Acetate Binding of Spinach Chloroplasts as a Facet of Fatty Acid Synthesis

A particulate fraction of spinach chloroplasts is the major site of binding when either acetate or acetyl-CoA is used as substrate. The acetate is linked covalently, and the binding is inhibited by reagents which react with sulfhydryl groups. The amount of acetate bound is lowered by both citrate and oxaloacetate; however, the binding is not reversed by oxaloacetate. Reversal of binding is also not brought about by the addition of unlabeled acetyl-CoA. If cofactors for fatty acid synthesis and cold acetyl-CoA are added, the binding of labeled acetate is reversed. Acyl carrier protein from E. coli increases the binding of labeled acetate.     

Fat Metabolism in Higher Plants: XLV. Some Factors Regulating Fatty Acid Synthesis by Isolated Spinach Chloroplasts

In the biosynthesis of fatty acids from 1-¹⁴C-acetate by intact spinach chloroplasts, ATP and Triton X-100 exert opposing effects on the conversion of palmitic acid to stearic acid; thus, ATP decreases the conversion and Triton X-100 increases the conversion. Changes in the availability of photosynthetically generated reduced nicotinamide adenine dinucleotide phosphate apparently does not markedly affect the C₁₆-C₁₈ ratio. Various H₂O₂-generating systems, such as viologen dyes, inhibit oleate synthesis from acetate and cause stearate to accumulate. Catalase partially reverses the effect of these days.     

Nucleic Acid Synthesis in the Chloroplasts of Acetabularia mediterranea

Radioautographic and radiochemical techniques were used to establish the presence of replicating DNA in the chloroplasts of Acetabularia mediterranea. These techniques also demonstrated that these chloroplasts synthesize RNA. It was found that label from thymine was also incorporated into DNA and RNA in these chloroplasts.

With the establishment of protein and nucleic acid synthesis in Acetabularia chloroplasts, it is clear that these chloroplasts carry out those metabolic processes which are most characteristic of autonomous cells.

     

Effects of Cyanide on Rates and Products of Fatty Acid Synthesis by Chloroplasts Isolated from Spinacia oleracea

Cyanide inhibited unesterified fatty acid synthesis but stimulated glyceride synthesis from [1-¹⁴C]acetate when Spinacia oleracea chloroplasts were incubated in basal media. Both unesterified fatty acid and glyceride accumulation were inhibited when chloroplasts were incubated in a diacylglycerol mode. Stimulation of chloroplast fatty acid synthesis by either exogenous coenzyme A or Triton X-100 was almost completely abolished in the presence of cyanide. Stearoyl-ACP desaturation is considered to be inhibited to a greater extent than is fatty acid synthesis de novo.     

Fatty Acids in Chloroplasts and Leaves

The fatty acid composition of green and white leaf tissue inAcer negundo, Zea mais, and Ilex aquifolium, of green and yellowtissue in Ligustrum ovatifolium and of etiolated and green tissuein Vicia faba has been determined. The mesophytic green leavesexamined show a general similarity in fatty acid composition,characterized by a high concentration of non-conjugated octadecatrienoicacid. Chloroplasts were isolated from Vicia and Acer and containan even higher concentration of this acid and only traces ofnon-conjugated octadecadienoic acid. Conjugated diene and trieneacids occur in traces in chioroplasts, but are also found innon-green leaf tissue. The fats of non-green leaves are in generalmore saturated than those from green tissue but vary considerablyin composition. The relationship between fat composition andplastid development is discussed.     

Control of Fatty Acid Synthesis in Bacteria

When glycerol-requiring auxotrophs of Bacillus subtilis are deprived of glycerol, the synthesis of fatty acids continues at an apparent rate of 20 to 50% that of supplemented cultures. The newly synthesized fatty acids are not incorporated into phospholipid and accumulate as free fatty acids. These molecules undergo a much more rapid turnover than phospholipid fatty acids, and the rate of turnover is sufficient to indicate that the rate of fatty acid synthesis in glycerol-deprived cultures is similar to that in supplemented ones. The average chain length of the free fatty acids is greater than that of the phospholipid fatty acids. Cells deprived of required amino acids also show a diminution in the apparent rate of fatty acid synthesis; however, in this case, the fatty acids accumulate in phospholipid, and no increase of the free fatty acid fraction is observed. It is argued on the basis of these findings that the control of lipid synthesis does not operate at the level of transacylation but must act on one or more of the reactions of the fatty acid synthetase.     

Overproduction of a Functional Fatty Acid Biosynthetic Enzyme Blocks Fatty Acid Synthesis in Escherichia coli

β-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.     

