A simple two-step purification of Vibrio harveyi fatty acyl-acyl carrier protein (acyl-ACP) synthetase, which is useful for the quantitative preparation and analysis of fatty-acylated derivatives of ACP, is described. Acyl-ACP synthetase can be partially purified from extracts of this bioluminescent bacterium by Cibacron blue chromatography and Sephacryl S-300 gel filtration and is stable for months at -20 degrees C in the presence of glycerol. Incubation of ACP from Escherichia coli with ATP and radiolabeled fatty acids (6 to 16 carbons in length) in the presence of the enzyme resulted in quantitative conversion to biologically active acylated derivatives. The enzyme reaction can be monitored by a filter disk assay to quantitate levels of ACP or by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography to detect ACP in cell extracts. With its broad fatty acid chain length specificity and optimal activity in mild nondenaturing buffers, the soluble V. harveyi acyl-ACP synthetase provides an attractive alternative to current chemical and enzymatic methods of acyl-ACP preparation and analysis.     

Purification and characterization of acyl carrier protein from two cyanobacteria species

The acyl carrier protein (ACP), an essential protein cofactor for fatty acid synthesis, has been isolated from two cyanobacteria: the filamentous, heterocystous, Anabaena variabilis (ATCC 29211) and the unicellular Synechocystis 6803 (ATCC 27184). Both ACPs have been purified to homogeneity utilizing a three-column procedure. Synechocystis 6803 ACP was purified 1800-fold with 67% yield, while A. variabilis ACP was purified 1040-fold with 50% yield. Yields of 13.0 micrograms ACP/g Synechocystis 6803 and 9.0 micrograms ACP/g A. variabilis were achieved. Amino acid analysis indicated that these ACPs were highly charged acidic proteins similar to other known ACPs. Sequence analysis revealed that both cyanobacterial ACPs were highly conserved with both spinach and Escherichia coli ACP at the phosphopantetheine prosthetic group region. Examining the probability of alpha-helix and beta-turn regions in various ACPs, showed that cyanobacterial ACPs were more closely related to E. coli ACP than spinach ACP I. Immunoblot analysis and a competitive binding assay for ACP illustrated that both ACPs bound poorly to spinach ACP I antibody. SDS/PAGE and native PAGE of Synechocystis 6803 ACP and A. variabilis ACP showed that cyanobacteria ACPs co-migrated with E. coli ACP and had relative molecular masses of 18,100 and 17,900 respectively. Both native and urea gel analysis of acyl-ACP products from fatty acid synthase reactions demonstrated that bacterial ACPs and plant ACP gave essentially the same metabolic products when assayed using either bacterial or plant fatty acid synthase. A. variabilis and Synechocystis 6803 ACP could be acylated using E. coli acyl ACP synthetase.     

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.     

Plant holo-(acyl carrier protein) synthase.

1. An improved method was developed for the assay of plant holo-(acyl carrier protein) synthase activity, using Escherichia coli acyl-(acyl carrier protein) synthetase as a coupling enzyme. 2. Holo-(acyl carrier protein) synthase was partially purified from spinach (Spinacia oleracea) leaves by a combination of (NH4)2SO4 fractionation and anion-exchange and gel-permeation chromatography. 3. The partially purified enzyme had a pH optimum of 8.2 and Km values of 2 microM, 72 microM and 3 mM for apo-(acyl carrier protein), CoA and Mg2+ respectively. Synthase activity was inhibited in vitro by the reaction product 3',5'-ADP. 4. Results from the fractionation of spinach leaf and developing castor-oil-seed (Ricinus communis) endosperm cells were consistent with a cytosolic localization of holo-(acyl carrier protein) synthase activity in plant cells.     

