Mycobacterium smegmatis fatty acid synthetase. Long chain transacylase chain length specificity

Long chain transacylase activity, acyl-CoA + enzyme in equilibrium acyl-enzyme + CoA, catalyzed by the multienzyme complex fatty acid synthetase from Mycobacterium smegmatis was measured by exchange of radioactive coenzyme A into even numbered fatty acyl-CoA substrates 14 to 24 carbon atoms long. This transacylase activity decreases sharply with increasing chain length. It is suggested that C24 transacylation may be rate-limiting in de novo fatty acid synthesis catalyzed by the myocobacterial system. Mycobacterial polysaccharides stimulate the rate of transacylation, and this enhancement becomes more marked as the chain length of the substrate increases. The magnitude of the effect is similar to polysaccharide stimulation of overall synthetase activity. It is therefore proposed that terminal transacylation is the specific and perhaps only partial reaction catalyzed by the M. smegmatis fatty acid synthetase which is facilitated by polysaccharide. The product distribution of the synthetase is distinctly bimodal, with peaks for acyl chains 16 and 24 carbon atoms long. A scheme based on nonoverlapping unimodal chain length specificities for the rates of two activities, elongation and terminal transacylation, is offered to explain this bimodal distribution.     

Overproduction of a functional fatty acid biosynthetic enzyme blocks fatty acid synthesis in Escherichia coli

beta-Ketoacyl-acyl carrier protein (ACP) synthetase II (KAS II) is one of three Escherichia coli isozymes that catalyze the elongation of growing fatty acid chains by condensation of acyl-ACP with malonyl-ACP. Overexpression of this enzyme has been found to be extremely toxic to E. coli, much more so than overproduction of either of the other KAS isozymes, KAS I or KAS III. The immediate effect of KAS II overproduction is the cessation of phospholipid synthesis, and this inhibition is specifically due to the blockage of fatty acid synthesis. To determine the cause of this inhibition, we examined the intracellular pools of ACP, coenzyme A (CoA), and their acyl thioesters. Although no significant changes were detected in the acyl-ACP pools, the CoA pools were dramatically altered by KAS II overproduction. Malonyl-CoA increased to about 40% of the total cellular CoA pool upon KAS II overproduction from a steady-state level of around 0.5% in the absence of KAS II overproduction. This finding indicated that the conversion of malonyl-CoA to fatty acids had been blocked and could be explained if either the conversion of malonyl-CoA to malonyl-ACP and/or the elongation reactions of fatty acid synthesis had been blocked. Overproduction of malonyl-CoA:ACP transacylase, the enzyme catalyzing the conversion of malonyl-CoA to malonyl-ACP, partially relieved the toxicity of KAS II overproduction, consistent with a model in which high levels of KAS II blocks access of the other KAS isozymes to malonyl-CoA:ACP transacylase.     

The Saccharomyces cerevisiae FAT1 gene encodes an acyl-CoA synthetase that is required for maintenance of very long chain fatty acid levels

