Diversity of Δ12 Fatty Acid Desaturases in Santalaceae and Their Role in Production of Seed Oil Acetylenic Fatty Acids

Plants in the Santalaceae family, including the native cherry Exocarpos cupressiformis and sweet quandong Santalum acuminatum, accumulate ximenynic acid (trans-11-octadecen-9-ynoic acid) in their seed oil and conjugated polyacetylenic fatty acids in root tissue. Twelve full-length genes coding for microsomal Δ12 fatty acid desaturases (FADs) from the two Santalaceae species were identified by degenerate PCR. Phylogenetic analysis of the predicted amino acid sequences placed five Santalaceae FADs with Δ12 FADs, which include Arabidopsis thaliana FAD2. When expressed in yeast, the major activity of these genes was Δ12 desaturation of oleic acid, but unusual activities were also observed: i.e. Δ15 desaturation of linoleic acid as well as trans-Δ12 and trans-Δ11 desaturations of stearolic acid (9-octadecynoic acid). The trans-12-octadecen-9-ynoic acid product was also detected in quandong seed oil. The two other FAD groups (FADX and FADY) were present in both species; in a phylogenetic tree of microsomal FAD enzymes, FADX and FADY formed a unique clade, suggesting that are highly divergent. The FADX group enzymes had no detectable Δ12 FAD activity but instead catalyzed cis-Δ13 desaturation of stearolic acid when expressed in yeast. No products were detected for the FADY group when expressed recombinantly. Quantitative PCR analysis showed that the FADY genes were expressed in leaf rather than developing seed of the native cherry. FADs with promiscuous and unique activities have been identified in Santalaceae and explain the origin of some of the unusual lipids found in this plant family.     

Influence of Fatty Acid Precursors,Including Food Preservatives,on the Growth and Fatty Acid Composition of Listeria monocytogenes at 37 and 10°C