A novel, general radioimmunoassay for acyl carrier proteins

A radioimmunoassay (RIA) for acyl carrier proteins (ACP) is described that is based on the competitive binding between [3H]acyl-ACP and unlabeled ACP of the same species. The radiolabeled antigen, [3H]palmitoyl-ACP, is enzymatically synthesized by Escherichia coli acyl-ACP synthetase. Because acyl-ACP synthetase can specifically radiolabel ACP in crude extracts from several plant sources, the use of this enzyme to prepare [3H]acyl-ACP obviates the need for pure preparations of each ACP. Preparation of [3H]acyl-ACP with a specific activity of 15 Ci/mmol allows RIA detection of total ACP in crude plant extracts at the nanogram level. Because antibodies against spinach ACP partially crossreact with ACP from many plant sources, RIAs for other plant species can be constructed using only one preparation of antibody. ACP preparations from safflower, soybean, avocado, corn, and E. coli show a decreasing order of partial immunocross-reactivity with spinach ACP-specific antiserum, as examined by RIA using spinach [3H]palmitoyl-ACP.     

Purification and biochemical characterization of the Mycobacterium tuberculosis beta-ketoacyl-acyl carrier protein synthases KasA and KasB

Mycolic acids are vital components of the Mycobacterium tuberculosis cell wall, and enzymes involved in their formation represent attractive targets for the discovery of novel anti-tuberculosis agents. Biosynthesis of the fatty acyl chains of mycolic acids involves two fatty acid synthetic systems, the multifunctional polypeptide fatty acid synthase I (FASI), which performs de novo fatty acid synthesis, and the dissociated FASII system, which consists of monofunctional enzymes, and acyl carrier protein (ACP) and elongates FASI products to long chain mycolic acid precursors. In this study, we present the initial characterization of purified KasA and KasB, two beta-ketoacyl-ACP synthase (KAS) enzymes of the M. tuberculosis FASII system. KasA and KasB were expressed in E. coli and purified by affinity chromatography. Both enzymes showed activity typical of bacterial KASs, condensing an acyl-ACP with malonyl-ACP. Consistent with the proposed role of FASII in mycolic acid synthesis, analysis of various acyl-ACP substrates indicated KasA and KasB had higher specificity for long chain acyl-ACPs containing at least 16 carbons. Activity of KasA and KasB increased with use of M. tuberculosis AcpM, suggesting that structural differences between AcpM and E. coli ACP may affect their recognition by the enzymes. Both enzymes were sensitive to KAS inhibitors cerulenin and thiolactomycin. These results represent important steps in characterizing KasA and KasB as targets for antimycobacterial drug discovery.     

Nonorganellar acyl carrier protein from oleaginous yeast is a homologue of ribosomal protein P2

Acyl carrier protein (ACP) is responsible for carrying the growing fatty acid chain from one enzyme active site to the next during fatty acid biosynthesis. Here we report the identification, purification, immunocytochemical localization, and cloning of ACP from the oleaginous yeast, Rhodotorula glutinis. The soluble fraction of this organism can synthesize triacylglycerol and is able to accept the acyl group from acyl-ACP for the synthesis. The ACP, cloned from the system, showed a significant similarity with ribosomal protein P2. Expression and characterization of the recombinant protein showed that the ACP was acylated in vitro. The recombinant protein was post-translationally modified, since it was observed in [14C]beta-alanine labeling and matrix-assisted laser desorption mass spectroscopic analysis. Site-directed mutants were generated to identify a serine residue responsible for phosphopantetheinylation and found that mutation of serine 59 to alanine abrogated the fatty acylation ability of the protein. These results demonstrate that a novel modification of ribosomal protein P2 allows it to act as an acyl carrier protein and participate in acylation reactions.     

