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1.
Because of their structures, phleic acids (general formula: CH3-(CH2)m-(CH=CH-CH2-CH2)n-CO2H; main component: m = 14, n = 5) cannot be synthesized by the same kinds of enzymatic systems as other natural polyunsaturated fatty acids. By using specifically labelled 14C compounds, we have tested the ability of different molecules to be incorporated in the phleate skeletons by Mycobacterium phlei. The localisation of radioactive carbon atoms has been studied by chemical degradation of labelled phleates, isolation and purification of the degradation products, and determination of their specific radioactivity. When M. phlei cells are incubated with labelled acetate, the unsaturated and saturated parts of the molecules of phleic acids are unequally labelled. The radioactivity of succinate monoester on the one hand and fatty acids (mixture of myristic and palmitic acids) on the other hand, measured after oxidative degradation of phleate esters, shows a constant ratio under definite conditions. Whether [1-14C]acetate or [2-14C]acetate is used for incubation, the same ratio is observed. Therefore acetate is the precursor of the unsaturated part as well as of the saturated part of the phleate molecules. By using labelled fatty acid esters, it has been found that palmitic acid is the precursor of phleates with m = 14, while myristic acid is the precursor of phleates with m = 12. Stearic and eicosanoic acids are not incorporated without degradation. The hypothesis of a condensation of a saturated fatty acid with a preformed polyunsaturated molecule was examined. Search for such a molecule in the lipids of M. phlei gives negative results. Pentaunsaturated phleate arising from palmitate is more abundant than pentaunsaturated phleate arising from myristate, while the reverse is true for hexaunsaturated phleates. These observations make very unlikely such an hypothesis. An elongation process fits well with the observed facts provided that this process involves elongation by two acetate units simultaneously, making elongation by four carbon atoms at a time. Such a requirement would be easily satisfied if two molecules of acetate are condensed together before their utilization in the elongation process. In such a hypothetical process, crotonate would be the most probable substrate of the elongation reaction.  相似文献   

2.
A 10,800 X g supernatant fraction from disrupted cells of Mycobacterium tuberculosis H37Ra was incubated with [2-14C]malonate to produce labeled long-chain fatty acids upon saponification. These acids were derivatized to the p-bromophenacyl ester and separated into the nonmycolic saturated, monounsaturated, and multiunsaturated esters by argentation thin-layer chromatography. Each of these fractions was then analyzed by high-performance liquid chromatography by using a C18-bonded silica cartridge and a mobile phase of a linear gradient of 0 to 70% p-dioxane in acetonitrile. The results showed that the cell-free system is able to synthesize both saturated and monounsaturated fatty acids of the sizes C30 to C40 and C48 to C56. This latter series was strikingly similar to meromycolic acid, a putative precursor of mycolic acid. When acetate or oleate was used as the labeled substrate, the major products were no longer than C32. When palmitate was used as the labeled substrate, the saturated acids ranged in size from C18 to C32, whereas the monounsaturated products contained C18, C26 to C30 and C40 fatty acids. Fatty acids greater than C40 were also detected. When methyl-labeled S-adenosyl-L-methionine was used as the substrate, the methyl group was incorporated into short-chain and C48 to C56 fatty acids. Unlabeled malonyl-coenzyme A was included in all of these reactions. This cell-free system was not able to synthesize mycolic acid (final product) or its keto derivative (intermediate product). However, since the meromycolate-like C48 to C56 fatty acids were synthesized, we suggest that the present system is able to take the synthesis to a point before the alpha-alkyl condensation reaction.  相似文献   

