Biosynthesis of triphosphoribosyl-dephospho-coenzyme A, the precursor of the prosthetic group of malonate decarboxylase

Malonate decarboxylase from Klebsiella pneumoniae consists of four subunits MdcA, D, E, and C and catalyzes the cleavage of malonate to acetate and CO(2). The smallest subunit MdcC is an acyl carrier protein to which acetyl and malonyl thioester residues are bound via a 2'-(5' '-phosphoribosyl)-3'-dephospho-CoA prosthetic group and turn over during the catalytic mechanism. We report here on the biosynthesis of holo acyl carrier protein from the unmodified apoprotein. The prosthetic group biosynthesis starts with the MdcB-catalyzed condensation of dephospho-CoA with ATP to 2'-(5' '-triphosphoribosyl)-3'-dephospho-CoA. In this reaction, a new alpha (1' ' --> 2') glycosidic bond between the two ribosyl moieties is formed, and thereby, the adenine moiety of ATP is displaced. MdcB therefore is an ATP:dephospho-CoA 5'-triphosphoribosyl transferase. The second protein involved in holo ACP synthesis is MdcG. This enzyme forms a strong complex with the 2'-(5' '-triphosphoribosyl)-3'-dephospho-CoA prosthetic group precursor. This complex, called MdcG(i), is readily separated from free MdcG by native polyacrylamide gel electrophoresis. Upon incubation of MdcG(i) with apo acyl carrier protein, holo acyl carrier protein is synthesized by forming the phosphodiester bond between the 2'-(5' '-phosphoribosyl)-3'-dephospho-CoA prosthetic group and serine 25 of the protein. MdcG corresponds to a 2'-(5' '-triphosphoribosyl)-3'-dephospho-CoA:apo ACP 2'-(5' '-phosphoribosyl)-3'-dephospho-CoA transferase. In absence of the prosthetic group precursor, MdcG catalyzes at a low rate the adenylylation of apo acyl carrier protein using ATP as substrate. The adenylyl ACP thus formed is an unphysiological side product and is not involved in the biosynthesis of holo ACP. The 2'-(5' '-triphosphoribosyl)-3'-dephospho-CoA precursor of the prosthetic group has been purified and its identity confirmed by mass spectrometry and enzymatic analysis.     

Phosphopantethenylated Precursor Acyl Carrier Protein Is Imported into Spinach (Spinacia oleracea) Chloroplasts

Acyl carrier protein (ACP) is an essential cofactor of fatty acid synthase. In plants, ACP is synthesized in the cytosol as a larger precursor protein and then is imported into the plastid where it is processed to a smaller mature form. The active form of ACP uses a covalently linked 4[prime]-phosphopantetheine prosthetic group derived from coenzyme A to covalently bind the acyl intermediates during fatty acid synthesis. The prosthetic group is added to ACP by holoACP synthase. This enzyme activity is associated with both the plastidial subcellular fraction and the soluble, or cytoplasmic, fraction. To gain further insight into potential in vivo pathways for the synthesis and maturation of ACP, in this study we examined whether precursor holoACP can be imported by isolated spinach (Spinacia oleracea) chloroplasts. Precursor holoACP containing a [35S]phosphopantetheine prosthetic group was prepared, and the radiolabel was used to demonstrate import of the phosphopantethenylated protein into isolated chloroplasts. In addition, timed chloroplast import assays indicated that in vitro import of the phosphopantethenylated protein is at least as efficient as import of the precursor apoprotein. Evidence was also obtained for a low level turnover of the prosthetic group among endogenous plastidial ACPs when coenzyme A was supplied exogenously.     

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.     

Biosynthesis and degradation both contribute to the regulation of coenzyme A content in Escherichia coli.

Escherichia coli mutants [coaA16(Fr); Fr indicates feedback resistance] were isolated which possessed a pantothenate kinase activity that was refractory to feedback inhibition by coenzyme A (CoA). Strains harboring this mutation had CoA levels that were significantly elevated compared with strains containing the wild-type kinase and also overproduced both intra- and extracellular 4'-phosphopantetheine. The origin of 4'-phosphopantetheine was investigated by using strain SJ135 [panD delta(aroP-aceEF)], in which synthesis of acetyl-CoA was dependent on the addition of an acetate growth supplement. Rapid degradation of CoA to 4'-phosphopantetheine was triggered by the conversion of acetyl-CoA to CoA following the removal of acetate from the media. CoA hydrolysis under these conditions appeared not to involve acyl carrier protein prosthetic group turnover since [acyl carrier protein] phosphodiesterase was inhibited equally well by acetyl-CoA or CoA. These data support the view that the total cellular CoA content is controlled by modulation of biosynthesis at the pantothenate kinase step and by degradation of CoA to 4'-phosphopantetheine.     

