Regulating effect of β-ketoacyl synthase domain of fatty acid synthase on fatty acyl chain length in de novo fatty acid synthesis

Fatty acid synthase (FAS) is a multifunctional homodimeric protein, and is the key enzyme required for the anabolic conversion of dietary carbohydrates to fatty acids. FAS synthesizes long-chain fatty acids from three substrates: acetyl-CoA as a primer, malonyl-CoA as a 2 carbon donor, and NADPH for reduction. The entire reaction is composed of numerous sequential steps, each catalyzed by a specific functional domain of the enzyme. FAS comprises seven different functional domains, among which the β-ketoacyl synthase (KS) domain carries out the key condensation reaction to elongate the length of fatty acid chain. Acyl tail length controlled fatty acid synthesis in eukaryotes is a classic example of how a chain building multienzyme works. Different hypotheses have been put forward to explain how those sub-units of FAS are orchestrated to produce fatty acids with proper molecular weight. In the present study, molecular dynamic simulation based binding free energy calculation and access tunnels analysis showed that the C16 acyl tail fatty acid, the major product of FAS, fits to the active site on KS domain better than any other substrates. These simulations supported a new hypothesis about the mechanism of fatty acid production ratio: the geometric shape of active site on KS domain might play a determinate role.     

Is the fatty acid composition of Daphnia galeata determined by the fatty acid composition of the ingested diet?

1. The fatty acid (FA) composition of Daphnia galeata and their algal food was analysed and showed many similarities, however, some significant differences were also found in the relative abundance of the FA C16 : 4ω3 and docosahexaenoic acid (DHA). Their relative abundances were much lower in daphnids than in their algal diet.
2. When daphnids were fed three distinct emulsion particles with DHA : eicosapentaenoic acid (EPA) ratios of c. 0.7, 2 and 4, the final DHA : EPA ratio in the daphnids always favoured EPA. The increase of the food DHA : EPA ratio resulted in a minor increase of DHA (to c. 2%). Feeding the animals on emulsion particles with increasing ratios of DHA : EPA, caused a minor ( c. 2%) increase of DHA level but EPA levels remained high ( c. 10%).
3. When labelled with [¹⁴C]linoleic acid and [¹⁴C]linolenic acid daphnids showed low conversion of both essential FA into C20 polyunsaturated fatty acids (PUFAs). This low conversion activity might explain the importance of C20 PUFAs as dietary compounds in the food of Daphnia.
4. The results indicate the insignificance of DHA and C16 : 4ω3 for daphnids. As EPA can be derived from C18 : 3ω3 it is not strictly essential, although it might be a significant factor in food quality for Daphnia.     

Probing the fatty acid binding site of β-lactoglobulins

The interactions of fatty acids with porcine and bovine β-lactoglobulins were measured using tryptophan fluorescence enhancement. In the case of bovine β-lactoglobulin, the apparent binding constants for most of the saturated and unsaturated fatty acids were in the range of 10^?7 M at neutralpH. Bovine β-lactoglobulin displays only one high affinity binding site for palmitate with an apparent dissociation constant of 1·10^?7 M. The strength of the binding was decreasing in the following way: palmitate > stearate > myristate > arachidate > laurate. Caprylic and capric acids are not bound at all. The affinity of β-lactoglobulin for palmitate decreased as thepH of the incubation medium was lowered and BLG/palmitate complex was not observed atpH's lower than 4.5. Surprisingly, chemically modified bovine β-lactoglobulin and porcine β-lactoglobulin did not bind fatty acids in the applied conditions.     

Lipase-catalyzed synthesis of vitamin C fatty acid esters

Fatty acid esters of vitamin C (ascorbic acid) where synthesized in a mainly solid-phase system in the presence of small amounts of organic solvent (acetone or t-butanol) catalyzed by immobilized lipase B from Candida antarctica.Highest reaction rates and yields of isolated products were obtained using fatty acid vinyl esters, e.g., 6-O-palmitoyl-l-ascorbic acid was obtained in 91% isolated yield after 48 h. As vitamin C and its esters are very sensitive to oxidation, a mild extraction method for the isolation of reaction products was developed.     