Functional analysis of an interspecies chimera of acyl carrier proteins indicates a specialized domain for protein recognition

The nodulation protein NodF of Rhizobium shows 25% identity to acyl carrier protein (ACP) from Escherichia coli (encoded by the gene acpP). However, NodF cannot be functionally replaced by AcpP. We have investigated whether NodF is a substrate for various E. coli enzymes which are involved in the synthesis of fatty acids. NodF is a substrate for the addition of the 4′-phosphopantetheine prosthetic group by holo-ACP synthase. The K_m value for NodF is 61?μM, as compared to 2?μM for AcpP. The resulting holo-NodF serves as a substrate for coupling of malonate by malonyl-CoA:ACP transacylase (MCAT) and for coupling of palmitic acid by acyl-ACP synthetase. NodF is not a substrate for β-keto-acyl ACP synthase III (KASIII), which catalyses the initial condensation reaction in fatty acid biosynthesis. A chimeric gene was constructed comprising part of the E.coliacpP gene and part of the nodF gene. Circular dichroism studies of the chimeric AcpP-NodF (residues 1–33 of AcpP fused to amino acids 43–93 of NodF) protein encoded by this gene indicate a similar folding pattern to that of the parental proteins. Enzymatic analysis shows that AcpP-NodF is a substrate for the enzymes holo-ACP synthase, MCAT and acyl-ACP synthetase. Biological complementation studies show that the chimeric AcpP-NodF gene is able functionally to replace NodF in the root nodulation process in Vicia sativa. We therefore conclude that NodF is a specialized acyl carrier protein whose specific features are encoded in the C-terminal region of the protein. The ability to exchange domains between such distantly related proteins without affecting conformation opens exciting possibilities for further mapping of the functional domains of acyl carrier proteins (i. e., their recognition sites for many enzymes).     

Glutamate-41 of Vibrio harveyi acyl carrier protein is essential for fatty acid synthase but not acyl-ACP synthetase activity

Bacterial acyl carrier protein (ACP) is a small, acidic, and highly conserved protein that supplies acyl groups for biosynthesis of a variety of lipid products. Recent modelling studies predict that residues primarily in helix II of Escherichia coli ACP (Glu-41, Ala-45) are involved in its interaction with the condensing enzyme FabH of fatty acid synthase. Using recombinant Vibrio harveyi ACP as a template for site-directed mutagenesis, we have shown that an acidic residue at position 41 is essential for V. harveyi fatty acid synthase (but not acyl-ACP synthetase) activity. In contrast, various replacements of Ala-45 were tolerated by both enzymes. None of the mutations introduced dramatic structural changes based on circular dichroism and native gel electrophoresis. These results confirm that Glu-41 of ACP is a critical residue for fatty acid synthase, but not for all enzymes that utilize ACP as a substrate.     

Functional analysis of an interspecies chimera of acyl carrier proteins indicates a specialized domain for protein recognition

The nodulation protein NodF of Rhizobium shows 25% identity to acyl carrier protein (ACP) from Escherichia coli (encoded by the gene acpP). However, NodF cannot be functionally replaced by AcpP. We have investigated whether NodF is a substrate for various E. coli enzymes which are involved in the synthesis of fatty acids. NodF is a substrate for the addition of the 4′-phosphopantetheine prosthetic group by holo-ACP synthase. The K_m value for NodF is 61 μM, as compared to 2 μM for AcpP. The resulting holo-NodF serves as a substrate for coupling of malonate by malonyl-CoA:ACP transacylase (MCAT) and for coupling of palmitic acid by acyl-ACP synthetase. NodF is not a substrate for β-keto-acyl ACP synthase III (KASIII), which catalyses the initial condensation reaction in fatty acid biosynthesis. A chimeric gene was constructed comprising part of the E.coliacpP gene and part of the nodF gene. Circular dichroism studies of the chimeric AcpP-NodF (residues 1–33 of AcpP fused to amino acids 43–93 of NodF) protein encoded by this gene indicate a similar folding pattern to that of the parental proteins. Enzymatic analysis shows that AcpP-NodF is a substrate for the enzymes holo-ACP synthase, MCAT and acyl-ACP synthetase. Biological complementation studies show that the chimeric AcpP-NodF gene is able functionally to replace NodF in the root nodulation process in Vicia sativa. We therefore conclude that NodF is a specialized acyl carrier protein whose specific features are encoded in the C-terminal region of the protein. The ability to exchange domains between such distantly related proteins without affecting conformation opens exciting possibilities for further mapping of the functional domains of acyl carrier proteins (i. e., their recognition sites for many enzymes). Received: 22 September 1997 / Accepted: 31 October 1997     

