acyl-ACP Synthetase Research Articles

Effects of fatty acid activation on photosynthetic production of fatty acid-based biofuels in Synechocystis sp. PCC6803

BackgroundDirect conversion of solar energy and carbon dioxide to drop in fuel molecules in a single biological system can be achieved from fatty acid-based biofuels such as fatty alcohols and alkanes. These molecules have similar properties to fossil fuels but can be produced by photosynthetic cyanobacteria.ResultsSynechocystis sp. PCC6803 mutant strains containing either overexpression or deletion of the slr1609 gene, which encodes an acyl-ACP synthetase (AAS), have been constructed. The complete segregation and deletion in all mutant strains was confirmed by PCR analysis. Blocking fatty acid activation by deleting slr1609 gene in wild-type Synechocystis sp. PCC6803 led to a doubling of the amount of free fatty acids and a decrease of alkane production by up to 90 percent. Overexpression of slr1609 gene in the wild-type Synechocystis sp. PCC6803 had no effect on the production of either free fatty acids or alkanes. Overexpression or deletion of slr1609 gene in the Synechocystis sp. PCC6803 mutant strain with the capability of making fatty alcohols by genetically introducing fatty acyl-CoA reductase respectively enhanced or reduced fatty alcohol production by 60 percent.ConclusionsFatty acid activation functionalized by the slr1609 gene is metabolically crucial for biosynthesis of fatty acid derivatives in Synechocystis sp. PCC6803. It is necessary but not sufficient for efficient production of alkanes. Fatty alcohol production can be significantly improved by the overexpression of slr1609 gene.

Expression of Vibrio harveyi Acyl-ACP Synthetase Allows Efficient Entry of Exogenous Fatty Acids into the Escherichia coli Fatty Acid and Lipid A Synthetic Pathways

Although the Escherichia coli fatty acid synthesis (FAS) pathway is the best studied type II fatty acid synthesis system, a major experimental limitation has been the inability to feed intermediates into the pathway in vivo because exogenously supplied free fatty acids are not efficiently converted to the acyl-acyl carrier protein (ACP) thioesters required by the pathway. We report that expression of Vibrio harveyi acyl-ACP synthetase (AasS), a soluble cytosolic enzyme that ligates free fatty acids to ACP to form acyl-ACPs, allows exogenous fatty acids to enter the E. coli fatty acid synthesis pathway. The free fatty acids are incorporated intact and can be elongated or directly incorporated into complex lipids by acyltransferases specific for acyl-ACPs. Moreover, expression of AasS strains and supplementation with the appropriate fatty acid restored growth to E. coli mutant strains that lack essential fatty acid synthesis enzymes. Thus, this strategy provides a new tool for circumventing the loss of enzymes essential for FAS function.

Neutralization of Acidic Residues in Helix II Stabilizes the Folded Conformation of Acyl Carrier Protein and Variably Alters Its Function with Different Enzymes

Acyl carrier protein (ACP), a small protein essential for bacterial growth and pathogenesis, interacts with diverse enzymes during the biosynthesis of fatty acids, phospholipids, and other specialized products such as lipid A. NMR and hydrodynamic studies have previously shown that divalent cations stabilize native helical ACP conformation by binding to conserved acidic residues at two sites (A and B) at either end of the "recognition" helix II. To examine the roles of these amino acids in ACP structure and function, site-directed mutagenesis was used to replace individual site A (Asp-30, Asp-35, Asp-38) and site B (Glu-47, Glu-53, Asp-56) residues in recombinant Vibrio harveyi ACP with the corresponding amides, along with combined mutations at each site (SA, SB) or both sites (SA/SB). Like native V. harveyi ACP, all individual mutants were unfolded at neutral pH but adopted a helical conformation in the presence of millimolar Mg(2+) or upon fatty acylation. Mg(2+) binding to sites A or B independently stabilized native ACP conformation, whereas mutant SA/SB was folded in the absence of Mg(2+), suggesting that charge neutralization is largely responsible for ACP stabilization by divalent cations. Asp-35 in site A was critical for holo-ACP synthase activity, while acyl-ACP synthetase and UDP-N-acetylglucosamine acyltransferase (LpxA) activities were more affected by mutations in site B. Both sites were required for fatty acid synthase activity. Overall, our results indicate that divalent cation binding site mutations have predicted effects on ACP conformation but unpredicted and variable consequences on ACP function with different enzymes.