Role of fatty acid uptake and fatty acid β-oxidation in mediating insulin resistance in heart and skeletal muscle

Fatty acids are a major fuel source used to sustain contractile function in heart and oxidative skeletal muscle. To meet the energy demands of these muscles, the uptake and β-oxidation of fatty acids must be coordinately regulated in order to ensure an adequate, but not excessive, supply for mitochondrial β-oxidation. However, imbalance between fatty acid uptake and β-oxidation has the potential to contribute to muscle insulin resistance. The action of insulin is initiated by binding to its receptor and activation of the intrinsic protein tyrosine kinase activity of the receptor, resulting in the initiation of an intracellular signaling cascade that eventually leads to insulin-mediated alterations in a number of cellular processes, including an increase in glucose transport. Accumulation of fatty acids and lipid metabolites (such as long chain acyl CoA, diacylglycerol, triacylglycerol, and/or ceramide) can lead to alterations in this insulin signaling pathway. An imbalance between fatty acid uptake and oxidation is believed to be responsible for this lipid accumulation, and is thought to be a major cause of insulin resistance in obesity and diabetes, due to lipid accumulation and inhibition of one or more steps in the insulin-signaling cascade. As a result, decreasing muscle fatty acid uptake can improve insulin sensitivity. However, the potential role of increasing fatty acid β-oxidation in the heart or skeletal muscle in order to prevent cytoplasmic lipid accumulation and decrease insulin resistance is controversial. While increased fatty acid β-oxidation may lower cytoplasmic lipid accumulation, increasing fatty acid β-oxidation can decrease muscle glucose metabolism, and incomplete fatty acid oxidation has the potential to also contribute to insulin resistance. In this review, we discuss the proposed mechanisms by which alterations in fatty acid uptake and oxidation contribute to insulin resistance, and how targeting fatty acid uptake and oxidation is a potential therapeutic approach to treat insulin resistance.     

Identification and evaluation of ω-3 fatty acid desaturase genes for hyperfortifying α-linolenic acid in transgenic rice seed

α-Linolenic acid (ALA) deficiency and a skewed of ω6:ω3 fatty acid ratio in the diet are a major explanation for the prevalence of cardiovascular diseases and inflammatory/autoimmune diseases. There is a need to enhance the ALA content and to reduce the ratio of linoleic acid (LA) to ALA. Six ω-3 (Δ-15) fatty acid desaturase (FAD) genes were cloned from rice and soybean. The subcellular localizations of the proteins were identified. The FAD genes were introduced into rice under the control of an endosperm-specific promoter, GluC, or a Ubi-1 promoter to evaluate their potential in increasing the ALA content in seeds. The ALA contents in the seeds of endoplasmic reticulum (ER)-localized GmFAD3-1 and OsFAD3 overexpression lines increased from 0.36 mg g?1 to 8.57 mg g?1 and 10.06 mg g?1, respectively, which was 23.8- and 27.9-fold higher than that of non-transformants. The trait of high ALA content was stably inheritable over three generations. Homologous OsFAD3 is more active than GmFAD3-1 in catalysing LA conversion to ALA in rice seeds. Overexpression of ER-localized GmFAD3-2/3 and chloroplast-localized OsFAD7/8 had less effect on increasing the ALA content in rice seeds. The GluC promoter is advantageous compared with Ubi-1 in this experimental system. The enhanced ALA was preferentially located at the sn-2 position in triacylglycerols. A meal-size portion of high ALA rice would meet >80% of the daily adult ALA requirement. The ALA-rich rice could be expected to ameliorate much of the global dietary ALA deficiency.     

Carotenol fatty acid esters: easy substrates for digestive enzymes?