Solubilisation and reconstitution of acylcoenzyme A:estradiol-17 beta acyltransferase

Acylcoenzyme A:estradiol-17 beta acyltransferase in microsomes of bovine placenta cotyledons was strongly membrane bound. The enzyme was solubilised from microsomes by sodium cholate and was reconstituted into phospholipid vesicles. The apparent Km for estradiol-17 beta was 11 microM which was close to the value of 8 microM previously found with the membrane-bound enzyme. Testosterone was also a substrate for the reconstituted enzyme (apparent Km 62 microM) and was a competitive inhibitor (Ki 74 microM) of the acylation of estradiol-17 beta. Although various long-chained fatty acyl CoAs acted as acyl donors, these proved to have widely differing apparent Km values with palmitoleoyl CoA having the highest affinity (Km 24 microM) and arachidonoyl CoA the lowest affinity (Km 330 microM).     

Site-directed mutagenesis of acyl carrier protein (ACP) reveals amino acid residues involved in ACP structure and acyl-ACP synthetase activity.

Acyl carrier protein (ACP) interacts with many different enzymes during the synthesis of fatty acids, phospholipids, and other specialized products in bacteria. To examine the structural and functional roles of amino acids previously implicated in interactions between the ACP polypeptide and fatty acids attached to the phosphopantetheine prosthetic group, recombinant Vibrio harveyi ACP and mutant derivatives of conserved residues Phe-50, Ile-54, Ala-59, and Tyr-71 were prepared from glutathione S-transferase fusion proteins. Circular dichroism revealed that, unlike Escherichia coli ACP, V. harveyi-derived ACPs are unfolded at neutral pH in the absence of divalent cations; all except F50A and I54A recovered native conformation upon addition of MgCl(2). Mutant I54A was not processed to the holo form by ACP synthase. Some mutations significantly decreased catalytic efficiency of ACP fatty acylation by V. harveyi acyl-ACP synthetase relative to recombinant ACP, e.g. F50A (4%), I54L (20%), and I54V (31%), whereas others (V12G, Y71A, and A59G) had less effect. By contrast, all myristoylated ACPs examined were effective substrates for the luminescence-specific V. harveyi myristoyl-ACP thioesterase. Conformationally sensitive gel electrophoresis at pH 9 indicated that fatty acid attachment stabilizes mutant ACPs in a chain length-dependent manner, although stabilization was decreased for mutants F50A and A59G. Our results indicate that (i) residues Ile-54 and Phe-50 are important in maintaining native ACP conformation, (ii) residue Ala-59 may be directly involved in stabilization of ACP structure by acyl chain binding, and (iii) acyl-ACP synthetase requires native ACP conformation and involves interaction with fatty acid binding pocket residues, whereas myristoyl-ACP thioesterase is insensitive to acyl donor structure.     

Evidence for interactions of acyl carrier protein with glycerol-3-phosphate acyltransferase, an inner membrane protein of Escherichia coli

We [(1989) FEBS Lett., in press] have previously shown that membrane vesicles from Escherichia coli contain protein-binding sites for the acyl carrier protein (ACP). We report now that membrane vesicles prepared from a strain amplified for glycerol-3-phosphate acyltransferase (GPAT) contain a higher number of ACP-binding sites than the membrane vesicles prepared from a wild type strain. In addition, we show that GPAT is retained specifically on an ACP-Sepharose affinity column and that [3H]ACP binds to the enzyme solubilized by detergent. We conclude that GPAT, an inner membrane protein which catalyses the transesterification of a fatty acyl group from acyl coenzyme A or acyl ACP to glycerol-3-phosphate, possesses a binding site for ACP.