Acyl-acyl Carrier Protein Synthetase Research Articles

A broad inhibitor of acyl-acyl carrier protein synthetases

The acyl-acyl carrier protein synthetase enzyme enables some bacteria to scavenge free fatty acids from the environment for direct use in lipids. This fatty acid recycling pathway can help pathogens circumvent fatty acid synthase (FAS) inhibition with established antibiotics and those in clinical development. AasS enzymes are surprisingly hard to identify as they show high sequence similarity to other adenylate forming enzymes, and only a handful have been correctly annotated to date. Four recently discovered AasS enzymes from Gram negative bacteria, Chlamydia trachomatis, Neisseria gonorrhoeae, and Alistipes finegoldii, form distinct clusters in protein sequence similarity networks and have varying substrate preferences. We previously synthesized C10-AMS, an inhibitor of AasS that mimics the acyl-AMP reaction intermediate. Here we tested its ability to be broadly applicable to enzymes in this class, and found it inhibits all four newly annotated AasS enzymes. C10-AMS therefore provides a tool to study the role of AasS in fatty acid recycling in pathogenic bacteria as well as offers a platform for antibiotic development.

Synthesis of an acyl-acyl carrier protein synthetase inhibitor to study fatty acid recycling

Fatty acids are essential to most organisms and are made endogenously by the fatty acid synthase (FAS). FAS is an attractive target for antibiotics and many inhibitors are in clinical development. However, some gram-negative bacteria harbor an enzyme known as the acyl-acyl carrier protein synthetase (AasS), which allows them to scavenge fatty acids from the environment and shuttle them into FAS and ultimately lipids. The ability of AasS to recycle fatty acids may help pathogenic gram-negative bacteria circumvent FAS inhibition. We therefore set out to design and synthesize an inhibitor of AasS and test its effectiveness on an AasS enzyme from Vibrio harveyi, the most well studied AasS to date, and from Vibrio cholerae, a pathogenic model. The inhibitor C10-AMS [5′-O-(N-decanylsulfamoyl)adenosine], which mimics the tightly bound acyl-AMP reaction intermediate, was able to effectively inhibit AasS catalytic activity in vitro. Additionally, C10-AMS stopped the ability of Vibrio cholerae to recycle fatty acids from media and survive when its endogenous FAS was inhibited with cerulenin. C10-AMS can be used to study fatty acid recycling in other bacteria as more AasS enzymes continue to be annotated and provides a platform for potential antibiotic development.

Shifting the Hydrolysis Equilibrium of Substrate Loaded Acyl Carrier Proteins.

Acyl carrier proteins (ACP)s transport intermediates through many primary and secondary metabolic pathways. Studying the effect of substrate identity on ACP structure has been hindered by the lability of the thioester bond that attaches acyl substrates to the 4'-phosphopantetheine cofactor of ACP. Here we show that an acyl acyl-carrier protein synthetase (AasS) can be used in real time to shift the hydrolysis equilibrium toward favoring acyl-ACP during solution NMR spectroscopy. Only 0.005 molar equivalents of AasS enables 1 week of stability to palmitoyl-AcpP from Escherichia coli. 2D NMR spectra enabled with this method revealed that the tethered palmitic acid perturbs nearly every secondary structural region of AcpP. This technique will allow previously unachievable structural studies of unstable acyl-ACP species, contributing to the understanding of these complex biosynthetic pathways.

Characterization of Acyl Carrier Protein-Dependent Glycosyltransferase in Mitomycin C Biosynthesis.

Mitomycins make up a group of antitumor natural products that are biosynthesized from aminohydroxybenzoic acid (AHBA) and N-acetylglucosamine (GlcNAc). While the biosynthetic gene cluster was reported two decades ago, the mechanism by which the two building blocks, AHBA and GlcNAc, are coupled during biosynthesis remained uncharacterized. Here we report evidence that AHBA is first loaded onto an MmcB acyl carrier protein (ACP) by a MitE acyl ACP synthetase, followed by a transfer of GlcNAc from UDP-GlcNAc by MitB. The results suggest that the early steps of mitomycin biosynthesis proceed via intermediates linked to MmcB.

A simple method for isolation and construction of markerless cyanobacterial mutants defective in acyl-acyl carrier protein synthetase.