3.
Mycolic acid synthesis by Mycobacterium aurum cell-free extracts   总被引:6,自引:0,他引:6  
The first cell-free system capable of synthesizing whole mycolic acids: (R1CH(OH)CH(R2)COOH, with 60 to 90 carbon atoms) from [1-14C]acetate is described and preliminary investigations into some of its requirements and properties are reported. Biosynthetic activity for mycolic acids occurred in an insoluble fraction (40 000 X g pellet) from disrupted cells of Mycobacterium aurum (ATCC 23366-type strain); it produced mycolic acids, but a very small amount of non-hydroxylated fatty acids. The predominant product was unsaturated mycolic acid (type I), while oxo- (type IV) and dicarboxy- (type VI) mycolic acids were synthesized to a lesser extent. When [1-14C]palmitic acid was used as a marker, no labelled mycolic acid was detected. The reaction required a divalent cation (Mg2+ or Mn2+), KHCO3 and O2. Neither CoA, NADH, NADPH nor ATP were necessary, but CoA rather increased the synthesis of non-hydroxylated fatty acids. Glucose or trehalose were not required. Avidin inhibited the biosynthesis of the three types of mycolic acid indicating the presence of a biotin-requiring enzyme in the reaction sequence and therefore a carboxylation step, but citrate had no allosteric effect. Iodoacetamide inhibited the system. These first data are in favor of a complex multienzyme system.  相似文献   

4.
Mycolic acids are 70-90 carbon, alpha-alkyl, beta-hydroxy fatty acids constituting a major component of the cell envelope of Mycobacterium tuberculosis. The fact that the mycolic acid biosynthetic pathway is both essential in mycobacteria and the target for many first-line anti-TB drugs necessitates a detailed understanding of its biochemistry. A whole cell-free, but cell particulate- and membrane-containing enzyme preparation for mycolic acid biosynthesis was developed a few years ago and studied extensively. This system was shown to catalyze the synthesis of mature mycolic acids from [14C]acetate, but allows only minimal deposition into the cell wall proper. In the meantime the sequence of the entire genome of M. tuberculosis has been elucidated and its analysis using numerous protein sequence-based algorithms predicted cytoplasmic localization and a soluble, not a particulate, nature for the enzymes involved in the mycolic acid synthetic pathway. Accordingly, we re-assessed the 'cell-free' system for mycolic acid synthesis and concluded that it is probably due to the presence of unbroken cells, since viable cells were recovered from the cell wall preparation. The amount of whole cells depended upon the efficiency of the cell disruption method and conditions, and the amount of mycolic acid synthesized by the putative cell-free system correlated with the content of whole cells. Thus, accumulated results from the use of this 'cell-free' cell wall-based system should be re-evaluated in the light of these new data.  相似文献   

5.
Methyl esters of normal fatty acids, corynomycolate and corynomycolenate were used as model compounds for thermospray mass spectrometric procedures for molecular weight determination of the related nocardial mycolic acids. By using ammonium acetate at the positive ion generator, in both cases, a family of ions was produced. The following members were found and corresponded to the adducts: (1) M + H; M + NH4 and M + H + NH4 for methyl esters of normal fatty acids, whereas M + H, M + 2H and M + H + NH4 were the adducts most frequently observed with methyl corynomycolates. The methyl esters of C40-C48 mycolic acids from Rhodococcus rhodochrous exhibited prominent peaks corresponding to adducts M + H + NH4 whereas those corresponding to M + 2H showed slightly lower intensities. The structure M + H had no significant representatives with this subclass of mycolic acids. A similar pattern was observed with methyl esters of C50-C54 mycolic acids from Nocardia asteroides GUH-2. Ion peaks C50-C54 representing adducts M + 2H and M + H + NH4 prevailed in the mass spectrum. In this case, the intensities of peaks corresponding to M + 2H were slightly higher than those of the M + H + NH4. Essentially three main species of nocardomycolic acids were detected: (1) monounsaturated C50:1, C52:1 and C54:1; (2) diunsaturated C50:2, C52:2 and C54:2 and (3) triunsaturated C52:3 and C54:3 mycolic acids. The most abundant mycolic acid was C52:2 followed in decreasing abundance by C52:1, C54:2, C50:2, C52:3 and C54:3 mycolic acids.  相似文献   

6.
1. Radioactivity from cyclopropane[(14)C]carboxylic acid is incorporated into fatty acids in vitro by rat and guinea-pig adipose tissue, by rat liver slices and by the supernatant fraction of rat liver homogenate. 2. The labelled acids are different from endogenous straight-chain fatty acids, and evidence is produced that they consist of a cyclopropyl ring in the omega-position, the remainder of the chain being built up from C(2) units (not derived from cyclopropanecarboxylic acid) in the normal way via the malonate pathway. 3. It is suggested that these unnatural acids have some metabolic effect related to the hypoglycaemic action of cyclopropanecarboxylic acid.  相似文献   