To study the specificity of gastric lipases on carotenoid mono- and diesters, an enzymatic assay was applied. Digestions were carried out in phosphate buffer at pH 7.4 and 37 °C. As substrates we employed oleoresins from marigold (Tagetes erecta L.; lutein diesters), red paprika (Capsicum annuum L., mainly capsanthin diesters), papaya (Carica papaya L.; β-cryptoxanthin esters), and loquat (Eriobotrya japonica Lindl.; β-cryptoxanthin esters) as well as retinyl palmitate. These were reacted with porcine pancreatic lipase, porcine pancreatin, porcine cholesterol esterase, and human pancreatic lipase. As reference enzyme a yeast lipase from Candida rugosa was applied. A high turnover could be observed with porcine pancreatic lipase and porcine cholesterol esterase, indicating cholesterol esterase to be a plausible candidate for generation of free carotenoids in the gut. Human pancreatic lipase accepted only retinyl palmitate as substrate, carotenoid mono- and diesters were not hydrolyzed. The assay permits an approach for calculation of enzymatic activities towards carotenoid esters as substrates for the first time, which is based on the amount of enzyme formulation, present in the assay (U/mg solid). Furthermore, these studies provide deeper insight into carotenoid ester bioaccessibility.     

Biotechnological production and applications of the ω-3 polyunsaturated fatty acid docosahexaenoic acid

Docosahexaenoic acid (DHA) is a polyunsaturated fatty acid composed of 22 carbon atoms and six double bonds. Because the first double bond, as counted from the methyl terminus, is at position three, DHA belongs to the so-called -3 group. In recent years, DHA has attracted much attention because of its beneficial effect on human health. At present, fish oil is the major source of DHA, but alternatively it may be produced by use of microorganisms. Marine microorganisms may contain large quantities of DHA and are considered a potential source of this important fatty acid. Some of these organisms can be grown heterotrophically on organic substrates without light. These processes can be well controlled and DHA with constant quality can be produced all year round. This paper reviews recent advances in the biotechnological production of DHA by marine microorganisms.     

In-vitro evidence for feed-back regulation of β-ketoacyl-acyl carrier protein synthase III in medium-chain fatty acid biosynthesis

The cerulenin-insensitive -ketoacyl-acyl carrier protein (ACP) synthase III (KAS III, EC 2.3.1.41) catalyzes the first condensing step of the fatty-acid synthase (FAS) reaction in plants and bacteria, using directly acetyl-CoA as substrate for condensation with malonyl-ACP. In order to identify a possible site for regulation of the biosynthesis of medium-chain fatty acids, the influence of acyl-ACPs of different chain-lengths (C₄,C₆,C₈ and C₁₀) on the activity of KAS III was investigated in vitro using an FAS preparation from seeds of Cuphea lanceolata Ait. (a crop accumulating up to 90% decanoic acid into triacylglycerols) that had been treated with 100 M cerulenin. All acyl-ACPs investigated led to a decrease in the activity of KAS III towards acetyl-CoA, an effect apparently related to the length of the acyl chain. Analysis of the reaction products of the assay revealed that short-chain acyl-ACPs elongated to a very small extent simultaneously with acetyl-CoA. This extent of elongation did not correlate with the decrease in KAS III-activity levels. These data excluded the possibility of competition between acetyl-CoA and acyl-ACPs, but indicated that acyl-ACPs inhibited the enzyme. Decanoyl-ACP caused the highest decrease in enzyme activity (IC₅₀ = 0.45 M), thus being a potent inhibitor of KAS III. Michaelis-Menten kinetics revealed that the inhibition of KAS III by decanoyl-ACP was non-competitive in relation to malonyl-ACP and uncompetitive in relation to acetyl-CoA. Moreover, our data indicate that KAS III has a strict specificity for the elongation of acetyl-CoA. An inhibition of KAS III by acyl-ACPs was observed in experiments using FAS preparations from rape seeds and spinach leaves, but the inhibition of KAS III from C. lanceolata seeds by decanoyl-ACP was approximately 1.5-fold higher. The data provide evidence that acyl-ACPs are involved in the modulation of plant fatty-acid biosynthesis by a feed-back mechanism.Abbreviations ACP acyl carrier protein - DTT dithiothreitol - TCA trichloroacetic acid - ecACP acyl carrier protein from Escherichia coli - FAS fatty-acid synthase - IC₅₀ concentration causing 50% inhibition - KAS -ketoacyl-ACP synthase - NEM N-ethylmaleimide In honour of Professor Hartmut K. Lichtenthaler's sixtieth birthdayThis work was supported by a grant from the German Ministry of Research and Technology (BMFT) and in part by the Fonds der Chemischen Industrie and the Ministry of Science and Research of the State Northrhine-Westfalia. The authors wish to thank Prof. G. Röbbelen (University of Göttingen, Göttingen, Germany) for kindly providing the plant material. This paper is part of the doctoral thesis of Fritzi Maike Brück.     