Cyanobacterial mutants defective in acyl-acyl carrier protein synthetase (Aas) secrete free fatty acids (FFAs) into the external medium and hence have been used for the studies aimed at photosynthetic production of biofuels. While the wild-type strain of Synechocystis sp. PCC 6803 is highly sensitive to exogenously added linolenic acid, mutants defective in the aas gene are known to be resistant to the externally provided fatty acid. In this study, the wild-type Synechocystis cells were shown to be sensitive to lauric, oleic, and linoleic acids as well, and the resistance to these fatty acids was shown to be enhanced by inactivation of the aas gene. On the basis of these observations, we developed an efficient method to isolate aas-deficient mutants from cultures of Synechocystis cells by counter selection using linoleic acid or linolenic acid as the selective agent. A variety of aas mutations were found in about 70 % of the FFA-resistant mutants thus selected. Various aas mutants were isolated also from Synechococcus sp. PCC 7002, using lauric acid as a selective agent. Selection using FFAs was useful also for construction of markerless aas knockout mutants from Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002. Thus, genetic engineering of FFA-producing cyanobacterial strains would be greatly facilitated by the use of the FFAs for counter selection.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-016-7850-8) contains supplementary material, which is available to authorized users.

Arabidopsis acyl-acyl carrier protein synthetase AAE15 with medium chain fatty acid specificity is functional in cyanobacteria.

Cyanobacteria are potential hosts for the biosynthesis of oleochemical compounds. The metabolic precursors for such compounds are fatty acids and their derivatives, which require chemical activation to become substrates in further conversion steps. We characterized the acyl activating enzyme AAE15 of Arabidopsis encoded by At4g14070, which is a homologue of a cyanobacterial acyl-ACP synthetase (AAS). We expressed AAE15 in insect cells and demonstrated its AAS activity with medium chain fatty acid (C10–C14) substrates in vitro. Furthermore, we used AAE15 to complement a Synechocystisaas deletion mutant and showed that the new strain preferentially incorporates supplied medium chain fatty acids into internal lipid molecules. Based on this data we propose that AAE15 can be utilized in metabolic engineering strategies for cyanobacteria that aim to produce compounds based on medium chain fatty acids.

Activation of Exogenous Fatty Acids to Acyl-Acyl Carrier Protein Cannot Bypass FabI Inhibition in Neisseria

Neisseria is a Gram-negative pathogen with phospholipids composed of straight chain saturated and monounsaturated fatty acids, the ability to incorporate exogenous fatty acids, and lipopolysaccharides that are not essential. The FabI inhibitor, AFN-1252, was deployed as a chemical biology tool to determine whether Neisseria can bypass the inhibition of fatty acid synthesis by incorporating exogenous fatty acids. Neisseria encodes a functional FabI that was potently inhibited by AFN-1252. AFN-1252 caused a dose-dependent inhibition of fatty acid synthesis in growing Neisseria, a delayed inhibition of growth phenotype, and minimal inhibition of DNA, RNA, and protein synthesis, showing that its mode of action is through inhibiting fatty acid synthesis. Isotopic fatty acid labeling experiments showed that Neisseria encodes the ability to incorporate exogenous fatty acids into its phospholipids by an acyl-acyl carrier protein-dependent pathway. However, AFN-1252 remained an effective antibacterial when Neisseria were supplemented with exogenous fatty acids. These results demonstrate that extracellular fatty acids are activated by an acyl-acyl carrier protein synthetase (AasN) and validate type II fatty acid synthesis (FabI) as a therapeutic target against Neisseria.

Identification of a Cyanobacterial RND-Type Efflux System Involved in Export of Free Fatty Acids.

An RND (resistance-nodulation-division)-type transporter having the capacity to export free fatty acids (FFAs) was identified in the cyanobacterium Synechococcus elongatus strain PCC 7942 during characterization of a mutant strain engineered to produce FFAs. The basic strategy for construction of the FFA-producing mutant was a commonly used one, involving inactivation of the endogenous acyl-acyl carrier protein synthetase gene (aas) and introduction of a foreign thioesterase gene ('tesA), but a nitrate transport mutant NA3 was used as the parental strain to achieve slow, nitrate-limited growth in batch cultures. Also, a nitrogen-regulated promoter PnirA was used to drive 'tesA to maximize thioesterase expression during the nitrate-limited growth. The resulting mutant (dAS2T) was, however, incapable of growth under the conditions of nitrate limitation, presumably due to toxicity associated with FFA overproduction. Incubation of the mutant culture under the non-permissive conditions allowed for isolation of a pseudorevertant (dAS2T-pr1) capable of growth on nitrate. Genome sequence and gene expression analyses of this strain suggested that expression of an RND-type efflux system had rescued growth on nitrate. Targeted inactivation of the RND-type transporter genes in the wild-type strain resulted in loss of tolerance to exogenously added FFAs including capric, lauric, myristic, oleic and linolenic acids. Overexpression of the genes in dAS2T, on the other hand, enhanced FFA excretion and cell growth in nitrate-containing medium, verifying that the genes encode an efflux pump for FFAs. These results demonstrate the importance of the efflux system in efficient FFA production using genetically engineered cyanobacteria.