7.
[14C]acetate was incorporated into mycolic acids by a cell-free, cell-wall fraction from Mycobacterium smegmatis . This activity was inhibited by methyl 4-(2-octadecylcyclopropen-1-y1) butanoate which was designed as a structural analogue of cis -tetracos-5-enoate, a precursor of mycolic acid biosynthesis. Other fatty acids and their methyl esters failed to inhibit mycolic acid biosynthesis at the concentration 1–2 mg ml-1, at which methyl 4-(2-octadecylcyclopropen-1-y1) butanoate was effective. Thus a novel agent was shown to act against an enzyme activity or target involved specifically in biosynthesis of a characteristic, mycobacterial, cell-wall component.  相似文献   

8.
The short-term effect of T3 both on de novo synthesized and on exogenously added fatty acids was studied in isolated rat hepatocytes. Lipogenesis from [14C] acetate or [3H] H2O was stimulated by the addition of T3. In contrast, the utilization of exogenous [14C] palmitate for the synthesis of longer chain fatty acids was markedly reduced. This T3-induced inhibition was removed by octanoylcarnitine, an inhibitor of carnitine palmitoyl-transferase I and of fatty acid oxidation. T3 also stimulated glycerolipid synthesis from acetate, neutral lipids being more influenced than phospholipids, but reduced the incorporation of palmitate in all the lipid fractions. It is suggested that T3 exerts opposing effects on the hepatic utilization of newly synthesized and exogenous fatty acids.  相似文献   

9.
Mycolic acids, the hallmark of mycobacteria and related bacteria, are major and specific components of their cell envelope and essential for the mycobacterial survival. Mycobacteria contain structurally related long-chain lipids, but the metabolic relationships between these various classes of compounds remain obscure. To address this question a series of C(35) to C(54) nonhydroxylated fatty acids (mycobacteric acids), ketones, and alcohols structurally related to the C(70-80) dicyclopropanated or diethylenic mycolic acids were characterized in three mycobacterial strains and their structures compared. The relationships between these long-chain acids and mycolic acids were established by following the in vivo traffic of (14)C labeled alpha-mycolic acids purified from the same mycobacterial species. The labeling was exclusively found in mycobacteric acids. The mechanism of this degradation was established by incorporation of (18)O(2) into long-chain lipids and shown to consist in the rupture of mycolic acids between carbon 3 and 4 by a Baeyer-Villiger-like reaction. We also demonstrated that mycobacteric acids occur exclusively in the triacylglycerol (TAG) fraction where one molecule of these acids esterifies one of the three hydroxyl groups of glycerol. Altogether, these data suggest that these compounds represent a pathway of metabolic energy that would be used by mycobacteria in particular circumstances.  相似文献   

10.
The nonmycolic C16 to C55 fatty acids obtained from Mycobacterium smegmatis ATCC 356 by saponification were enriched with respect to the C28 to C55 acids by successive chromatography on silicic acid and Sephadex LH-20 columns. These partially purified fatty acids were then derivatized to the p-bromophenacyl ester and further fractionated by argentation thin-layer chromatography and reverse-phase high-performance liquid chromatography into their individual components. The esters were characterized by electron impact mass spectrometry. Two structural series of C28:1 to C42:1 and C45:2 to C55:2 fatty acids were identified as possible precursors of the monoenyl and dienyl mycolic acids, respectively. These acids were structurally related to the alpha-alkylhydroxyl group of the corresponding mycolic acid. The results suggest that these C28 to C55 fatty acids (meromycolic acids) of M. smegmatis might be precursors of mycolic acids.  相似文献   