Role of helix II residues in the conformation and substrate properties of acyl carrier protein

Acyl carrier protein (ACP) and its fatty acylated derivatives are substrates for a variety of enzymes involved in the synthesis of bacterial lipids, endotoxins, and other products. ACP is believed to interact with most of these enzymes through a central acidic α-helix (helix II) containing weak divalent cation binding sites at its N- and C-termini. Site-directed mutagenesis was used to examine the roles of specific helix II residues on the conformation of Vibrio harveyi ACP and its ability to support holo-ACP synthase (ACPS), acyl-ACP synthetase (AAS), fatty acid synthase (FAS) and UDP-GlcNAc acyltransferase (LpxA) activities. Neutralization of acidic residues at either end of helix II stabilized the helical conformation of ACP but had distinct effects on ACP-dependent enzymes: N-terminal mutations (eg. D35N) affected ACPS activity, while C-terminal mutations had more impact on AAS and LpxA activities. FAS was sensitive to neutralizing mutations at both ends of helix II. In contrast, replacement of selected residues along helix II with tryptophan (eg. A45W, L46W, and F50W) had no major effect either on the conformation of ACP or on its substrate properties with AAS or FAS, although LpxA activity was affected. Our results support helix II as the major recognition domain for ACP-dependent enzymes, but suggest that different enzymes might have distinct binding determinants in this region (supported by CIHR and NSERC).

Identification of a plastid acyl-acyl carrier protein synthetase in Arabidopsis and its role in the activation and elongation of exogenous fatty acids.

Plant cells are known to elongate exogenously provided fatty acid (FA), but the subcellular sites and mechanisms for this process are not currently understood. When Arabidopsis leaves were incubated with 14C-FAs with <or=16 carbons, the label appeared in elongated and desaturated products. Laurate elongation was 85% inhibited by 50 microm cerulenin, an inhibitor of ketoacyl-acyl carrier protein (ACP) synthetase I/II. In contrast, haloxyfop, an inhibitor of cytosolic acetyl-coenzyme A (CoA) carboxylase, inhibited only elongation into very long chain FAs (>or=20 carbons) but not synthesis of 14C-unsaturated 18-carbon or 16-carbon FAs. Isolated pea chloroplasts were also able to elongate 14C-FAs (<or=16 carbons) in the light. No detectable 14C-acyl-CoA intermediates were formed during 14C-laurate elongation, whereas 14C-acyl-ACP accumulated to 2.3 microm. These data indicate that the elongation of exogenous medium-chain FAs to 16- and 18-carbon FAs occurs primarily in the chloroplasts, most likely via the enzymes of de novo FA synthesis. An Arabidopsis mutant with a T-DNA insertion in At4g14070 (AAE15) was reduced 80% in 14C-laurate elongation into 16- and 18-carbon FAs. AAE15 has sequence similarity to long-chain acyl-CoA synthetases and a predicted N-terminal plastidial targeting sequence. Direct acyl-ACP-forming activity from FA and ACP was observed in extracts of Arabidopsis leaves and isolated chloroplasts but aae15 plants had markedly reduced in vitro acyl-ACP synthesis activity. Together these results demonstrate that plants possess a mechanism for direct activation of FA to ACP in the plastid via an acyl-ACP synthetase encoded by At4g14070.

A novel approach for over-expression, characterization, and isotopic enrichment of a homogeneous species of acyl carrier protein from Plasmodium falciparum

Acyl carrier protein (ACP) plays a central role in fatty acid biosynthesis by transferring the acyl groups from one enzyme to another for the completion of the fatty acid synthesis cycle. Holo-ACP is the obligatory substrate for the synthesis of acyl-ACPs which act as the carrier and donor for various metabolic reactions. Despite its interactions with numerous proteins in the cell, its mode of interaction is poorly understood. Here, we report the over-expression of PfACP in minimal medium solely in its holo form and in high yield. Expression in minimal media provides a means to isotopically label PfACP for high resolution multi-nuclear and multi-dimensional NMR studies. Indeed, the proton–nitrogen correlated NMR spectrum exhibits very high chemical shift dispersion and resolution. We also show that holo-PfACP thus expressed is amenable to acylation reactions using Escherichia coli acyl-ACP synthetase as well as by standard chemical methods.