Identification and characterization of new Δ-17 fatty acid desaturases

ω-3 fatty acid desaturase is a key enzyme for the biosynthesis of ω-3 polyunsaturated fatty acids via the oxidative desaturase/elongase pathways. Here we report the identification of three ω-3 desaturases from oomycetes, Pythium aphanidermatum, Phytophthora sojae, and Phytophthora ramorum. These new ω-3 desaturases share 55 % identity at the amino acid level with the known Δ-17 desaturase of Saprolegnia diclina, and about 31 % identity with the bifunctional Δ-12/Δ-15 desaturase of Fusarium monoliforme. The three enzymes were expressed in either wild-type or codon optimized form in an engineered arachidonic acid producing strain of Yarrowia lipolytica to study their activity and substrate specificity. All three were able to convert the ω-6 arachidonic acid to the ω-3 eicosapentanoic acid, with a substrate conversion efficiency of 54–65 %. These enzymes have a broad ω-6 fatty acid substrate spectrum, including both C18 and C20 ω-6 fatty acids although they prefer the C20 substrates, and have strong Δ-17 desaturase activity but weaker Δ-15 desaturase activity. Thus, they belong to the Δ-17 desaturase class. Unlike the previously identified bifunctional Δ-12/Δ-15 desaturase from F. monoliforme, they lack Δ-12 desaturase activity. The newly identified Δ-17 desaturases could use fatty acids in both acyl-CoA and phospholipid fraction as substrates. The identification of these Δ-17 desaturases provides a set of powerful new tools for genetic engineering of microbes and plants to produce ω-3 fatty acids, such as eicosapentanoic acid and docosahexanoic acid, at high levels.     

Synthesis of the α,ω-dicarboxylic acid precursor of biotin by the canonical fatty acid biosynthetic pathway

Biotin synthesis requires the C7 α,ω-dicarboxylic acid, pimelic acid. Although pimelic acid was known to be primarily synthesized by a head to tail incorporation of acetate units, the synthetic mechanism was unknown. It has recently been demonstrated that in most bacteria the biotin pimelate moiety is synthesized by a modified fatty acid synthetic pathway in which the biotin synthetic intermediates are O-methyl esters disguised to resemble the canonical intermediates of the fatty acid synthetic pathway. Upon completion of the pimelate moiety, the methyl ester is cleaved. A very restricted set of bacteria have a different pathway in which the pimelate moiety is formed by cleavage of fatty acid synthetic intermediates by BioI, a member of the cytochrome P450 family.     

Is bacterial fatty acid synthesis a valid target for antibacterial drug discovery?

The emergence of resistance against most current drugs emphasizes the need to develop new approaches to control bacterial pathogens, particularly Staphylococcus aureus. Bacterial fatty acid synthesis is one such target that is being actively pursued by several research groups to develop anti-Staphylococcal agents. Recently, the wisdom of this approach has been challenged based on the ability of a Gram-positive bacterium to incorporate extracellular fatty acids and thus circumvent the inhibition of de novo fatty acid synthesis. The generality of this conclusion has been challenged, and there is enough diversity in the enzymes and regulation of fatty acid synthesis in bacteria to conclude that there is not a single organism that can be considered typical and representative of bacteria as a whole. We are left without a clear resolution to this ongoing debate and await new basic research to define the pathways for fatty acid uptake and that determine the biochemical and genetic mechanisms for the regulation of fatty acid synthesis in Gram-positive bacteria. These crucial experiments will determine whether diversity in the control of this important pathway accounts for the apparently different responses of Gram-positive bacteria to the inhibition of de novo fatty acid synthesis in presence of extracellular fatty acid supplements.     

