Inhibition of fatty acid synthetases by the antibiotic cerulenin

The antibiotic cerulenin is a potent and apparently non-competitive inhibitor of fatty acid synthetase systems isolated from various microorganisms and from rat liver. Cerulenin specifically blocks the activity of β-keto acyl thioester synthetase (condensing enzyme). This effect may account for the inhibition of overall fatty acid synthesis by the antibiotic.     

Target of inhibition by the anti-lipogenic antibiotic cerulenin of sterol synthesis in yeast

The antibiotic cerulenin inhibited the incorporation of ¹⁴C-acetyl-CoA by 67% at a concentration of 9 × 10^?6 M but not that of ¹⁴C-HMG-CoA into the non-saponifiable fraction in a cell-free extract of Saccharomyces cerevisiae. Cerulenin markedly inhibited the activity of partially purified HMG-CoA synthase. No inhibition of acetoacetyl-CoA thiolase activity was observed in the same preparation of HMG-CoA synthase. Therefore, cerulenin inhibition of overall sterol synthesis may be accounted for by the specific inhibition of HMG-CoA synthase activity.     

Inhibition of unsaturated fatty acid synthesis in escherichia coli by the antibiotic cerulenin.

Low concentrations of cerulenin inhibit the growth of Escherichia coli by selectively blocking unsaturated fatty acid synthesis. This inhibition was relieved by unsaturated fatty acid supplements alone but not by saturated fatty acid supplements. The utilization of exogenous unsaturated fatty acids to sustain growth in the presence of cerulenin was confirmed by the analysis of bulk lipid composition. The effects of cerulenin on fatty acid synthesis were examined in vivo by pulse labeling with [14C]acetate and in vitro using [14C]malonyl-coenzyme A. In both cases, unsaturated fatty acid synthesis was inhibited by low concentrations of cerulenin with a stimulation of saturated fatty acid synthesis. Using mutant strains deficient in fatty acid synthesis, the effects of cerulenin on beta-ketoacyl-[acyl-carrier-protein] synthetases I and II were examined. Our results indicate that beta-ketoacyl-[acyl-carrier-protein] synthetase I is more sensitive to inhibition by cerulenin than beta-ketoacyl-[acyl-carrier-protein] synthetase II.     

A study of the influence of the growth media on the fatty acid composition inCandida lipolytica diddens and lodder

Candida lipolytica YB 423-12 is able to incorporate fatty acid from the culture medium when lipids are used as carbon substrate. The composition of cell lipids is largely dependent on that of the culture medium. An important 9 desaturase activity acts on incorporated palmitic and stearic acids; and 11-eicosenoic and erucic acids are shortened to oleic acid.     

Effect of a Teucrium polium L. extract on the growth and fatty acid composition of Saccharomyces cerevisiae and Yarrowia lipolytica

Aqueous Teucrium polium extract slightly inhibits the growth of Saccharomyces cerevisiae (K_i=0.029 [g/l]^-1) and Yarrovia lipolytica (K_i=0.061 [g/l]^-1). However, this extract causes important changes in the unsaturation degree (/mol) of the cellular lipids. It moreover favours the increase of the linolenic acid concentration and the decrease of the oleic one in both species.     

Binding site of cerulenin in fatty acid synthetase

An antibiotic cerulenin, (2R, 3S)-2,3-epoxy-4-oxo-7,10-trans,trans- dodecadienamide, irreversibly inhibits fatty acid synthetase from Saccharomyces cerevisiae. Three moles of cerulenin were bound to 1 mol of the enzyme with concomitant loss of its activity. Pretreatment of the enzyme with iodoacetamide reduced the amount of cerulenin bound to the enzyme. Since iodoacetamide is known to specifically bind to the cysteine residue on the condensing reaction domain, cerulenin is considered to bind to the same domain. Tryptic digestion of the [3H] cerulenin-treated enzyme gave a radioactive peptide; its amino acid composition was Asx 1, Thr 1, Ser 1, Glx 2, Pro 1, Gly 1, Ala 1, Val 1, Ile 1, and Leu 2. This composition included all the amino acids of the condensing reaction site (Thr-Pro-Val-Gly-Ala-Cys) previously reported by Kresze et al. (Eur. J. Biochem., 79, 181 [1977] except for Cys. When the enzyme was treated with [3H]cerulenin and digested successively with trypsin and carboxypeptidase P, a [3H] cerulenin-cysteine adduct was isolated as the sole product. This was identified with the adduct chemically synthesized from non-labeled cerulenin and cysteine, and its structure was elucidated by 1H-, 13C-NMR, and fast atom bombardment mass spectrometry. These results indicate that cerulenin, forming a hydroxylactam ring, reacts at its epoxide carbon (C-2 position) with the SH-group of the cysteine residue in the condensing reaction domain of yeast fatty acid synthetase.     

Inhibition of 6-methylsalicyclic acid synthesis by the antibiotic cerulenin.