11.
The metabolism of 1-11C-labelled derivatives of palmitic (C16:0), arachidonic (C20:4,n-6) lignoceric (C21:0) and tetracosatetraenoic (C24:4,n-6) acids was studied in normal skin fibroblast cultures and in cultures of fibroblasts from peroxisome-deficient (Zellweger's syndrome) patients. Radiolabelled products of the fatty acids included carbon dioxide. C14-24 saturated and mono-unsaturated fatty acids formed from released acetate either by synthesis de novo or by elongation of endogenous fatty acids, fatty acids formed by 2-6-carbon elongation of added substrates, and a number of water-soluble compounds, some of which were tentatively identified as the amino acids glutamine, glutamic acid and asparagine. The labelled amino acids were found predominantly in the culture medium. Zellweger's syndrome fibroblasts showed a marked decrease in radiolabelled carbon dioxide and water-soluble-product formation from (I-14C)-labelled arachidonic, tetracosatetraenoic and lignoceric acids but not from [I-14C]palmitic acid, and the production of radiolabelled C14-18 fatty acids was also diminished. However, the elongation of individual fatty acids was either normal or above normal. Our data support the view that the oxidation of 20:4, 24:4 and 24:0 fatty acids in cultured skin fibroblasts takes place largely in peroxisomes, and further that the acetyl-CoA released by the beta-oxidation process is available for the synthesis of fatty acids and amino acids. We speculate that the generation of C2 units used for synthesis is a major peroxisomal function and that this function is absent or greatly impaired in Zellweger's syndrome cells.  相似文献   

12.
Overproduction of glutamate by Corynebacterium glutamicum is induced by biotin limitation or by the supplementation of specific detergents, sublethal amounts of penicillin, or cerulenin. But, it remains unclear why these different treatments, which have different sites of primary action, produce similar effects.In this study, it was found that the cellular content of mycolic acids--characteristic constituents of Corynebacterineae that are synthesized from fatty acids and form a cell surface layer--decreased under all conditions that induced glutamate overproduction. Furthermore, short mycolic acids increased under conditions of biotin limitation and cerulenin supplementation. These results suggest that different treatments produce the same effect that causes defects in the mycolic acid layer. This is perhaps one of the key factors in overproduction of glutamate by C. glutamicum.  相似文献   

13.
1. The patterns of incorporation of (14)C into glycerolipid fatty acids of developing maize leaf lamina from supplied [1-(14)C]acetate and from (14)CO(2) during steady-state photosynthesis were similar. Oleate of phosphatidylcholine and palmitate of phosphatidylglycerol attained linear rates of labelling more rapidly than did other fatty acids, particularly the linoleate and linolenate of monogalactosyl diacylglycerol. 2. After the transfer of lamina from labelled to unlabelled acetate, there was a decrease in labelled oleate and linoleate of phosphatidylcholine and a concomitant increase in the amount of radioactivity in the linoleate and linolenate of monogalactosyl diacylglycerol. 3. The rapidly labelled phospholipids, phosphatidylcholine and phosphatidylglycerol, were shown by differential and sucrose-density-gradient centrifugation to be associated with different organelles, the former being mainly in a low-density membrane fraction, probably microsomal, and the latter mainly in chloroplasts. 4. During a 48h period after supplying spinach leaves with [(14)C]acetate, radioactivity was lost from the oleate of phosphatidylcholine present in fractions sedimented at 12000g and 105000g, and accumulated in the linolenate of monogalactosyl diacylglycerol of the chloroplast. 5. It is proposed that the phosphatidylcholine of some non-plastid membranes is intimately involved in the process of oleate desaturation and that this lipid serves as a donor of unsaturated C(18) fatty acids to other lipids, principally monogalactosyl diacylglycerol, of the chloroplasts.  相似文献   