Essential Role of Acyl-ACP Synthetase in Acclimation of the Cyanobacterium Synechococcus elongatus Strain PCC 7942 to High-Light Conditions.

Most organisms capable of oxygenic photosynthesis have an aas gene encoding an acyl-acyl carrier protein synthetase (Aas), which activates free fatty acids (FFAs) via esterification to acyl carrier protein. Cyanobacterial aas mutants are often used for studies aimed at photosynthetic production of biofuels because the mutation leads to intracellular accumulation of FFAs and their secretion into the external medium, but the physiological significance of the production of FFAs and their recycling involving Aas has remained unclear. Using an aas-deficient mutant of Synechococcus elongatus strain PCC 7942, we show here that remodeling of membrane lipids is activated by high-intensity light and that the recycling of FFAs is essential for acclimation to high-light conditions. Unlike wild-type cells, the mutant cells could not increase their growth rate as the light intensity was increased from 50 to 400 µmol photons m(-2) s(-1), and the high-light-grown mutant cells accumulated FFAs and the lysolipids derived from all the four major classes of membrane lipids, revealing high-light-induced lipid deacylation. The high-light-grown mutant cells showed much lower PSII activity and Chl contents as compared with the wild-type cells or low-light-grown mutant cells. The loss of Aas accelerated photodamage of PSII but did not affect the repair process of PSII, indicating that PSII is destabilized in the mutant. Thus, Aas is essential for acclimation of the cyanobacterium to high-light conditions. The relevance of the present finding s to biofuel production using cyanobacteria is discussed.

Diarylcoumarins inhibit mycolic acid biosynthesis and kill Mycobacterium tuberculosis by targeting FadD32

Infection with the bacterial pathogen Mycobacterium tuberculosis imposes an enormous burden on global public health. New antibiotics are urgently needed to combat the global tuberculosis pandemic; however, the development of new small molecules is hindered by a lack of validated drug targets. Here, we describe the identification of a 4,6-diaryl-5,7-dimethyl coumarin series that kills M. tuberculosis by inhibiting fatty acid degradation protein D32 (FadD32), an enzyme that is required for biosynthesis of cell-wall mycolic acids. These substituted coumarin inhibitors directly inhibit the acyl-acyl carrier protein synthetase activity of FadD32. They effectively block bacterial replication both in vitro and in animal models of tuberculosis, validating FadD32 as a target for antibiotic development that works in the same pathway as the established antibiotic isoniazid. Targeting new steps in well-validated biosynthetic pathways in antitubercular therapy is a powerful strategy that removes much of the usual uncertainty surrounding new targets and in vivo clinical efficacy, while circumventing existing resistance to established targets.

The Acyl-Acyl Carrier Protein Synthetase from Synechocystis sp. PCC 6803 Mediates Fatty Acid Import

The transfer of fatty acids across biological membranes is a largely uncharacterized process, although it is essential at membranes of several higher plant organelles like chloroplasts, peroxisomes, or the endoplasmic reticulum. Here, we analyzed loss-of-function mutants of the unicellular cyanobacterium Synechocystis sp. PCC 6803 as a model system to circumvent redundancy problems encountered in eukaryotic organisms. Cells deficient in the only cytoplasmic Synechocystis acyl-acyl carrier protein synthetase (SynAas) were highly resistant to externally provided α-linolenic acid, whereas wild-type cells bleached upon this treatment. Bleaching of wild-type cells was accompanied by a continuous increase of α-linolenic acid in total lipids, whereas no such accumulation could be observed in SynAas-deficient cells (Δsynaas). When SynAas was disrupted in the tocopherol-deficient, α-linolenic acid-hypersensitive Synechocystis mutant Δslr1736, double mutant cells displayed the same resistance phenotype as Δsynaas. Moreover, heterologous expression of SynAas in yeast (Saccharomyces cerevisiae) mutants lacking the major yeast fatty acid import protein Fat1p (Δfat1) led to the restoration of wild-type sensitivity against exogenous α-linolenic acid of the otherwise resistant Δfat1 mutant, indicating that SynAas is functionally equivalent to Fat1p. In addition, liposome assays provided direct evidence for the ability of purified SynAas protein to mediate α-[(14)C]linolenic acid retrieval from preloaded liposome membranes via the synthesis of [(14)C]linolenoyl-acyl carrier protein. Taken together, our data show that an acyl-activating enzyme like SynAas is necessary and sufficient to mediate the transfer of fatty acids across a biological membrane.