Cerulenin, a specific inhibitor of beta-ketoacyl-(acyl carrier protein) synthetase [EC 2.3.1.41] and 3-hydroxy-3-methylglutaryl-CoA synthetase [EC 4.1.3.5], was studied to determine whether it inhibits 6-methylsalicylic acid synthesis, in which so-called "polyketide" formation, a condensation step similar to that in fatty acid synthesis, is involved. In fact, 100 mug/ml (4.5 X 10(-4) M) of cerulenin inhibited 60% of 6-methylsalicylic acid synthetase activity and 68% of fatty acid synthetase activity of Penicillium urticae.     

Effects of cerulenin, an inhibitor of fatty acid synthesis on reconstitution of the dental basement membrane

When trypsin-dissociated enamel organs and dental papillae were recombined in the presence of cerulenin--an antibiotic which is a potent inhibitor of fatty acid synthesis--the newly synthesized basement membrane seemed defective in a dose-dependent manner. The three-dimensional relationship between the basement membrane components and the plasma membrane appears to be regulated in part by lipids.     

Effect of growth temperature on the fatty acid composition of Mycobacterium phlei

In Mycobacterium phlei, fatty acid unsaturation increased with decreasing temperature. The 10-hexadecenoic acid content increased as the temperature was reduced from 35°C to 26–20°C. At lower temperatures tuberculostearic acid decreased while oleic and linoleic acids increased, the latter being found in M. phlei for the first time. Concomitantly palmitic acid content decreased, and the 6- and 9-hexadecenoic acids increased slightly on reducing the temperature from 35 to 10°C. Thus, down to 26–20°C palmitic acid was mainly replaced by 10-hexadecenoic acid. From this range down to 10°C, palmitic and tuberculostearic acids were replaced by oleic and linoleic acids. Consequently, fatty acid branching decreased and mean chain length increased, as the temperature was reduced. These observations support the view that regulation of membrane fatty acid composition is part of microbial temperature adaptation, and that themechanism behind the responses might be more complex than generally believed.Abbreviations ACP acyl carrier protein - FAS I (Type I) fatty acid synthetase I - FAS II (Type II) fatty acid synthetase II - MGLP methylglucose containing lipopolysaccharide - MMP methylmannose containning polysaccharide     

Effects of conservation method on fatty acid composition of silage

This experiment was conducted to investigate effects of wilting and additives on the fatty acid (FA) composition of grass silage. The crop used was timothy (Phelum pratense L., cv. Grindstad), and the additives were Proens? (formic acid and propionic acid, 60–66 g/100 g and 25–30 g/100 g, respectively), the bacterial inoculant Siloferm® Plus (Pediococcus acidilactici and Lactobacillus plantarum) and water (control). The wilted material reached a dry matter (DM) content of 336 g/kg at the first cut and 350 g/kg at the second cut. Neither wilting nor the additives had any major effect on the FA proportions, with the only differences in the concentrations of C16:0 and C18:3. Silage treated with bacterial inoculant contained a higher proportion of C16:0 (P<0.05) than silage treated with acid, and a lower (P<0.05) concentration of C18:3 than silage treated with either of the other two additives. In the silages, there were lower (P<0.05) proportions of C16:0, C18:0, C18:1 and C18:3, and higher (P<0.05) proportions of C16:1, C18:2 and other identified FAs, than in the fresh material. A wilting process shorter than 24 h, to a DM content of 330–350 g/kg, did not have any effect on the proportions of FAs in P. pratense L., cv. Grindstad. Since the different additives and wilting strategies tested in this study did not affect the proportions of FAs in silage to a major extent, the results indicate that such a process offers a robust means to avoid losses of FAs that can occur during wilting, while retaining the positive effects of wilting, such as reduced losses of nutrients through effluents.     

Effects of linoleic acid and mitogenic stimulation on the fatty acid composition of human lymphocytes

Perturbation of the fatty acid composition of human lymphocytes in vitro was investigated by addition of linoleic acid complexed to bovine serum albumin (BSA-LA) and by mitogenic stimulation with phytohaemagglutinin (PHA). BSA-LA resulted in a 45% increase in linoleic acid in phosphatidylethanolamine (PE) and over 100% in phosphatidylcholine (PC) in peripheral blood cells. Supplementation with BSA-LA in PHA-stimulated lymphocytes produced even greater changes: 100% increase in linoleic acid content for PE and over 300% for PC. There was a large decrease in oleic acid: 40% for PE and almost 100% in PC. Significant decreases in arachidonic acid occurred in both phospholipid fractions. PHA alone also altered membrane phospholipid fatty acid composition, with reductions in palmitic, stearic and linoleic acid for PE and increases in oleic acid and arachidonic acid (almost 100%). For PC, there were large decreases in stearic (40%), linoleic (30%) and arachidonic (40%) acids, together with an increase in oleic acid (65%). Cells supplemented with linoleic acid grown in the presence of PHA, compared with those grown in linoleic acid-supplemented medium alone, showed a 40% decrease in palmitic acid and a 55% increase in arachidonic acid in PE. For PC, there were large decreases in stearic acid (40%) and arachidonic acid (57%). Antibody-induced redistribution of surface molecules ('capping') was inhibited by some 14% after incubation with BSA-LA. However, no consistent alterations in PHA-induced cell proliferation were observed. These data suggest that profound alterations of membrane fatty acid composition occur spontaneously during the mitotic cycle, and may be further induced by experimental manipulation, without gross perturbation of cell function.     