The Saccharomyces cerevisiae FAT1 gene appears to encode an acyl-CoA synthetase that is involved in the regulation of very long chain (C20-C26) fatty acids. Fat1p, has homology to a rat peroxisomal very long chain fatty acyl-CoA synthetase. Very long chain acyl-CoA synthetase activity is reduced in strains containing a disrupted FAT1 gene and is increased when FAT1 is expressed in insect cells under control of a baculovirus promoter. Fat1p accounts for approximately 90% of the C24-specific acyl-CoA synthetase activity in glucose-grown cells and approximately 66% of the total activity in cells grown under peroxisomal induction conditions. Localization of functional Fat1p:green fluorescent protein gene fusions and subcellular fractionation of C24 acyl-CoA synthetase activities indicate that the majority of Fat1p is located in internal cellular locations. Disruption of the FAT1 gene results in the accumulation of very long chain fatty acids in the sphingolipid and phospholipid fractions. This includes a 10-fold increase in C24 acids and a 6-fold increase in C22 acids. These abnormal accumulations are further increased by perturbation of very long chain fatty acid synthesis. Overexpression of Elo2p, a component of the fatty acid elongation system, in fat1Delta cells causes C20-C26 levels to rise to approximately 20% of the total fatty acids. These data suggest that Fat1p is involved in the maintenance of cellular very long chain fatty acid levels, apparently by facilitating beta-oxidation of excess intermediate length (C20-C24) species. Although fat1Delta cells were reported to grow poorly in oleic acid-supplemented medium when fatty acid synthase activity is inactivated by cerulenin, fatty acid import is not significantly affected in cells containing disrupted alleles of FAT1 and FAS2 (a subunit of fatty acid synthase). These results suggest that the primary cause of the growth-defective phenotype is a failure to metabolize the incorporated fatty acid rather than a defect in fatty acid transport. Certain fatty acyl-CoA synthetase activities, however, do appear to be essential for bulk fatty acid transport in Saccharomyces. Simultaneous disruption of FAA1 and FAA4, which encode long chain (C14-C18) fatty acyl-CoA synthetases, effectively blocks the import of long chain saturated and unsaturated fatty acids.     

Regulation of enzymatic activity involved in sex pheromone production in the housefly, Musca domestica

Ovarian produced ecdysteroids regulate sex pheromone production in the female housefly, inducing the synthesis of (Z)-9-tricosene (Z9-23:Hy), cis-9,10-epoxytricosane, (Z)-14-tricosen-10-one and methylalkanes. Experiments were performed to gain a detailed understanding of the processes affected by 20-hydroxyecdysone (20-HE) that result in sex pheromone production as the female becomes reproductively mature. A novel microsomal fatty acid synthetase (FAS) is present in the epidermal tissue and plays a role in producing the methyl-branched fatty acid precursors to the methylalkanes. This FAS is released from the microsomes in the presence of 3 M KCl. A major enzyme activity influenced by 20-HE is the fatty acyl-CoA elongation system. A shift in the chain length specificity of the products of the elongation system causes the change in the chain lengths of the alkenes produced to switch from C27 and longer in the previtellogenic female to C23 in the mature female. Data is presented indicating that it is the condensation activity of the elongation system that is affected. Z9-23:Hy arises from a 24 carbon acyl group which is reduced to an aldehyde, and then converted to the hydrocarbon. Data is presented demonstrating that it is the fatty acyl-CoA derivative and not the free fatty acid that is the substrate. There does not appear to be a chain length specificity which regulates the conversion of fatty acyl-CoAs to hydrocarbons as both 24 and 28 carbon fatty acyl-CoAs are converted to hydrocarbon by both males and females of all ages.     

Partial purification and properties of the fatty acid elongation systems in the outer and inner membranes of beef liver mitochondria

Purified outer membrane of beef liver mitochondria was found to elongate medium chain fatty acyl-CoA primer by the incorporation of [1-14C]acetyl-CoA. This enzymic activity, extracted by Triton X-100, was purified 8-fold by ammonium sulfate fractionation followed by chromatography on a Sephadex column. Purified inner membrane, when processed through an identical purification procedure, yielded a second enzyme system which incorporated [1-14C]acetyl-CoA into long chain fatty acids in the presence of medium chain fatty acyl-CoA primer. This enzyme preparation was about four times as active as the preparation from the outer membrane, and used NADH as the reductant for the synthesis. The molecular weights of the inner and the outer membrane enzyme systems, estimated by gel filtration as well as sucrose density gradient centrifugation, were approx. 57 000 and 126 000, respectively. The partially purified outer membrane enzyme system required NADH and a medium chain acyl-CoA primer for the incorporation of [1-14C]acetyl-CoA into long chain fatty acids. KNC stimulated the reaction. NADPH could substitute for NADH only to a limited extent. Malonyl-CoA was ineffective as a substrate in this reaction. The optimum pH of the reaction was 7.2-7.6 in 0.1 M potassium phosphate buffer. Dithiothreitol, beta-mercaptoethanol, N-ethylmaleimide and high concentrations of ATP and acyl-CoA primer inhibited the reaction. The specificity for the acyl-CoA primer in the reaction was very broad. All the primers tested, C8 to C16, incorporated acetyl-CoA significantly. However, maximum incorporation was observed with dodecanoyl-CoA. Decanoyl-CoA was the best primer for the enzyme system isolated from the inner membrane. About 42% of the radioactivity in the fatty acids synthesized by the outer membrane enzyme system, from myristoyl-CoA and [1-C14]acetyl-CoA, was in palmitic acid. Of the remaining activity, 41% was in stearic acid and about 38% in longer-chain acids. Hence, the elongation of the primer fatty acid by one C2 unit appeared to be the predominant process in this synthesis. In the elongation of myristoyl-C0A by the inner membrane enzyme system, palmitic acid which constituted nearly 78% of the fatty acids synthesized, was the primary product.     