14.
Biosynthetic activity for mycolic acid occurred in the fluffy layer fraction but not in the 5000g supernatant of Bacterionema matruchotii. With [1-14C]palmitic acid as precursor for the in vitro system, the predominant product was identified as C32:0 mycolic acid by radio-gas-liquid chromatographie (radio-GLC) and gas chromatographic/mass spectroscopic analyses; if [1-14C]stearic acid was used, two major radioactive peaks appeared on GLC: one corresponding to the peak of (C34:0 + C34:1) mycolic acids and the other to (C36:0 + C36:1) mycolic acids. By pyrolysis/radio-GLC analysis, C32:0 mycolic acid synthesized by [1-14C]palmitic acid was pyrolyzed at 300 °C to form palmitaldehyde (the mero moiety) and methyl palmitate (the branch moiety). The pH optimum for the incorporation of [1-14C]palmitate into bacterionema mycolic acids was 6.4 and the reaction required a divalent cation. The in vitro system utilized myristic, palmitic, stearic and oleic acids (probably via their activated forms) well as precursors, among which myristic and palmitic acids were more effective than the rest. Avidin showed no effect on the biosynthesis of mycolic acid from 14C-palmitate whereas cerulenin, a specific inhibitor of β-ketoacyl synthetase in de novo fatty acid synthesis, inhibited the reaction at a relatively higher concentration. Thin-layer chromatographic analysis of lipids extracted from the reacting mixture without alkaline hydrolysis showed that both exogenous [1-14] fatty acid and synthesized mycolic acids were bound to an unknown compound by an alkali-labile linkage and this association seemed to occur prior to the condensation of two molecules of fatty acid.  相似文献   

15.
Trans mycolic acid content is directly related to cell wall fluidity and permeability in mycobacteria. Carbon-13 NMR spectroscopy of mycolic acids isolated from Mycobacterium tuberculosis (MTB) and Mycobacterium smegmatis (MSM) fed 13C-labeled precursor molecules was used to probe the biosynthetic pathways that modify mycolic acids. Heteronuclear correlation spectroscopy (HMQC) of ketomycolic acid from MTB allowed assignment of the complete 13C-NMR spectrum. Incorporation patterns from [1-13C]-acetate and [2-13C]-acetate feeding experiments suggested that the mero chain and alpha branch of mycolic acids are both synthesized by standard fatty acid biosynthetic reactions. [13C-methyl]-L-methionine was used to specifically label carbon atoms derived from the action of the methyl transferases involved in meromycolate modification. To enrich for trans mycolic acids a strain of MTB overexpressing the mma1 gene was labeled. Carbon-carbon coupling was observed in mycolate samples doubly labeled with 13C-acetate and [13C-methyl]-L-methionine and this information was used to assess positional specificity of methyl transfer. In MTB such methyl groups were found to occur exclusively on carbons derived from the 2 position of acetate, while in MSM they occurred only on carbons derived from the 1 position. These results suggest that the MSM methyltransferase MMAS-1 operates in an inverted manner to that of MTB.  相似文献   

16.
1. The use of labelled acetate for studying the synthesis of long-chain fatty acids in rat adipose tissue in vitro has been examined, with special reference to the effect of acetate concentration. 2. The incorporation of acetate into fatty acids is proportional to the concentration of acetate in the medium when the latter does not exceed about 10mum. Above this concentration, the relative incorporation becomes progressively less, and reasons for this are discussed. 3. In particular it is shown that this is not necessarily due to disturbance of the endogenous rate of fatty acid synthesis by a relatively large amount of acetyl-CoA derived from added acetate. 4. However, to ensure that the added acetate does not cause such a disturbance its concentration must be kept sufficiently low. For labelled acetate used under present conditions, this concentration should not be more than about 10mum.  相似文献   

17.
Mixed rumen microorganisms (MRM) or suspensions of rumen Holotrich protozoa obtained from a sheep were incubated anaerobically with [1-(14)C]linoleic acid, [U-(14)C]glucose, or [1-(14)C]acetate. With MRM, the total amount of fatty acids present did not change after incubation. An increase in fatty acids esterified into sterolesters (SE) and polar lipids at the expense of free fatty acids was observed. This effect was intensified by the addition of fermentable carbohydrate to the incubations. Radioactivity from [1-(14)C]linoleic acid was incorporated into SE and polar lipids with both MRM and Holotrich protozoa. With MRM the order of incorporation of radioactivity was as follows: SE > phosphatidylethanolamine > phosphatidylcholine. With Holotrich protozoa, the order of incorporation was phosphatidylcholine > phosphatidylethanolamine > SE. With MRM the radioactivity remaining in the free fatty acids and that incorporated into SE was mainly associated with saturated fatty acids, but a considerable part of the radioactivity in the polar lipids was associated with dienoic fatty acids. This effect of hydrogenation prior to incorporation was also noted with Holotrich protozoa but to a much lesser extent. Small amounts of radioactivity from [U-(14)C]glucose and [1-(14)C]acetate were incorporated into rumen microbial lipids. With protozoa incubated with [U-(14)C]glucose, the major part of incorporated radioactivity was present in the glycerol moiety of the lipids. From the amounts of lipid classes present, their radioactivity, and fatty acid composition, estimates were made of the amounts of higher fatty acids directly incorporated into microbial lipids and the amounts synthesized de novo from glucose or acetate. It is concluded that the amounts directly incorporated may be greater than the amounts synthesized de novo.  相似文献   