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.

Identification of a key residue in the conformational stability of acyl carrier protein

Conformational flexibility of acyl carrier protein (ACP) is important for its ability to interact with multiple enzymes in bacterial fatty acid metabolism. We have recently shown that, unlike the prototypical ACP from Escherichia coli, the more acidic Vibrio harveyi ACP is largely unfolded at physiological pH. Mutations D18K, A75H and A75H/D18K were made in recombinant V. harveyi ACP (rACP) to determine the importance of basic residues Lys-18 and His-75 in maintaining the native conformation of E. coli ACP. Both D18K and A75H ACPs were fatty acylated by acyl-ACP synthetase, showing that neither mutation grossly alters tertiary structure. Circular dichroism (CD) indicated that rACP refolded upon addition of MgCl 2 at 100-fold lower concentrations (<1 mM) than KCl, suggesting that divalent cations stabilize rACP by interaction at specific sites. Surprisingly, mutants A75H and A75H/D18K exhibited native-like conformation in the absence of MgCl 2, while the D18K mutant was comparable to rACP. Moreover, the α-helical content of A75H, A75H/D18K and E. coli ACPs was more sensitive than that of rACP or D18K ACP to modification by the histidine-selective reagent diethylpyrocarbonate. Together, these results suggest that the partial positive charge of His-75 may be important in maintaining the conformational stability of E. coli ACP at a neutral pH.

Purification and characterization of acyl-acyl carrier protein synthetase from oleaginous yeast and its role in triacylglycerol biosynthesis

Fatty acids are activated in an ATP-dependent manner before they are utilized. We describe here how the 10S triacylglycerol biosynthetic multienzyme complex from Rhodotorula glutinis is capable of activating non-esterified fatty acids for the synthesis of triacylglycerol. The photolabelling of the complex with [32P]azido-ATP showed labelling of a 35kDa polypeptide. The labelled polypeptide was identified as acyl-acyl carrier protein (ACP) synthetase, which catalyses the ATP-dependent ligation of fatty acid with ACP to form acyl-ACP. The enzyme was purified by successive PAGE separations to apparent homogeneity from the soluble fraction of oleaginous yeast and its apparent molecular mass was 35kDa under denaturing and reducing conditions. Acyl-ACP synthetase was specific for ATP. The Km values for palmitic, stearic, oleic and linoleic acids were found to be 42.9, 30.4, 25.1 and 22.7μM, respectively. The antibodies to acyl-ACP synthetase cross-reacted with Escherichia coli acyl-ACP synthetase. Anti-ACP antibodies showed no cross-reactivity with the purified acyl-ACP synthetase, indicating no bound ACP with the enzyme. Immunoprecipitations with antibodies to acyl-ACP synthetase revealed that this enzyme is a part of the 10S triacylglycerol biosynthetic complex. These results demonstrate that the soluble acyl-ACP synthetase plays a novel role in activating fatty acids for triacylglycerol biosynthesis in oleaginous yeast.

Purification and characterization of acyl-acyl carrier protein synthetase from oleaginous yeast and its role in triacylglycerol biosynthesis

Fatty acids are activated in an ATP-dependent manner before they are utilized. We describe here how the 10 S triacylglycerol biosynthetic multienzyme complex from Rhodotorula glutinis is capable of activating non-esterified fatty acids for the synthesis of triacylglycerol. The photolabelling of the complex with [(32)P]azido-ATP showed labelling of a 35 kDa polypeptide. The labelled polypeptide was identified as acyl-acyl carrier protein (ACP) synthetase, which catalyses the ATP-dependent ligation of fatty acid with ACP to form acyl-ACP. The enzyme was purified by successive PAGE separations to apparent homogeneity from the soluble fraction of oleaginous yeast and its apparent molecular mass was 35 kDa under denaturing and reducing conditions. Acyl-ACP synthetase was specific for ATP. The K(m) values for palmitic, stearic, oleic and linoleic acids were found to be 42.9, 30.4, 25.1 and 22.7 microM, respectively. The antibodies to acyl-ACP synthetase cross-reacted with Escherichia coli acyl-ACP synthetase. Anti-ACP antibodies showed no cross-reactivity with the purified acyl-ACP synthetase, indicating no bound ACP with the enzyme. Immunoprecipitations with antibodies to acyl-ACP synthetase revealed that this enzyme is a part of the 10 S triacylglycerol biosynthetic complex. These results demonstrate that the soluble acyl-ACP synthetase plays a novel role in activating fatty acids for triacylglycerol biosynthesis in oleaginous yeast.