The prosthetic group of citrate-lyase acyl-carrier protein.

The acyl carrier protein of citrate lyase contains adenine, phosphate, sugar, cysteamine, beta-alanine and pantoic acid in a molar ratio of 1:2:2:1:1:1. Peptides containing these components in the same stoichiometric relationship were isolated after proteolytic digestion of acyl carrier protein. All components were linked together in a single prosthetic group. This was released from the peptide by mild alkaline hydrolysis. Under these conditions a phosphodiester bond is cleaved which links the prosthetic group to a serine residue of the peptide. Incubation of the prosthetic-group-containing peptide with phosphodiesterase I yielded 4'-phosphopantetheine and adenylic acid. The 5'-AMP was not free but was substituted by presumably an acidic sugar residue, which was released by mild acid hydrolysis yielding free 5'-AMP. It was concluded from these results that the prosthetic group of citrate lyase acyl carrier protein consists of a substituted isomeric dephospho-CoA. This is bound to the protein by the 5'-phosphate group of adenylic acid. The 4'-phosphopantetheine residue is bound by a phosphodiester linkage to the 2' or 3' position of ribose and the remaining hydroxyl group of ribose is substituted with presumably an acidic sugar residue. The structural similarities of this prothetic group and coenzyme A are discussed and related to the catalytic properties of citrate lyase.     

A continuous coupled enzyme assay for bacterial malonyl-CoA:acyl carrier protein transacylase (FabD)

Bacterial malonyl-CoA:acyl carrier protein transacylase catalyzes the transfer of a malonyl moiety from malonyl-CoA to the free thiol group of the phosphopantetheine arm of acyl carrier protein. Malonyl-ACP, the product of this enzymatic reaction, is the key building block for de novo fatty acid biosynthesis. Here, we describe a continuous enzyme assay based on the coupling of the malonyl-CoA:acyl carrier protein transacylase reaction to alpha-ketoglutarate dehydrogenase (KDH). KDH-dependent consumption of the coenzyme A generated by malonyl-CoA:acyl carrier protein transacylase is accompanied by a reduction of nicotinamide adenine dinucleotide, oxidized (NAD(+)) to nicotinamide adenine dinucleotide, reduced. The rate of NAD(+) reduction is continuously monitored as a change in fluorescence using a microtiter plate reader. We show that this coupled enzyme assay is amenable to routine chemical compound screening.     

Acyl carrier protein is a cellular target for the antibacterial action of the pantothenamide class of pantothenate antimetabolites

Pantothenate is the precursor of the essential cofactor coenzyme A (CoA). Pantothenate kinase (CoaA) catalyzes the first and regulatory step in the CoA biosynthetic pathway. The pantothenate analogs N-pentylpantothenamide and N-heptylpantothenamide possess antibiotic activity against Escherichia coli. Both compounds are substrates for E. coli CoaA and competitively inhibit the phosphorylation of pantothenate. The phosphorylated pantothenamides are further converted to CoA analogs, which were previously predicted to act as inhibitors of CoA-dependent enzymes. Here we show that the mechanism for the toxicity of the pantothenamides is due to the inhibition of fatty acid biosynthesis through the formation and accumulation of the inactive acyl carrier protein (ACP), which was easily observed as a faster migrating protein using conformationally sensitive gel electrophoresis. E. coli treated with the pantothenamides lost the ability to incorporate [1-(14)C]acetate to its membrane lipids, indicative of the inhibition of fatty acid synthesis. Cellular CoA was maintained at the level sufficient for bacterial protein synthesis. Electrospray ionization time-of-flight mass spectrometry confirmed that the inactive ACP was the product of the transfer of the inactive phosphopantothenamide moiety of the CoA analog to apo-ACP, forming the ACP analog that lacks the sulfhydryl group for the attachment of acyl chains for fatty acid synthesis. Inactive ACP accumulated in pantothenamide-treated cells because of the active hydrolysis of regular ACP and the slow turnover of the inactive prosthetic group. Thus, the pantothenamides are pro-antibiotics that inhibit fatty acid synthesis and bacterial growth because of the covalent modification of ACP.     