Effects of artificial ultraviolet-B radiation on growth and fatty acid composition of duckweed (Lemna minor)

1. Duckweed (Lemna minor), collected either in summer or early fall was exposed under laboratory conditions to control (photosynthetically active and UV‐A radiation) or experimental (control plus UV‐B radiation) conditions. 2. Growth and survival were determined by counting the number of green, and brown/white fronds following 1–5 or 11 days of irradiation. Growth of duckweed was impaired by exposure to UV‐B radiation in the fall experiment but not in the summer. 3. Fatty acid compositions were analysed following 5 or 11 days of irradiation and a recovery period of 0, 5, 29 or 40 h. Concentrations of the major fatty acids, palmitic, linoleic (LA) and α‐linolenic (ALA) acids were similar in the summer and fall duckweed collections, but the summer samples had higher concentrations of the desaturation products of LA and ALA. 4. UV‐B exposure had small, but significant, and contrasting effects on duckweed fatty acid concentrations. In the summer experiment, duckweed exposed to UV‐B had slightly lower concentrations of major fatty acids than control duckweed, while the reverse was true in the fall experiments. 5. These minor effects of UV‐B on concentrations of LA and ALA would be unlikely to have a major impact on the supply of these essential fatty acids from duckweed to freshwater food webs.     

Sterol and fatty acid composition of polyene macrolide antibiotic resistant Torulopsis glabrata.

Successive reculturing of Torulopsis glabrata on media containing increasing concentration of the polyene macrolide antibiotics nystalin or lucensomycin resulted in the segregation of cultures resistant to these antibiotics. Isolates resistant to lucensomycin showed good resistance to nystatin, and vice versa. Analysis of the sterols and fatty acids of sensitive and polyene resistant T. glabrata revealed that compositional changes occurred in both classes of lipids upon acquistion of resistance. The sterol composition of nystatin and lucensomycin resistant cultures possessed reduced amounts of, or no ergosterol (the major sterol of the sensitive parent culture), and increased amounts of sterols which were biogenetically more primitive than ergosterol. Resistant cultures in which ergosterol was absent possessed a fatty acid composition that did not differ significantly from the parent sensitive culture grown under identical conditions. Resistant cultures containing significantly reduced amounts of ergosterol were found to possess altered fatty acid compositions. Generally it was observed that these latter cultures possessed fatty acids containing shorter and more saturated chains. These results are considered to indicate that alteration in both lipid and sterol composition is involved in determination of culture resistance to polyene macrolides.     

Influence of selected sugars and temperature on fatty acids composition in Candida lipolytica

Summary An investigation was made of the effects of temperature and of mono- and disaccharides on lipids, biomass, odd-chain and unsaturated fatty acids production of Candida lipolytica. With different sugars as carbon source at 30°C, the order for biomass production was: fructose > glucose > sucrose > lactose > galactose, while lipids production/g biomass decreased as follows: lactose, sucrose, galactose, fructose and glucose. On the other hand, the odd-chain fatty acids contents decreased in the following order: fructose, lactose, glucose, sucrose and galactose. Lowering the temperature of cultivation to 15°C, biomass, lipids and unsaturated fatty acids decreased. However, a notable decrease in the content of odd-chain fatty acids was detected.     

Effect of growth temperature and media composition on the fatty acid composition of Bacillus stearothermophilus AN 002

The influence of growth temperature, media composition and cell age on the chemical composition of Bacillus stearothermophilus strain AN 002 has been determined. The total cellular protein decreased and the free amino acid content increased with growth temperature, in both exponential and stationary growth phase. The protein and free amino acid contents of cells were higher in the stationary phase than in the exponential phase, irrespective of growth temperature and media composition. The RNA content was only reduced in cells grown at 55° C. No significant variations were observed in the DNA and carbohydrate contents with respect to growth temperature and cell age. The total lipid and fatty acid compositions on the other hand varied as a function of growth temperature, cell age and media composition. Differences in the relative concentrations of even, odd and branched chain fatty acids were noticed. Novariation was observed in the antiiso and unsaturated fatty acids with respect to growth temperature. The unique variations in the fatty acid composition and total lipids at the growth temperature of 50° C and their variations in the stationary growth phase seem to be characteristic for B. stearothermophilus AN 002.     

Effect of growth phase on the fatty acid composition of Thermus spp.

Two yellow and two red pigmented strains of Thermus were monitored for changes in fatty acid content and composition with reference to growth phase at the optimum temperature. Fatty acid content per mg of dry weight increased as the cultures aged. In addition the quantities of iso C 15:0, iso C 17:0 and iso C 16:0 increased in yellow pigmented strains, but in red pigmented strains, an increase was seen in iso C 15:0, but C 16:0 and iso C 16:0 levels decreased. Thus the fatty acid composition of these organisms varies with growth phase, and shows also strain specific variability.