Utilization of methylmalonate for the synthesis of branched-chain fatty acids by preparations of chicken liver and sheep adipose tissue

1. The utilization of methyl[2-14C]malonyl-CoA for fatty acid synthesis was investigated using synthetase preparations from chicken liver and sheep adipose tissue. 2. The rate of fatty acid synthesis from acetyl-CoA and malonyl-CoA was greatly diminished in the presence of methylmalonyl-CoA. 3. In the absence of malonyl-CoA, methylmalonyl-CoA was utilized for fatty acid synthesis only very slowly by the synthetase from sheep adipose tissue and not at all by that from chicken liver. 4. Despite the inhibitory effect of methylmalonyl-CoA on fatty acid synthesis from malonyl-CoA, it was utilized by the synthetase preparations from both species to produce a complex mixture of methyl-branched fatty acids.     

Fatty acid synthetase from the Harderian gland of guinea pig: biosynthesis of methyl-branched fatty acids

Fatty acid synthetase was isolated from guinea pig Harderian gland. This enzyme complex exhibited a unique character as compared with the fatty acid synthetase from the liver of the same animal. The former enzyme produced many odd-numbered and methyl-branched fatty acids in the presence of methylmalonyl-CoA. These fatty acids are characteristic components of the lipid secreted from this gland. The chemical structure of this lipid has been identified as 1-O-alkyl-2,3-diacylglycerol by previous work from this laboratory (Yamazaki, T., Seyama, Y., Otsuka, H., Ogawa, H., & Yamakawa, T. (1981) J. Biochem. 89, 683-691). Apparent Km values (5 X 10(-6) M) for acetyl-CoA and propionyl-CoA were the same, but the Vmax for propionyl-CoA was much higher than that for acetyl-CoA. The pI value of the fatty acid synthetase from Harderian gland was 5.3, and the molecular weight of the enzyme was 9 X 10(5) daltons. The beta-ketoacyl reductase had pro-S stereospecificity and the enoly reductase had pro-R stereospecificity for NADPH. The results presented in this paper indicate that the fatty acid synthetase from guinea pig Harderian gland can produce a set of fatty acids needed for the synthesis of the lipid secreted from this gland, and that the fatty acid synthetase has a characteristic organ specificity.     

Hypolipidemic activity of boronated nucleosides and nucleotides in rodents

Base-boronated nucleoside and phosphate-boronated nucleotides were potent hypolipidemic agents in rodents, lowering both serum cholesterol and triglyceride levels. Rat VLDL and LDL cholesterol levels were generally reduced and HDL cholesterol levels were significantly elevated after 14 days dosing at 8 mg/kg/day. Tissue cholesterol, triglyceride and phospholipid levels were reduced by selected derivatives. Increased fecal excretion of lipids did not appear to be a mechanism by which these derivatives lowered serum lipids in rodents. Rather, the agents suppressed appetite and reduced the activities of rate-limiting enzymes for de novo lipid synthesis, specifically cytoplasmic acetyl CoA synthetase, squalene synthetase, and phosphatidylate phosphohydrolase with IC50 values of approximately 10(-5) m.     