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.

Metabolic control analysis of de novo sunflower fatty acid biosynthesis

We have obtained a simulation of the final steps of de novo fatty acid biosynthesis in sunflower control line RHA-274. For this simulation, we have used data from the evolution of fatty acids during seed formation and from the biochemical characterization of beta-keto-acyl-ACP synthetase II (FASII), stearoyl-ACP desaturase (SAD) and acyl-ACP thioesterase activities and the program GEPASI (based on the metabolic control-analysis theory). When physiological data from high- and medium-stearic acid mutants seed development were used with this model the predicted changes in SAD and TE were very similar to those actually found in the biochemical characterization of these mutants. However, the model had to be modified when results from high-palmitic mutants, accumulating unusual fatty acids like palmitoleic, asclepic and palmitolinoleic acids, were used. The emerging model, that fits all of our results, predicts the existence of a dynamic channelling between the FASII complex and SAD, that channelling being responsible for the alternative pathway starting with the desaturation of palmitic acid by the stearoyl-ACP desaturase. This channelling is consistent with our previous results. For instance, the determination of SAD activity on sunflower seed crude extracts only rendered oleic acid when the stearic acid used as a substrate was obtained from a KASII assay, but not when the stearic acid came from in vitro synthesis using acyl-ACP synthetase from Escherichia coli. This theoretical approximation will be very useful in predicting the evolution of the system when introducing new or modified activities; similar approximations in other oil-seed crops could be of great interest.

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 Km value for NodF is 61 microM, as compared to 2 microM 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 beta-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. coli acpP 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).

Sequence and function of the aas gene in Escherichia coli.

2-Acylglycerophosphoethanolamine (2-acyl-GPE) acyltransferase and acyl-acyl carrier protein (acyl-ACP) synthase activities are encoded by the aas gene in Escherichia coli. The aas gene was cloned, and the DNA sequence of the aas gene and the region between aas and galR established the clockwise gene order in the 61.2 min of the E. coli chromosome as aas-orf-galR-lysA-lysR-orf-araE. The aas gene consists of a single open reading frame of 2,157 base pairs predicted to encode a protein of 80.6 kDa. Strains harboring multiple copies of the aas gene overproduced both 2-acyl-GPE acyltransferase and acyl-ACP synthetase activities in vitro and had higher specific activities for the incorporation of exogenous fatty acids and lysophospholipids into the membrane in vivo. Specific expression of the aas gene yielded a protein with an apparent molecular mass of 81 kDa. Comparison of the predicted amino acid sequence of the aas gene with mammalian, yeast, and bacterial long chain acyl-coenzyme A synthetases revealed three domains of high similarity which are postulated to form the acyl-AMP binding pocket. These data verify that 2-acyl-GPE acyltransferase and acyl-ACP synthetase are reactions carried out by the same gene product, verify the role of 2-acyl-GPE acyltransferase/acyl-ACP synthetase in the acylation of endogenous 2-acyl-GPE, and establish the product of the aas gene as the rate-limiting enzyme in the uptake and incorporation of exogenous 2-acyllysophospholipids.

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.

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 −20δC 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.

13] 2-Acylglycerophosphoethanolamine acyltransferase/ acyl-[acyl-carrier-protein] synthetase from Escherichia coli

2-acylglycerophosphoethanolamine (2-acyl-GPE) acyltransferase is a membrane-bound enzyme that either activates fatty acids for acyl transfer in the presence of ATP and Mg 2+ or transfers fatty acids from acyl–acyl carrier protein (ACP) to the 1-position of lysophospholipids. 2-acyl-GPE acyltransferase is a heterodimer composed of a hydrophobic membrane-bound subunit and ACP. The catalytic cycle of the acyltransferase/synthetase is described in this chapter. The first step is the ligation of a fatty acid to the 4 ' -phosphopantetheine sulfhydryl group of the ACP subunit. The second step in the acyltransferase reaction is the transfer of the acyl moiety from ACP to the 2-acyl-GPE substrate to form phosphatidylethanolamine (PtdEtn). The assay method for 2-acyl-GPE acyltransferase is based on measuring the formation of [14C]PtdEtn from 2-acyl-GPE and [14C]palmitic acid in the presence of ATP, Mg 2+ , and ACP. A replica print procedure is used to isolate mutants lacking both 2-acyl-GPE acyltransferase and acyl-ACP synthetase activities. The acyltransferase/synthetase functions only as an acyltransferase in vivo ; its ability to synthesize acyl-ACP in vitro has made it a useful tool for the preparation of defined acyl-ACPs to be used as substrates for other enzymes.