Fatty Acid Activation in Cyanobacteria Mediated by Acyl-Acyl Carrier Protein Synthetase Enables Fatty Acid Recycling

In cyanobacteria fatty acids destined for lipid synthesis can be synthesized de novo, but also exogenous free fatty acids from the culture medium can be directly incorporated into lipids. Activation of exogenous fatty acids is likely required prior to their utilization. To identify the enzymatic activity responsible for activation we cloned candidate genes from Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942 and identified the encoded proteins as acyl-acyl carrier protein synthetases (Aas). The enzymes catalyze the ATP-dependent esterification of fatty acids to the thiol of acyl carrier protein. The two protein sequences are only distantly related to known prokaryotic Aas proteins but they display strong similarity to sequences that can be found in almost all organisms that perform oxygenic photosynthesis. To investigate the biological role of Aas activity in cyanobacteria, aas knockout mutants were generated in the background of Synechocystis sp. PCC 6803 and S. elongatus PCC 7942. The mutant strains showed two phenotypes characterized by the inability to utilize exogenous fatty acids and by the secretion of endogenous fatty acids into the culture medium. The analyses of extracellular and intracellular fatty acid profiles of aas mutant strains as well as labeling experiments indicated that the detected free fatty acids are released from membrane lipids. The data suggest a considerable turnover of lipid molecules and a role for Aas activity in recycling the released fatty acids. In this model, lipid degradation represents a third supply of fatty acids for lipid synthesis in cyanobacteria.

The Soluble Acyl-Acyl Carrier Protein Synthetase of Vibrio harveyi B392 Is a Member of the Medium Chain Acyl-CoA Synthetase Family

The gene encoding the unique soluble acyl-acyl carrier protein synthetase (AasS) of the bioluminescent Vibrio harveyi strain B392 has been isolated by expression cloning in Escherichia coli. This enzyme catalyzes the ATP-dependent acylation of the thiol of acyl carrier protein (ACP) with fatty acids with chain lengths from C4 to C18. The gene (called aasS) encodes a protein of 60 kDa, a hexahistidine-tagged version of which was readily expressed in E. coli and purified in large quantities. Surprisingly, the sequence of the encoded protein was significantly more similar to that of an acyl-CoA synthetase of the distantly related bacterium, Thermus thermophilus, than to that of the membrane-bound acyl-acyl carrier protein synthetase of E. coli, an enzyme that catalyzes the same reaction from a more closely related organism. Indeed, the AasS sequence can readily be modeled on the known crystal structures of the T. thermophilus acyl-CoA synthetase with remarkably high levels of conservation of the catalytic site residues. To test the possible role of AasS in the fatty aldehyde-dependent bioluminescence pathway of V. harveyi, the chromosomal aasS gene of the organism was disrupted by insertion of a kanamycin cassette by homologous recombination. The resulting aasS::kan strains retained low levels of acyl-acyl carrier protein synthetase consistent with prior indications of a second such activity in this bacterium. The mutant strains grew normally and had normal levels of bioluminescence but were deficient in the incorporation of exogenous octanoic acid into the cellular phospholipids of V. harveyi, particularly at low octanoate concentrations. These data indicate that AasS is responsible for a high-affinity and high-capacity uptake system that efficiently converts exogenous fatty acids into acyl-ACP species competent to enter the fatty acid biosynthetic cycle.

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.

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.

Isolation and Localization of a Cytosolic 10 S Triacylglycerol Biosynthetic Multienzyme Complex from Oleaginous Yeast

Triacylglycerol is one of the major storage forms of metabolic energy in eukaryotic cells. Biosynthesis of triacylglycerol is known to occur in membranes. We report here the isolation, purification, and characterization of a catalytically active cytosolic 10 S multienzyme complex for triacylglycerol biosynthesis from Rhodotorula glutinis during exponential growth. The complex was characterized and was found to contain lysophosphatidic acid acyltransferase, phosphatidic acid phosphatase, diacylglycerol acyltransferase, acyl-acyl carrier protein synthetase, and acyl carrier protein. The 10 S triacylglycerol biosynthetic complex rapidly incorporates free fatty acids as well as fatty acyl-coenzyme A into triacylglycerol and its biosynthetic intermediates. Lysophosphatidic acid acyltransferase, phosphatidic acid phosphatase, and diacylglycerol acyltransferase from the complex were microsequenced. Antibodies were raised against the synthetic peptides corresponding to lysophosphatidic acid acyltransferase and phosphatidic acid phosphatase sequences. Immunoprecipitation and immunolocalization studies show the presence of a cytosolic multienzyme complex for triacylglycerol biosynthesis. Chemical cross-linking studies revealed that the 10 S multienzyme complex was held together by protein-protein interactions. These results demonstrate that the cytosol is one of the sites for triacylglycerol biosynthesis in oleaginous yeast.