Identification of a gene encoding an acyl CoA:diacylglycerol acyltransferase, a key enzyme in triacylglycerol synthesis

Triacylglycerols are quantitatively the most important storage form of energy for eukaryotic cells. Acyl CoA:diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) catalyzes the terminal and only committed step in triacylglycerol synthesis, by using diacylglycerol and fatty acyl CoA as substrates. DGAT plays a fundamental role in the metabolism of cellular diacylglycerol and is important in higher eukaryotes for physiologic processes involving triacylglycerol metabolism such as intestinal fat absorption, lipoprotein assembly, adipose tissue formation, and lactation. DGAT is an integral membrane protein that has never been purified to homogeneity, nor has its gene been cloned. We identified an expressed sequence tag clone that shared regions of similarity with acyl CoA:cholesterol acyltransferase, an enzyme that also uses fatty acyl CoA as a substrate. Expression of a mouse cDNA for this expressed sequence tag in insect cells resulted in high levels of DGAT activity in cell membranes. No other acyltransferase activity was detected when a variety of substrates, including cholesterol, were used as acyl acceptors. The gene was expressed in all tissues examined; during differentiation of NIH 3T3-L1 cells into adipocytes, its expression increased markedly in parallel with increases in DGAT activity. The identification of this cDNA encoding a DGAT will greatly facilitate studies of cellular glycerolipid metabolism and its regulation.     

Characterization of fatty acid elongase enzymes from germinating pea seeds

In vitro biosynthesis of radioactive arachidate and behenate was observed when microsomal fractions of germinating pea seeds were incubated with exogenous stearoyl-CoA (18:0-CoA) or arachidoyl-CA (20:0-CoA) in the presence of NADPH, [2-14C]malonyl-CoA and ATP. Characterization of parameters required for optimal stearoyl- and arachidoyl-CoA elongation revealed that, at least, two chain-length-specific elongases are necessary for very-long-chain fatty acid synthesis. Both enzymes were found to be sensitive to the group-selective reagents, p-CMB, NEM, iodoacetate, arsenite and phenylglyoxal. Subcellular fractionation studies indicated that both of these elongases were localized mainly in the endoplasmic reticulum.     

Transport of activated fatty acids by the peroxisomal ATP-binding-cassette transporter Pxa2 in a semi-intact yeast cell system

In the yeast Saccharomyces cerevisiae, fatty acid beta-oxidation is restricted to peroxisomes. Previous studies have shown two possible routes by which fatty acids enter the peroxisome. The first route involves transport of medium-chain fatty acids across the peroxisomal membrane as free fatty acids, followed by activation within the peroxisome by Faa2p, an acyl-CoA synthetase. The second route involves transport of long-chain fatty acids. Long-chain fatty acids enter the peroxisome via a route that involves activation in the extraperoxisomal space, followed by transport across the peroxisomal membrane. It has been suggested that this transport is dependent upon the peroxisomal ATP-binding-cassette transporters Pxa1p and Pxa2p. In this paper we investigated whether Pxa2p is directly responsible for the transport of C18:1-CoA, a long-chain acyl-CoA ester. Using protoplasts in which the plasma membrane has been selectively permeabilised by digitonin, we show that C18:1-CoA, but not C8:0-CoA, enters the peroxisome via Pxa2p, in an ATP-dependent fashion. The results obtained may contribute to the elucidation of the primary defect in the human disease X-linked adrenoleukodystrophy.     

13C NMR study of the effects of leptin treatment on kinetics of hepatic intermediary metabolism