Positional Specificity and Fatty Acid Selectivity of Purified sn-Glycerol 3-Phosphate Acyltransferases from Chloroplasts

Soluble acyl-CoA:sn-glycerol 3-phosphate acyltransferases (EC 2.3.1.15) which are localized in chloroplasts were purified from leaves of Pisum sativum and Spinacia oleracea and obtained free from interfering activities. The purification raised the specific activities by factors of about 1,000 for pea and 200 for spinach preparations. In pea chloroplasts, acyltransferase activity occurs in two soluble forms with apparent isoelectric points of 6.3 and 6.6. For both forms, the same molecular weight of about 42,000 was determined. The enzyme from spinach chloroplasts showed a slightly higher molecular weight and a lower isoelectric point of 5.2.     

Envelope Membranes from Spinach Chloroplasts Are a Site of Metabolism of Fatty Acid Hydroperoxides

Enzymes in envelope membranes from spinach (Spinacia oleracea L.) chloroplasts were found to catalyze the rapid breakdown of fatty acid hydroperoxides. In contrast, no such activities were detected in the stroma or in thylakoids. In preparations of envelope membranes, 9S-hydroperoxy-10(E),12(Z)-octadecadienoic acid, 13S-hydroperoxy-9(Z),11(E)-octadecadienoic acid, or 13S-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid were transformed at almost the same rates (1-2 [mu]mol min-1 mg-1 protein). The products formed were separated by reversed-phase high-pressure liquid chromatography and further characterized by gas chromatography-mass spectrometry. Fatty acid hydroperoxides were cleaved (a) into aldehydes and oxoacid fragments, corresponding to the functioning of a hydroperoxide lyase, (b) into ketols that were spontaneously formed from allene oxide synthesized by a hydroperoxide dehydratase, (c) into hydroxy compounds synthesized enzymatically by a system that has not yet been characterized, and (d) into oxoenes resulting from the hydroperoxidase activity of a lipoxygenase. Chloroplast envelope membranes therefore contain a whole set of enzymes that catalyze the synthesis of a variety of fatty acid derivatives, some of which may act as regulatory molecules. The results presented demonstrate a new role for the plastid envelope within the plant cell.     

Fatty Acid Specificity in Lipase-Catalyzed Synthesis of Glucoside Esters

The fatty acid specificity of the B-lipase derived from Candida antarctica was investigated in the synthesis of esters of ethyl D-glucopyranoside. The specificity was almost identical with respect to straight-chain fatty acids with 10 to 18 carbon atoms. However, lower fatty acids such as hexanoic and octanoic acid and the unsaturated 9-cis-octadecenoic acid were found to be poor substrates of the enzyme. As a consequence of this selectivity, these fatty acids were accumulated in the unconverted fraction when ethyl D-glucopyranoside was esterified with an excess of a mixture of fatty acids. This accumulation can reduce the overall effectiveness of the process as the activity of the lipase was found to be reduced when exposed to high concentrations of short-chain fatty acids. Finally, using a simplified experimental set-up, the specificity of the C. antarctica B-lipase was compared to the specificity of lipases derived from C. rugosa, Mucor miehei, Humicola, and Pseudomonas. Apart from the C. rugosa lipase, which exhibited a very poor performance, all the enzymes showed a very similar specificity with respect to fatty acids longer than octanoic acid while only the C. antarctica B-lipase showed activity towards sort-chain fatty acids.     

Evidence for Light-Stimulated Fatty Acid Synthesis in Soybean Fruit

In leaves, the light reactions of photosynthesis support fatty acid synthesis but disagreement exists as to whether this occurs in green oilseeds. To address this question, simultaneous measurements of the rates of CO₂ and O₂ exchange (CER and OER, respectively) were made in soybean (Glycine max L.) fruits. The imbalance between CER and OER was used to estimate the diverted reductant utilization rate (DRUR) in the equation: DRUR = 4 × (OER + CER). This yielded a quantitative measure of the rate of synthesis of biomass that is more reduced per unit carbon than glucose (in photosynthesizing tissues) or than the substrates of metabolism (in respiring tissues). The DRUR increased by about 2.2-fold when fruits were illuminated due to a greater increase in OER than decrease in CER. This characteristic was shown to be a property of the seed (not the pod wall), to be present in fruits at all developmental stages, and to reach a maximal response at relatively low light. When seeds were provided with ¹³CO₂, light reduced ¹²CO₂ production but had little effect on ¹³CO₂ fixation. When they were provided with ¹⁸O₂, light stimulated ¹⁶O₂ production but had no effect on ¹⁸O₂ uptake. Together, these findings indicate that light stimulates fatty acid synthesis in photosynthetic oilseeds, probably by providing both ATP and carbon skeletons.     