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

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

Turnover of fatty acids in the 1-position of phosphatidylethanolamine in Escherichia coli

Phosphatidylethanolamine is the major membrane phospholipid of Escherichia coli, and two experimental approaches were used to investigate the metabolic activity of the fatty acids occupying the 1-position of this phospholipid. [3H]Acetate pulse-chase experiments with logarithmically growing cells indicated that 3-5% of the acyl groups were removed from the phosphatidylethanolamine pool/generation. The reacylation aspect of the turnover cycle was demonstrated by the incorporation of fatty acids into the 1-position of pre-existing phosphatidylethanolamine when de novo phospholipid biosynthesis was inhibited using the plsB acyltransferase mutant. 2- Acylglycerophosphoethanolamine would be the intermediate in a 1-position turnover cycle, and this lysophospholipid was identified as a membrane component that could re-esterified by a membrane-bound acyltransferase. The acyltransferase either utilized acyl-acyl carrier protein directly as an acyl donor or activated fatty acids for acyl transfer in the presence of ATP and Mg2+. Acyl-acyl carrier protein was also indicated as an intermediate in the latter reacylation reaction by the complete inhibition of phosphatidylethanolamine formation from fatty acids by acyl carrier protein-specific antibodies and by the observation that the inhibition of the acyltransferase by LiCl was reversed by the addition of acyl carrier protein. Coenzyme A thioesters were not substrates for this acyltransferase. These results suggest the existence of a metabolic cycle for the utilization of 1-position acyl moieties of phosphatidylethanolamine followed by the resynthesis of this membrane phospholipid from 2- acylglycerophosphoethanolamine by an acyl carrier protein-dependent 1-position acyltransferase.     

On the significance of the prosthetic group composition of citrate lyase.

1. Klebsiella aerogenes contains two different acyl carrier proteins, one specific for citrate lyase, the other for fatty acid synthetase. 2. The acyl carrier protein of fatty acid synthetase from K. aerogenes was isolated and compared with the corresponding protein from Escherichia coli and with the acyl carrier protein of citrate lyase from K. aerogenes. 3. As judged from prosthetic group compositions as well as amino acid and fingerprint analyses, the acyl carrier proteins of the two fatty acid synthetases are nearly identical but different from that of citrate lyase from K. aerogenes. 4. Therefore, the different prosthetic groups alone cannot be responsible for the different specificities of the acyl carrier proteins of fatty acid synthetase and citrate lyase in K. aerogenes. 5. The prosthetic group of citrate lyase, phosphoribosyl dephospho-CoA, apparently represents no incidental, phosphopantetheine-replacing aberration. The requirement of citrate lyase for the CoA-like prosthetic group may arise from the substrate requirement of both subunit enzymes of the enzyme complex.     

Mixed disulfides of acyl carrier protein and coenzyme A with specific soluble proteins in Escherichia coli.

Three soluble proteins in Escherichia coli specifically from mixed disulfides with either acyl carrier protein or coenzyme A. Coenzyme A was attached to one of these proteins, and the amount bound depended on the cellular coenzyme A concentration. The other two proteins were mixed disulfides between acyl carrier protein and each of the two 3-ketoacyl-acyl carrier protein synthases.     

Complete amino acid sequence of chicken liver acyl carrier protein derived from the fatty acid synthase

The acyl carrier protein domain of the chicken liver fatty acid synthase has been isolated after tryptic treatment of the synthase. The isolated domain functions as an acceptor of acetyl and malonyl moieties in the synthase-catalyzed transfer of these groups from their coenzyme A esters and therefore indicates that the acyl carrier protein domain exists in the complex as a discrete entity. The amino acid sequence of the acyl carrier protein was derived from analyses of peptide fragments produced by cyanogen bromide cleavage and trypsin and Staphylococcus aureus V8 protease digestions of the molecule. The isolated acyl carrier protein domain consists of 89 amino acid residues and has a calculated molecular weight of 10,127. The protein contains the phosphopantetheine group attached to the serine residue at position 38. The isolated acyl carrier protein peptide shows some sequence homology with the acyl carrier protein of Escherichia coli, particularly in the vicinity of the site of phosphopantetheine attachment, and shows extensive sequence homology with the acyl carrier protein from the uropygial gland of goose.     

Purification and characterization of the acyl carrier protein of the Streptomyces glaucescens tetracenomycin C polyketide synthase.

The acyl carrier protein (ACP) of the tetracenomycin C polyketide synthase, encoded by the tcmM gene, has been expressed in both Streptomyces glaucescens and Escherichia coli and purified to homogeneity. Expression of the tcmM gene in E. coli results mainly in the TcmM apo-ACP, whereas expression in S. glaucescens yields solely the holo-ACP. The purified holo-TcmM is active in a malonyl coenzyme A:ACP transacylase assay and is labeled by radioactive beta-alanine, confirming that it carries a 4'-phosphopantetheine prosthetic group.     