18.
Adult Drosophila melanogaster synthesizes dodecanoic and tetradecanoic acids in vivo, along with the more common 16- and 18-carbon fatty acids. The radiolabeled C12 and C14 fatty acids synthesized from sodium [1-14C]acetate are found primarily in the diacylglycerol and triacylglycerol fractions. Partially purified fatty acid synthetase (FAS) synthesizes C14, C16, and C18 fatty acids (as the free acids) at 0.2 M ionic strength. Increasing the ionic strength to 2.0 M causes partially purified FAS to synthesize primarily C12 and C14 fatty acids. Addition of aliquots of the microsomal pellet and other soluble protein fractions does not alter the pattern of fatty acids synthesized by FAS. The percentage of C12 and C14 fatty acids synthesized at high ionic strength by individual fractions from the FAS peak (Sepharose 6B column) is constant across the peak. None of the soluble protein fractions is able to relieve the inhibition of FAS by phenylmethylsulfonyl fluoride. These results indicate that the FAS of D. melanogaster has the inherent capability to form C12 and C14 fatty acids and that no other soluble protein appears to be involved in their synthesis.  相似文献   

19.
The cell wall of Mycobacterium spp. consists predominately of arabinogalactan chains linked at the reducing ends to peptidoglycan via a P-GlcNAc-(alpha1-3)-Rha linkage unit (LU) and esterified to a variety of mycolic acids at the nonreducing ends. Several aspects of the biosynthesis of this complex have been defined, including the initial formation of the LU on a polyprenyl phosphate (Pol-P) molecule followed by the sequential addition of galactofuranosyl (Galf) units to generate Pol-P-P-LU-(Galf)1,2,3, etc. and Pol-P-P-LU-galactan, catalyzed by a bifunctional galactosyltransferase (Rv3808c) capable of adding alternating 5- and 6-linked Galf units. By applying cell-free extracts of Mycobacterium smegmatis, containing cell wall and membrane fragments, and differential labeling with UDP-[14C]Galp and recombinant UDP-Galp mutase as the source of [14C]Galf for galactan biosynthesis and 5-P-[14C]ribosyl-P-P as a donor of [14C]Araf for arabinan synthesis, we now demonstrate sequential synthesis of the simpler Pol-P-P-LU-(Galf)n glycolipid intermediates followed by the Pol-P-P-LU-arabinogalactan and, finally, ligation of the P-LU-arabinogalactan to peptidoglycan. This first time demonstration of in vitro ligation of newly synthesized P-LU-arabinogalactan to newly synthesized peptidoglycan is a necessary forerunner to defining the genetics and enzymology of cell wall polymer-peptidoglycan ligation in Mycobacterium spp. and examining this step as a target for new antibacterial drugs.  相似文献   

20.
S C Cunnane 《FEBS letters》1992,306(2-3):273-275
Quantitative carbon-by-carbon analysis would be useful in determining the origin and fate of carbons involved in fatty acid metabolism. Incorporation of 13C from 2-[13C]acetate into specific carbons of liver fatty acids was lowest at the n-2 carbon of saturates and monoenes but was 47% greater at acyl C1 than at C2, suggesting substantial redistribution of the 13C from C2 to C1 of acetyl CoA or malonyl CoA prior to 13C incorporation into fatty acids during de novo synthesis or during elongation. Thus, 13C derived from exogenous acetate can be quantitatively measured and is differentially incorporated into individual carbons depending on position in the fatty acid molecule.  相似文献   

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