Fatty Acid Specificity in Lipase-Catalyzed Synthesis of Glucoside Esters

The fatty acid specificity of the B-lipase derived from Candida antarctica was investigated in the synthesis of esters of ethyl D-glucopyranoside. The specificity was almost identical with respect to straight-chain fatty acids with 10 to 18 carbon atoms. However, lower fatty acids such as hexanoic and octanoic acid and the unsaturated 9-cis-octadecenoic acid were found to be poor substrates of the enzyme. As a consequence of this selectivity, these fatty acids were accumulated in the unconverted fraction when ethyl D-glucopyranoside was esterified with an excess of a mixture of fatty acids. This accumulation can reduce the overall effectiveness of the process as the activity of the lipase was found to be reduced when exposed to high concentrations of short-chain fatty acids. Finally, using a simplified experimental set-up, the specificity of the C. antarctica B-lipase was compared to the specificity of lipases derived from C. rugosa, Mucor miehei, Humicola, and Pseudomonas. Apart from the C. rugosa lipase, which exhibited a very poor performance, all the enzymes showed a very similar specificity with respect to fatty acids longer than octanoic acid while only the C. antarctica B-lipase showed activity towards sort-chain fatty acids.     

In Vitro Fatty Acid Synthesis and Complex Lipid Metabolism in the Cyanobacterium Anabaena variabilis: I. Some Characteristics of Fatty Acid Synthesis

In vitro fatty acid synthesis was examined in crude cell extracts, soluble fractions, and 80% (NH₄)₂SO₄ fractions from Anabaena variabilis M3. Fatty acid synthesis was absolutely dependent upon acyl carrier protein and required NADPH and NADH. Moreover, fatty acid synthesis and elongation occurred in the cytoplasm of the cell. The major fatty acid products were palmitic acid (16:0) and stearic acid (18:0). Of considerable interest, both stearoyl-acyl carrier protein and stearoyl-coenzyme A desaturases were not detected in any of the fractions from A. variabilis. The similarities and differences in fatty acid synthesis between A. variabilis and higher plant tissues are discussed with respect to the endosymbiotic theory of chloroplast evolution.     

Effect of Thiolactomycin on Fatty Acid Synthesis in Higher Plants

Thiolactomycin (TLM), an antibiotic from Nocardia sp. No. 2-200,inhibited fatty acid synthesis in Avena leaves, with the concentrationcausing 50% inhibition being 0.38µg/ml. This antibioticis more inhibitory to the elongation of palmitic to oleic acidthan to the de novo synthesis of palmitic acid in both spinachchloroplasts and Avena leaves, in contrast to the effect ofcerulenin which inhibits de novo synthesis but not fatty acidelongation. On the other hand, TLM is less inhibitory to furtherelongation of stearic acid to very long chain fatty acids inpea seeds. The inhibition rate decreased in the order of synthesisof arachidic, behenic and lignoceric acid. (Received December 26, 1986; Accepted April 24, 1987)     

The Use of Fatty Acid Esters to Enhance Free Acid Sophorolipid Synthesis

Fatty acid esters were prepared by transesterification of soy oil with methanol (methyl-soyate, Me-Soy), ethanol (ethyl-soyate, Et-Soy) and propanol (propyl-soyate, Pro-Soy) and used with glycerol as fermentation substrates to enhance production of free-acid sophorolipids (SLs). Fed-batch fermentations of Candida bombicola resulted in SL yields of 46 ± 4 g/l, 42 ± 7 g/l and 18 ± 6 g/l from Me-Soy, Et-Soy, and Pro-Soy, respectively. Liquid chromatography with atmospheric pressure ionization mass spectrometry (LC/API-MS) showed that Me-Soy resulted in 71% open-chain SLs with 59% of those molecules remaining esterified at the carboxyl end of the fatty acids. Et-Soy and Pro-Soy resulted in 43% and 80% open-chain free-acid SLs, respectively (containing linoleic acid and oleic acid as the principal fatty acid species linked to the sophorose sugar at the omega-1 position), with no evidence of residual esterification. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

Lipase-Catalyzed Synthesis of Fatty Acid Esters Useful in the Food Industry

Enzyme reactions are very attractive in food technology because they can be carried out under mild conditions and without toxic solvents and other catalysts. Lipases can esterify various alcohols with fatty acids. There are opportunities to synthesize useful compounds with special functions as food materials by using the catalytic function of lipase. Reverse micellar systems are discussed as reaction systems for lipases in organic solvents, especially in triacylglycerol synthesis using phosphatidylcholine as the surfactant. Syntheses of some amphiphilic substances including O-acyl-L-homoserine are also discussed.     

Activation of Fatty Acid Synthesis in Cell-free Extracts of Saccharomyces cerevisiae

Fatty acid synthesis from acetate in extracts of Saccharomyces cerevisiae strain LK2G12 was shown to be stimulated by alpha-glycerophosphate and citrate, and by a number of compounds related to them. Magnesium was shown to stimulate fatty acid synthesis from acetyl-coenzyme A but not from malonyl-coenzyme A, thus indicating the site of stimulation of fatty acid synthesis to be the acetyl-coenzyme A step.