Occurrence of apoAcyl carrier protein and an S-alkylation-resistant form of holoAcyl carrier protein in Escherichia coli.

The 4′-phosphopantetheine prosthetic group of holoacyl carrier protein (holoACP) in Escherichia coli turns over independently of the apoprotein, due to the activities of holoACP hydrolase and holoACP synthetase. There is no measurable pool of apoACP in pantothenate-supplemented cells of a pantothenate-requiring mutant, but extended incubation on deficient medium, with exhaustion of cellular coenzyme A (CoA), leads to slow accumulation of the apoprotein. It is concluded that, although the activities of the synthetase and hydrolase are about equal in crude extracts, in the cells an excess synthetase activity maintains ACP completely as holoACP unless cells are artifically depleted of CoA, the donor of the 4′-phosphopantetheine group. About 20% of the holoACP in normal cells was designated as “holoACP esters,” being resistant to S-alkylation unless first treated with neutral hydroxylamine; this proportion increased to about 80% in pantothenate starvation. A preliminary attempt to identify acyl portions from this material was unsuccessful. The proportion of this material was not elevated in other strains under conditions which show feedback inhibition of fatty acid biosynthesis in vivo.     

Fatty acid metabolism in sn-glycerol-3-phosphate acyltransferase (plsB) mutants.

Fatty acid metabolism was examined in Escherichia coli plsB mutants that were conditionally defective in sn-glycerol-3-phosphate acyltransferase activity. The fatty acids synthesized when acyl transfer to glycerol-3-phosphate was inhibited were preferentially transferred to phosphatidylglycerol. A comparison of the ratio of phospholipid species labeled with 32Pi and [3H]acetate in the presence and absence of glycerol-3-phosphate indicated that [3H]acetate incorporation into phosphatidylglycerol was due to fatty acid turnover. A significant contraction of the acetyl coenzyme A pool after glycerol-3-phosphate starvation of the plsB mutant precluded the quantitative assessment of the rate of phosphatidylglycerol fatty acid labeling. Fatty acid chain length in membrane phospholipids increased as the concentration of the glycerol-3-phosphate growth supplement decreased, and after the abrupt cessation of phospholipid biosynthesis abnormally long chain fatty acids were excreted into the growth medium. These data suggest that the acyl moieties of phosphatidylglycerol are metabolically active, and that competition between fatty acid elongation and acyl transfer is an important determinant of the acyl chain length in membrane phospholipids.     

Correlation of Certain Alterations in Metabolic Activity with Alkaloid Production by Submerged Claviceps

Alkaloid biosynthesis in Claviceps paspali MG-6 was favored by unbalanced growth. A positive correlation between the rate of protein turnover and alkaloid formation was noted. The pattern of the orthophosphate content in the mycelium resembled that in the ripening sclerotia of the parasitic strains. Alkaloids were revealed as potentially effective in energy metabolism. Reduced adenosine triphosphate (ATP) utilization and an increase in the ATP pool were found to be favorable for alkaloid production. Acetyl-coenzyme A carboxylase activity and the level of cell lipids were directly related to the intensity of alkaloid biosynthesis. An inverse relationship was observed between the activities of the tricarboxylic acid and glycoxylate cycles and the rate of alkaloid formation.     

Purification of a malonyltransferase from Streptomyces coelicolor A3(2) and analysis of its genetic determinant.

Streptomyces coelicolor A3(2) synthesizes each half molecule of the dimeric polyketide antibiotic actinorhodin (Act) from one acetyl and seven malonyl building units, catalyzed by the Act polyketide synthase (PKS). The synthesis is analogous to fatty acid biosynthesis, and there is evident structural similarity between PKSs of Streptomyces spp. and fatty acid synthases (FASs). Each system should depend on a malonyl coenzyme A:acyl carrier protein malonyltransferase, which charges the FAS or PKS with the malonyl units for carbon chain extension. We have purified the Act acyl carrier protein-dependent malonyltransferase from stationary-phase, Act-producing cultures and have determined the N-terminal amino acid sequence and cloned the structural gene. The deduced amino acid sequence resembles those of known malonyltransferases of FASs and PKSs. The gene lies some 2.8 Mb from the rest of the act cluster, adjacent to an open reading frame whose gene product resembles ketoacylsynthase III of Escherichia coli FAS. The malonyltransferase was expressed equally as well during vegetative growth (when other components of the act PKS were not expressed) as in the stationary phase, suggesting that the malonyltransferase may be shared between the FAS and PKS of S. coelicolor. Disruption of the operon containing the malonyltransferase gene proved to be impossible, supporting the idea that the malonyltransferase plays an essential role in fatty acid biosynthesis.     