Effect of γ irradiation on the fatty acid composition of soybean and soybean oil

Food irradiation is a form of food processing to extend the shelf life and reduce spoilage of food. We examined the effects of γ radiation on the fatty acid composition, lipid peroxidation level, and antioxidative activity of soybean and soybean oil which both contain a large amount of unsaturated fatty acids. Irradiation at 10 to 80 kGy under aerobic conditions did not markedly change the fatty acid composition of soybean. While 10-kGy irradiation did not markedly affect the fatty acid composition of soybean oil under either aerobic or anaerobic conditions, 40-kGy irradiation considerably altered the fatty acid composition of soybean oil under aerobic conditions, but not under anaerobic conditions. Moreover, 40-kGy irradiation produced a significant amount of trans fatty acids under aerobic conditions, but not under anaerobic conditions. Irradiating soybean oil induced lipid peroxidation and reduced the radical scavenging activity under aerobic conditions, but had no effect under anaerobic conditions. These results indicate that the fatty acid composition of soybean was not markedly affected by radiation at 10 kGy, and that anaerobic conditions reduced the degradation of soybean oil that occurred with high doses of γ radiation.     

Role of ω-3 fatty acid desaturases in the regulation of the level of trienoic fatty acids during leaf cell maturation

Trienoic fatty acids, namely -linolenic acid and hexadecatrienoic acid, present in leaf lipids are produced by -3 fatty acid desaturases located in the endoplasmic reticulum and plastid membranes. The changes in the level of trienoic fatty acids during leaf maturation were investigated in wild-type plants of Arabidopsis thaliana (L.) Heynh. and in the fad7 mutant deficient in the activity of a plastid -3 desaturase. The levels of trienoic fatty acids increased in 26 °C- and 15 °C-grown wild-type plants with maturation of leaves. The increase in trienoic fatty acids was mainly due to galactolipids enriched in plastid membranes. In addition, the relative levels of trienoic fatty acids in major glycerolipids, including phospholipids enriched in the endoplasmic reticulum membranes, also increased with leaf maturation. By contrast, when the fad7 mutant was grown at 26 °C, the relative levels of trienoic fatty acids in individual lipids decreased with leaf maturation. The decreases in the levels of trienoic fatty acids, however, were alleviated when the fad7 mutant was grown at 15 °C. These results suggest that the plastid -3 desaturase plays a major role in increasing the levels of trienoic fatty acids with leaf maturation.Abbreviations 163 hexadecatrienoic acid - 183 -linolenic acid - DGD digalactosyldiacylglycerol - MGD monogalactosyldiacylglycerol - PC phosphatidylcholine - PE phosphatidylethanolamine - TA trienoic fatty acid - WT wild type - -3 refers to the position of the double bond from the methyl end of a fatty acid This research was supported in part by Grants-in-Aid for Scientific research (#07251214 and #06804050 to K.I.) from the Ministry of Education, Science and Culture, Japan, and by the research grant from Shorai Foundation.     

Comparison of derivatization and chromatographic methods for GC–MS analysis of amino acid enantiomers in physiological samples

GC–MS analysis of fluorinated and non-fluorinated chloroformate and anhydride derivatives of amino acid (AA) enantiomers on two different chiral columns was compared for the direct quantification of free l- and d-AAs in human serum and urine in a single analytical run. Best sensitivity was achieved with pentafluoropropionic anhydride/heptafluorobutanol derivatives separated on a Chirasil-l-Val column. However, the occurrence of racemization during derivatization precluded accurate quantification of AA enantiomers. Derivatization with methyl chloroformate/methanol and separation on an Rt-γDEXsa column did not exhibit racemization and yielded ten baseline separated racemates of proteinogenic AAs with resolution values greater than 2.4. However, protein and peptide hydrolysis occurred in serum and urine during the highly exothermal derivatization reaction under alkaline conditions. Removing serum proteins by precipitation before derivatization and performing the reaction at neutral pH enabled the determination of accurate free AA enantiomer concentrations. Accuracy of quantification was validated by an established nonchiral GC–MS method for AA analysis. Reliable quantification was achieved using stable-isotope labeled l-AAs as internal standards. Limits of detection (LOD) and lower limits of quantification (LLOQ) for the d-AAs were in the range of 3.2–446 nM and 0.031–1.95 μM, respectively. Relative standard deviations (N = 6) for the measurement of AAs in urine and serum ranged from 0.49–11.10% to 0.70–3.87%, respectively. The method was applied to the analysis of urine from 19 patients with renal insufficiency. In comparison to healthy probands, D-ratios of Ala, Val, Pro, Thr, Asp, and Asn were significantly increased.     