The recent discovery of leptin receptors in peripheral tissue raises questions about which of leptin's biological actions arise from direct effects of the hormone on extraneural tissues and what intracellular mechanisms are responsible for leptin's effects on carbohydrate and lipid metabolism. The present study is focused on the action of leptin on hepatic metabolism. Nondestructive 13C NMR methodology was used to follow the kinetics of intermediary metabolism by monitoring flux of 13C-labeled substrate through several multistep pathways. In perfused liver from either ob/ob or lean mice, we found that acute treatment with leptin in vitro modulates pathways controlling carbohydrate flux into 13C-labeled glycogen, thereby rapidly enhancing synthesis by an insulin-independent mechanism. Acute treatment of ob/ob liver also caused a rapid stimulation of long-chain fatty acid synthesis from 13C-labeled acetyl-CoA by the de novo synthesis route. Chronic leptin treatment in vivo induced homeostatic changes that resulted in a tripling of the rate of glycogen synthesis via the gluconeogenic pathway from [2-13C]pyruvate in ob/ob mouse liver perfused in the absence of the hormone. Consistent with the 13C NMR results, leptin treatment of the ob/ob mouse in vivo resulted in significantly increased hepatic glycogen synthase activity. Chronic treatment with leptin in vivo exerted the opposite effect of acute treatment in vitro and markedly decreased hepatic de novo synthesis of fatty acids in ob/ob mouse liver. In agreement with the 13C NMR findings, activities of hepatic acetyl-CoA carboxylase and fatty acid synthase were significantly reduced by chronic treatment of the ob/ob mouse with leptin. Our data represent a demonstration of direct effects of leptin in the regulation of metabolism in the intact functioning liver.     

Acetyl-C0A hydrolase; activity, regulation and physiological significance of the enzyme in brown adipose tissue from hamster

1. Acetate production in hamster brown adipose tissue is a consequence of the existence of an acetyl-CoA hydrolase. The enzyme is soluble and is localised to the mitochondrial matrix. 2. Acetyl-CoA hydrolase has an apparent Km for acetyl-CoA of 51 muM and a specific acitivyty at 30 degrees C of 870 nmol of acetate formed/min per mg 100 000 X g supernatant protein. 3. The enzyme is noncompetitively activated by ADP and inhibited by NADH and the effect of these nucleotides may well serve to regulate the enzyme activity in vivo. 4. A strong product inhibition by CoA is observed. The inhibition is of S-linear-I-hyperbolic noncompetitive nature. 5. The hydrolase has a q10 of 2.0, which represents a 7.3% change in the rat of acetate production per degrees C. The energy of activation is12 200 cal/mol (53905 J/mol). 6. The regulatory role of acetyl-CoA hydrolase for fatty acid oxidation in brown adipose tissue of the hamster (a hibernator) at low as well as at normal body temperature is discussed.     

The biosynthetic gene cluster for the macrolactone ring of the immunosuppressant FK506

Biosynthesis of the macrolactone ring of FK506 involves 10 elongation cycles that mechanistically resemble the steps in fatty acid synthesis. Sequencing of a 40-kb DNA segment of the FK506 gene cluster from Streptomyces sp. MA6548 has revealed two additional polyketide synthases (PKS) genes fkbB and fkbC which lie upstream of fkbA, a PKS gene recently shown to be responsible for the last four condensation steps of the FK506 biosynthesis [Motamedi, H., Cai, S. J., Shafiee, A. & Elliston, K. O. (1997) Eur. J. Biochem. 244, 74-80]. fkbB and fkbC are contiguous and encode respectively, the first (790129 Da) and the second (374438 Da) components of the FK506 polyketide synthase, a complex of three multidomain polypeptides. The predicted domain structures of FkbB and FkbC are analogous to that of FkbA and comprise 30 fatty-acid-synthase(FAS)-like domains arranged in 6 modules. Each module performs a specific extension cycle in the assembly of the carbon skeleton of the FK506 macrolactone ring. The component activities for the initiation of the polyketide chain consisting of a dihydrocyclohexenylcarbonyl coenzyme A (CoA) synthetase and a dihydrocyclohexenylcarbonyl CoA reductase required for the formation of the dihydrocyclohexylcarbonyl CoA starter unit and an acyl-carrier-protein to which the starter unit is anchored and translocated to the appropriate site on the PKS multienzyme are located at the N-terminal region of the FkbB polypeptide. A third gene, fkbL, lies at one end of the cluster and encodes lysine cyclodeaminase which catalyzes alpha-deamination and cyclization of the lysine into pipecolate. A fourth gene fkbP located at the other end of the sequence reported here encodes a peptide synthetase required for the activation and incorporation of the pipecolate moiety into the completed acyl chain. Finally the cluster carries a gene, fkbO, whose product is presumed to carry out a post-polyketide oxidation step of the FK506 marocycle.     