The phosphopantetheinyl transferase KirP activates the ACP and PCP domains of the kirromycin NRPS/PKS of Streptomyces collinus Tü 365

The main steps in the biosynthesis of complex secondary metabolites such as the antibiotic kirromycin are catalyzed by modular polyketide synthases (PKS) and/or nonribosomal peptide synthetases (NRPS). During antibiotic assembly, the biosynthetic intermediates are attached to carrier protein domains of these megaenzymes via a phosphopantetheinyl arm. This functional group of the carrier proteins is attached post-translationally by a phosphopantetheinyl transferase (PPTase). No experimental evidence exists about how such an activation of the carrier proteins of the kirromycin PKS/NRPS is accomplished. Here we report on the characterization of the PPTase KirP, which is encoded by a gene located in the kirromycin biosynthetic gene cluster. An inactivation of the kirP gene resulted in a 90% decrease in kirromycin production, indicating a substantial role for KirP in the biosynthesis of the antibiotic. In enzymatic assays, KirP was able to activate both acyl carrier protein and petidyl carrier domains of the kirromycin PKS/NRPS. In addition to coenzyme A (CoA), which is the natural substrate of KirP, the enzyme was able to transfer acyl-phosphopantetheinyl groups to the apo forms of the carrier proteins. Thus, KirP is very flexible in terms of both CoA substrate and carrier protein specificity. Our results indicate that KirP is the main PPTases that activates the carrier proteins in kirromycin biosynthesis.     

Holo-(acyl carrier protein) synthase and phosphopantetheinyl transfer in Escherichia coli

Holo-(acyl carrier protein) synthase (AcpS) post-translationally modifies apoacyl carrier protein (apoACP) via transfer of 4'-phosphopantetheine from coenzyme A (CoA) to the conserved serine 36 gamma-OH of apoACP. The resulting holo-acyl carrier protein (holo-ACP) is then active as the central coenzyme of fatty acid biosynthesis. The acpS gene has previously been identified and shown to be essential for Escherichia coli growth. Earlier mutagenic studies isolated the E. coli MP4 strain, whose elevated growth requirement for CoA was ascribed to a deficiency in holoACP synthesis. Sequencing of the acpS gene from the E. coli MP4 strain (denoted acpS1) showed that the AcpS1 protein contains a G4D mutation. AcpS1 exhibited a approximately 5-fold reduction in its catalytic efficiency when compared with wild type AcpS, accounting for the E. coli MP4 strain phenotype. It is shown that a conditional acpS mutant accumulates apoACP in vivo under nonpermissive conditions in a manner similar to the E. coli MP4 strain. In addition, it is demonstrated that the gene product, YhhU, of a previously identified E. coli open reading frame can completely suppress the acpS conditional, lethal phenotype upon overexpression of the protein, suggesting that YhhU may be involved in an alternative pathway for phosphopantetheinyl transfer and holoACP synthesis in E. coli.     

Consequences of reduced intracellular coenzyme A content in Escherichia coli.

Escherichia coli beta-alanine auxotrophs (panD2) were used to manipulate the specific cellular content of coenzyme A (CoA) and assess the associated physiological effects. Growth-limiting concentrations of CoA resulted in an increase in phospholipid/protein ratio in relA1 mutants, but not in their rel+ counterparts, indicating that protein biosynthesis was more severely affected by CoA deprivation than phospholipid biosynthesis. Acetyl-CoA was the dominant component (79.8%) of the CoA pool in cells exponentially growing in glucose-minimal medium, with significant concentrations of CoA (13.8%) and succinyl-CoA (5.9%) also detected. Malonyl-CoA was a minor species (0.5%), and the mixed disulfide of CoA and glutathione was not present. Acetyl-CoA was also the major constituent in cells depleted of CoA. On the other hand, succinyl-CoA was absent, suggesting that the protein synthesis defect may be due to the inability to generate sufficient quantities of precursors via the tricarboxylic acid cycle to support amino acid biosynthesis. Production of acyl carrier protein was controlled in part by the availability of CoA, and the lower concentration of acyl carrier protein in CoA-depleted cells was associated with a concomitant decrease in the saturated/unsaturated fatty acid ratio.