Non-β-oxidizable ω-[18F]fluoro long chain fatty acid analogs show cytochrome P-450-mediated defluorination: Implications for the design of PET tracers of myocardial fatty acid utilization

The nature of the in vivo defluorination of non-β-oxidizable no-carrier-added ω-[¹⁸F]fluoro long chain fatty acid (LCFA) analogs was studied with the aim of developing PET tracers of LCFA utilization. Extensive defluorination of 15-[¹⁸F]fluoro-3-thia-pentadecanoic acid (FTPA) in mouse was evidenced by radioactivity uptake by bone. [¹⁸F]Fluoride in the blood was verified analytically. Incubations of FTPA in rat-liver homogenates and subcellular fractions thereof showed a strong defluorination process in microsomes which was O₂- and NADPH-dependent. In contrast, defluorination of FTPA was relatively slow in Langendorff perfused rat heart. High bone uptake in mouse was also observed with 14-[¹⁸F]fluoro-13, 13-dimethyl-3-thia-tetradecanoic acid, where gem-dimethyl substitution precludes direct elimination of H¹⁸F. These data indicate that the defluorination of non-β-oxidizable ω-[¹⁸F]fluoro LCFA analogs is primarily governed by cytochrome P-450-mediated ω-oxidation.Therefore, labeling at the (ω-3) carbon was proposed to provide a more stabile ¹⁸F-label. Defluorination of the (ω-3)-labeled 13 (R,S)-[¹⁸F]fluoro-3-thia-hexadecanoic acid was lower than that of FTPA in mouse and was independent of O₂ and NADPH in vitro. Thus, (ω-3) labeling with ¹⁸F is preferable to ω labeling of non-β-oxidizable LCFA analogs.     

Optimization of extraction method for LC–MS based determination of phenolic acid profiles in different Impatiens species

The main aim of presented study was the comparison of various extraction methods for the quantitative and qualitative analysis (LC-ESI–MS/MS) of phenolic acids present in extracts obtained from leaves, flowers, and roots of Impatiens glandulifera. The accelerated solvent extraction (ASE) at three temperature ranges (80° C, 100° C, and 120° C), ultrasound assisted extraction (USAE) at 60° C, and traditional extraction in Soxhlet apparatus were used. Taking into account the extraction yield, and the diversity of the individual compounds, ultrasound assisted extraction proved to be the most efficient method, and it was used to determine the content of phenolic acids in leaves of four other Impatiens species, including I. balsamina, I. noli-tangere, I. parviflora, and I. walleriana. Eleven phenolic acids were identified in all examined species. These were protocatechuic, gentisic, 4- hydroxybenzoic, vanillic, trans-caffeic, syringic, trans-p-coumaric, trans- and cis-ferulic, salicylic, and 3-hydroxycinnamic acids. In the extract from the leaves of I. balsamina and I. walleriana, gallic and cis-p-coumaric acids were found additionally. The most abundant compounds in all examined extracts were protocatechuic and 3-hydroxycinnamic acids. The latest acid was found in the highest yield in I. noli-tangere (266.12 μg/g DW). In the leaves of I. glandulifera a great amount of 4-hydroxybenzoic (41.44 μg/g DW), vanillic (61.50 μg/g DW), and trans-p-coumaric (58.42 μg/g DW) acids was also observed. Our results indicate that protocatechuic, 4-hydroxybenzoic, vanillic, trans-p-coumaric, trans-ferulic, and 3-hydroxycinnamic acids were most characteristic of Impatiens species.Additionally, various phenolic-rich extracts from leaves, flowers, and roots of Impatiens glandulifera were tested for antioxidant activity. The highest antiradical activity was detected for roots using Soxhlet extraction (EC50 = 0.055 mg [DE/ml]).The study demonstrated that members of the genus Impatiens, and in particular Impatiens glandulifera, and Impatiens noli-tangere, contain significant amounts of phenolic acids. In addition, extracts from various parts of I. glandulifera could be interesting as novel sources of natural antioxidants.