Expression of Constitutive and Tissue-Specific Acyl Carrier Protein Isoforms in Arabidopsis

We have characterized the occurrence and expression of multiple acyl carrier protein (ACP) isoforms in Arabidopsis thaliana (L.) Heynh ecotype Columbia. Immunoblot analysis of ACPs from Arabidopsis tissues separated by native polyacrylamide gel electrophoresis and 1 molar urea polyacrylamide gel electrophoresis revealed a complex pattern of multiple ACP isoforms. All tissues examined (leaves, roots, and seeds) expressed at least three forms of ACP. The immunoblot identifications of ACP bands were confirmed by acylation of ACP extracts with Escherichia coli acyl-ACP synthetase. A full-length cDNA clone has been isolated that has 70% identity with a previously characterized Arabidopsis genomic ACP clone (ACP-1) (MA Post-Beittenmiller, A Hlousek-Radojcić, JB Ohlrogge [1989] Nucleic Acids Res 17: 1777). Based on RNA blot analysis, the cDNA clone represents an ACP that is expressed in leaves, seeds, and roots. In order to identify the protein products of each known ACP gene, their mature coding sequences have been expressed in E. coli. Using polymerase chain reactions, exons II and III of the genomic ACP-1 clone and the mature coding sequences of the ACP-2 cDNA clone were subcloned into E. coli expression vectors. Site-directed mutagenesis was used to convert the amino acid sequence of the ACP-2 cDNA clone to that of the A2 clone of Lamppa and Jacks ([1991] Plant Mol Biol 16: 469-474), ACP-3. The three E. coli-expressed proteins have different mobilities on polyacrylamide gel electrophoresis gels and each comigrates with a different Arabidopsis ACP isoform expressed in leaves, seeds, and roots. Thus, all of the three cloned ACPs appear to be constitutively expressed Arabidopsis ACPs. In addition to these three ACP isoforms, protein blots indicate that seed, leaf, and root each express one or more tissue-specific isoforms.

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.

2-Acylglycerolphosphoethanolamine acyltransferase/acyl-acyl carrier protein synthetase is a membrane-associated acyl carrier protein binding protein

Two enzymatic activities, 2-acylglycerolphosphoethanolamine (2-acyl-GPE) acyltransferase and acyl-acyl carrier protein (acyl-ACP) synthetase, were solubilized and purified from Escherichia coli membranes. Electrophoretic analysis of the final product of the purification procedure revealed a single protein species with an apparent molecular mass of 27 kilodaltons. The ratio of acyltransferase to synthetase activities remained the same throughout the purification scheme indicating that both activities were catalyzed by the same enzyme. 2-Acyl-GPE acyltransferase exhibited an apparent ACP Km of 64 nM under standard assay conditions that increased to 10 microM when the assay was conducted in the presence of 0.4 M LiCl. Acyl-ACP synthetase activity was not detected in the absence of 0.4 M LiCl, and the apparent ACP Km for this reaction was 16 microM. Direct evidence that ACP was a subunit of the acyltransferase/synthetase was obtained by the adsorption of both catalytic activities to an ACP-Sepharose affinity column and by the binding of [3H]ACP to the purified enzyme preparation. The apparent Km for acyl-ACP was 13 microM, and the rate of acyl transfer from this acyl donor was enhanced by the addition of 0.4 M LiCl indicating that the exchange of enzyme-bound ACP for acyl-ACP was a determinant factor in the rate of phosphatidylethanolamine formation from acyl-ACP. These data indicate that the 2-acyl-GPE acyltransferase and acyl-ACP synthetase reactions are catalyzed by the same membrane protein that possesses a high affinity binding site for soluble ACP.