A conserved histidine is essential for glycerolipid acyltransferase catalysis.

Sequence analysis of membrane-bound glycerolipid acyltransferases revealed that proteins from the bacterial, plant, and animal kingdoms share a highly conserved domain containing invariant histidine and aspartic acid residues separated by four less conserved residues in an HX4D configuration. We investigated the role of the invariant histidine residue in acyltransferase catalysis by site-directed mutagenesis of two representative members of this family, the sn-glycerol-3-phosphate acyltransferase (PlsB) and the bifunctional 2-acyl-glycerophosphoethanolamine acyltransferase/acyl-acyl carrier protein synthetase (Aas) of Escherichia coli. Both the PlsB[H306A] and Aas[H36A] mutants lacked acyltransferase activity. However, the Aas[H36A] mutant retained significant acyl-acyl carrier protein synthetase activity, illustrating that the lack of acyltransferase activity was specifically associated with the H36A substitution. The invariant aspartic acid residue in the HX4D pattern was also important. The substitution of aspartic acid 311 with glutamic acid in PlsB resulted in an enzyme with significantly reduced catalytic activity. Substitution of an alanine at this position eliminated acyltransferase activity; however, the PlsB[D311A] mutant protein did not assemble into the membrane, indicating that aspartic acid 311 is also important for the proper folding and membrane insertion of the acyltransferases. These data are consistent with a mechanism for glycerolipid acyltransferase catalysis where the invariant histidine functions as a general base to deprotonate the hydroxyl moiety of the acyl acceptor.

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.

Use of Escherichia coli strains containing fad mutations plus a triple plasmid expression system to study the import of myristate, its activation by Saccharomyces cerevisiae acyl-CoA synthetase, and its utilization by S. cerevisiae myristoyl-CoA:protein N-myristoyltransferase.

A system is described for studying protein N-myristoylation, a eukaryotic protein modification, in Escherichia coli strains containing components of eukaryotic metabolic pathways that regulate metabolism of myristoyl-CoA:protein N-myristoyltransferase (Nmt1p) substrates. Three recombinant plasmids were used to simultaneously direct synthesis of Saccharomyces cerevisiae Nmt1p, a substrate protein (S. cerevisiae ADP-ribosylation factor 1, Arf1p), and one of the acyl-CoA synthetases produced by S. cerevisiae (Faa1p) in isogenic strains of bacteria with wild type or mutant alleles of genes comprising the regulon for fatty acid degradation (FadR, FadE, FadL and FadD). Incorporation of exogenous tritiated myristate into Arf1p and bacterial phospholipid biosynthetic pathways was analyzed. Removal of FadL, a 448-residue protein necessary for efficient transport of fatty acids across the outer membrane, had no detectable effect on Nmt1p-dependent N-myristoylation of Arf1p. This finding is consistent with the notion that permeation of C14:0 across the bacterial inner membrane can occur by simple diffusion. Studies of strains that contain a mutation in FadE which inhibits beta-oxidation of exogenous fatty acids, confirm that Nmt1p retains its specificity for myristoyl-CoA over palmitoyl-CoA in E. coli. A mutation that inactivates FadD, a 580-residue protein which is the only acyl-CoA synthetase produced by this bacterium, completely blocks incorporation of exogenous myristate into Arf1p. This failure to be incorporated indicates that myristoyl-acyl carrier protein, generated by inner membrane acyl-acyl carrier protein synthetase, is not a substrate for Nmt1p. S. cerevisiae Faa1p can partially complement this mutant fadD allele. It can fully "restore" N-myristoylation of Arf1p. Faa1p can also rescue growth at 37 degrees C of fadD- strains on minimal media supplemented with C12:0, although this rescue becomes less efficient as the chain length of the supplemental fatty acid increases. In addition, S. cerevisiae Faa1p is better able to direct myristoyl-CoA to the bacteria's phospholipid biosynthetic pathways than FadD, while FadD is more efficient at directing myristoyl-CoA to the genetically engineered protein N-myristoylation pathway. Since cellular acyl-CoA synthetase activity in S. cerevisiae has been distributed to at least two functionally differentiated proteins, this system should be useful for comparing their structure-activity relationships as well as their interactions with Nmt1p in an organelle-free environment.