Structure of a mycobacterial polysaccharide-fatty acyl-CoA complex: nuclear magnetic resonance studies

MMP, a linear alpha 1 leads to 4 linked polymer of 3-O-methylmannose, regulates the fatty acid synthetase from Mycobacterium smegmatis by forming stoichiometric complexes with the long-chain acyl-CoA synthetase products. In agreement with previous proposals [Bloch, K. (1977) in Advances in Enzymology and Related Areas of Molecular Biology, ed. Meister, A. (Wiley, New York), Vol. 45, pp. 1-84], nuclear magnetic resonance studies show that the polysaccharide, a random coil in its free form, undergoes a major conformational transition upon enclosing long-chain acyl-CoA. The polysaccharide, probably in helical conformation in the complexed form, interacts with both the paraffinic chain and the CoA moieties of the included fatty acyl thioester.     

Effect of oxaloacetate and phosphorylation on ATP-citrate lyase activity

ATP-citrate lyase (CL) catalyzes the conversion of citrate and CoA to oxaloacetate (OA) and acetyl-CoA. As the coupled malic dehydrogenase (MDH) assay is not able either to study the effect of oxaloacetate (OA) on CL activity or to measure accurately CL activity in biological samples, a new assay was developed. The CL-citrate coupled CAT assay measures the amount of acetyl-CoA formed by transferring radiolabeled acetyl-CoA synthesized from [14C]citrate to chloramphenicol with chloramphenicol acetyltransferase (CAT). Employing this assay, the rate of increase in acetyl-CoA synthesis from citrate is linear with respect to added CL. Kinetic values for ATP, CoA and citrate are similar to those obtained using the MDH assay. The effect of CL phosphorylation on enzyme activity was determined. CL phosphorylated by cAMP-dependent protein kinase or by this kinase and glycogen synthase kinase-3 (GSK-3) decreases the apparent Vmax without changing the apparent Km. The effect of OA, a product of the enzyme reaction, on CL activity was also determined. Computational analysis of the data obtained without added OA and at three concentrations of OA indicate that the apparent Km for the substrate is not altered even though the apparent Vmax is decreased. The effect of OA on the activity of phosphorylated enzyme was also determined. OA decreases the apparent Vmax of the phosphorylated enzyme to the same extent as in control CL. This assay is able to measure CL activity in cytosol from 3T3-L1 adipocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fatty acid binding protein. Role in esterification of absorbed long chain fatty acid in rat intestine

Fatty acid binding protein (FABP) is a protein of 12,000 mol wt found in cytosol of intestinal mucosa and other tissues, which exhibits high affinity for long chain fatty acids. It has been suggested that FABP (which may comprise a group of closely related proteins of 12,000 mol wt) participates in cellular fatty acid transport and metabolism. Although earlier findings were consistent with this concept, the present studies were designed to examine its physiological function more directly. Everted jejunal sacs were incubated in mixed fatty acid-monoglyceride-bile acid micelles, in the presence or absence of equimolar concentrations of either of two compounds which inhibit oleate binding to FABP:flavaspidic acid-N-methyl-glucaminate and alpha-bromopalmitate. Oleate uptake, mucosal morphology, and oxidation of [14C]acetate remained unaffected by these agents, but oleate incorporation into triglyceride was inhibited by 62-64% after 4 min. The inhibition by flavaspidic acid was reversible with higher oleate concentrations. The effect of these compounds on enzymes of triglyceride biosynthesis was examined in intestinal microsomes. Neither flavaspidic acid nor alpha-bromopalmitate inhibited acyl CoA:monoglyceride acyl-transferase. Fatty acid:coenzyme A ligase activity was significantly enhanced in the presence of partially purified FABP, probably reflecting a physical effect on the fatty acid substrate or on the formation of the enzyme-substrate complex. Activity of the enzyme in the presence of 0.1 mM oleate was only modestly inhibited by equimolar flavaspidic acid and alpha-bromopalmitate, and this effect was blunted or prevented by FABP. We conclude that in everted gut sacs, inhibition of triglyceride synthesis by flavaspidic acid and alpha-bromopalmitate could not be explained as an effect on fatty acid uptake or on esterifying enzymes in the endoplasmic reticulum but rather can be interpreted as reflecting inhibition of fatty acid binding to FABP. These findings lend further support to the concept that FABP participates in cellular fatty acid transport and metabolism. It is also possible that FABP, by effecting an intracellular compartmentalization of fatty acids and acyl CoA, may play a broader role in cellular lipid metabolism.     

Pleiotropic phenotype of acetyl-CoA-carboxylase-defective yeast cells--viability of a BPL1-amber mutation depending on its readthrough by normal tRNA(Gln)(CAG)

The Saccharomyces cerevisiae gene BPL1 encodes the enzyme biotin:protein ligase (BPL), which is required for acetyl-CoA carboxylase (ACC) holoenzyme formation. Disruption of one of the two BPL1 alleles present in diploid cells results, upon sporulation, in a 2+:2(0) segregation of cell viability, with none of the two viable spores being BPL1 negative. In contrast to BPL1 deletants, BPL1 base-substitution mutants are potentially viable and may be isolated as long-chain-fatty-acid-requiring auxotrophs. In addition to ACC pyruvate carboxylase and an additional biotin-containing protein of unknown function fail to be biotinylated in BPL1-defective yeast mutants. In this study, one of these mutants, bpl1-C25/17, is shown to contain an amber stop codon at position 151 of the 689-amino-acid BPL sequence. In bpl1-C25/17 cells, de novo fatty acid synthesis is almost absent (< 2% of the wild type), while very-long-chain fatty acid (VLCFA) synthesis and, to some extent, medium-long-chain fatty acid elongation are still active. Hence, endogenous malonyl-CoA synthesis is reduced but not abolished by the translational stop mutation. A low rate of intact-BPL synthesis is accomplished in the mutant by occasional readthrough of the bpl1-C25/17 UAG nonsense triplet by normal yeast tRNA(Gln)(CAG). Correspondingly, ACC biotinylation is severely reduced though not completely absent in the two bpl1 mutants studied in this work. Residual BPL1 expression in bpl1-C25/17 cells is increased to a level allowing wild-type-like growth by transformation with high copy numbers of either the wild-type tRNA(Gln)(CAG) or the mutant bpl1-C25/17 genes. It is concluded that the lethality of BPL1 deletants is due to the lack of malonyl-CoA-dependent VLCFA synthesis and that the viability of distinct ACC-defective point mutants is due to their maintenance of a critical level of malonyl-CoA and, hence, VLCFA production. The residual capacity of malonyl-CoA synthesis, though, is inadequate to allow cytoplasmic bulk de novo fatty acid synthesis, nor does it support mutant growth on 13:0 as the only dietary fatty acid. ACC-defective mutants are respiratory deficient, which is attributed to the failure of mitochondrial fatty acid synthesis. Since lipoic acid levels of ACC1 and BPL1 mutants are essentially normal, an unknown product of mitochondrial fatty acid synthesis appears to be critically reduced in malonyl-CoA-deficient yeast cells.     

Identification of human acetyl-CoA carboxylase isozymes in tissue and in breast cancer cells

1. In the rat, acetyl-CoA carboxylase (ACC), a rate-limiting enzyme in fatty acid metabolism, exists as at least two different isozymes (M(r) 265,000 and 280,000) that display distinct tissue-specific distribution and regulation. 2. Based on the study of human tissue and human-derived breast cancer cell lines by enzyme isolation and protein blotting techniques, we have now identified two human isoforms of M(r) 265,000 (HACC 265) and 275,000 (HACC 275), each of which is homologous to one of the rat isozymes. 3. Human breast carcinoma cell lines show variable expression of these two isoforms, mirrored in the estimation of ACC acetyl-CoA kinetics.