Listeria monocytogenes is a food-borne pathogen that grows at refrigeration temperatures and increases its content of anteiso-C_15:0 fatty acid, which is believed to be a homeoviscous adaptation to ensure membrane fluidity, at these temperatures. As a possible novel approach for control of the growth of the organism, the influences of various fatty acid precursors, including branched-chain amino acids and branched- and straight-chain carboxylic acids, some of which are also well-established food preservatives, on the growth and fatty acid composition of the organism at 37°C and 10°C were studied in order to investigate whether the organism could be made to synthesize fatty acids that would result in impaired growth at low temperatures. The results indicate that the fatty acid composition of L. monocytogenes could be modulated by the feeding of branched-chain amino acid, C₄, C₅, and C₆ branched-chain carboxylic acid, and C₃ and C₄ straight-chain carboxylic acid fatty acid precursors, but the growth-inhibitory effects of several preservatives were independent of effects on fatty acid composition, which were minor in the case of preservatives metabolized via acetyl coenzyme A. The ability of a precursor to modify fatty acid composition was probably a reflection of the substrate specificities of the first enzyme, FabH, in the condensation of primers of fatty acid biosynthesis with malonyl acyl carrier protein.Listeriosis is a severe and life-threatening human infection encompassing meningoencephalitis, meningitis, focal infections in the immunocompromised, and stillbirths and neonatal sepsis due to infection of pregnant women (2). The disease is caused by the Gram-positive food-borne pathogen Listeria monocytogenes, which is responsible for common-source and sporadic disease involving a variety of different foods (27). Listeriosis has a high fatality rate (24). The U.S. Department of Agriculture has a zero tolerance policy for L. monocytogenes in ready-to-eat products, and high costs are associated with product recalls.L. monocytogenes has a remarkably low minimum growth temperature, e.g., −0.1°C (34), and thus the organism can multiply to dangerous levels when food is kept at refrigeration temperatures. We are interested in the molecular mechanisms of L. monocytogenes psychrotolerance, with a view to applying this knowledge to improve the control of the growth of the organism. Although the adaptations involved in low-temperature tolerance are global in scope, we have focused on changes in fatty acid composition that result in homeoviscous adjustments of membrane fluidity (31, 36). L. monocytogenes has a fatty acid composition that is dominated to an unusual extent (90% or more) by branched-chain fatty acids (BCFAs); the major fatty acids are anteiso-C_15:0, anteiso-C_17:0, and iso-C_15:0. Numerous studies have shown that the major change in fatty acid composition when L. monocytogenes is grown at low temperatures is an increase in the content of anteiso-C_15:0 fatty acid to 65% or more of the total (1, 12, 23, 25, 26, 28). Two cold-sensitive mutants with Tn917 insertions in the branched-chain α-keto acid dehydrogenase gene complex (bkd) were deficient in BCFAs, grew poorly at low temperatures, and had decreased membrane fluidity; all of these defects could be restored by growth in the presence of 2-methylbutyrate (2-MB), a precursor of odd-numbered anteiso fatty acids, including anteiso-C_15:0 fatty acid (1, 7, 13, 37). We believe that anteiso-C_15:0 fatty acid imparts fluidity to the cytoplasmic membrane, as revealed by its low phase transition temperature in model phospholipids (18) and disruption of the close packing of fatty acyl chains (21, 35).The amino acids isoleucine, leucine, and valine are the starting points for the biosynthesis of odd-numbered anteiso, odd-numbered iso, and even-numbered iso fatty acids, respectively (18, 37). The amino acids are converted to their corresponding α-keto acid derivatives through the activity of branched-chain amino acid transaminase. Branched-chain α-keto acid dehydrogenase (Bkd) then converts these α-keto compounds to branched-chain acyl coenzyme A (acyl-CoA) primers of fatty acid biosynthesis (18). These primers are then used to initiate fatty acid biosynthesis through the activity of β-ketoacyl-acyl carrier protein synthase III (FabH), which prefers branched-chain acyl-CoAs to acetyl-CoA as substrates (4, 22, 32). β-Keto-acyl carrier protein synthase II (FabF) is responsible for subsequent rounds of elongation until the acyl chain reaches 14 to 17 carbon atoms (36).We wished to ascertain whether we could manipulate the fatty acid composition of L. monocytogenes by feeding precursors that favored the production of fatty acids other than anteiso-C_15:0 and thereby inhibit the growth of the organism, especially at low temperatures. Kaneda (15, 16) has grouped Bacillus subtilis fatty acids into four pairs based on the precursors from which they are generated, i.e., anteiso-C_15:0 and C_17:0 from isoleucine, iso-C_15:0 and C_17:0 from leucine, iso-C_14:0 and C_16:0 from valine, and n-C_14:0 and n-C_16:0 from acetate or butyrate. The proportions of the fatty acids could be modulated by precursor feeding. We have studied the effects of feeding the potential fatty acid precursors branched-chain amino acids, branched-chain α-keto acids, short branched-chain carboxylic acids, short straight-chain carboxylic acids, medium-length straight-chain carboxylic acids, branched-chain C₆ carboxylic acids, and sodium diacetate (Fig. ​(Fig.1)1) on the growth and fatty acid composition of L. monocytogenes. Various short-chain carboxylic acids are used as food preservatives (5, 8, 29), and it was of interest to see whether any of them had an effect on the fatty acid composition of L. monocytogenes. Precursors giving rise to C₅ and C₆ branched-chain acyl-CoA derivatives, propionate, and butyrate had significant impacts on growth and fatty acid composition. Acetate and precursors that were metabolized to acetyl-CoA had minor effects on fatty acid composition, indicating that their preservative action is not due to effects on fatty acid composition.Open in a separate windowFIG. 1.Structures of potential fatty acid precursors.     

Are Fatty Acid Patterns Characteristic of Essential Fatty Acid Deficiency Indicative of Oxidative Stress?

Several unrelated diseases show plasma and tissue fatty acid patterns characteristic of those seen in Essential Fatty Acid Deficiency Disease (EFADD). A common feature occurring in all these diseases is oxidative stress. We hypothesize that reactive oxygen species or products of oxidative damage, particularly those derived from lipids, act as signal molecules to alter desaturase enzymes and induce the fatty acid patterns characteristic of EFADD.     

Structural and Functional Analysis of E. coli Cyclopropane Fatty Acid Synthase

1. Download high-res image (198KB)
2. Download full-size image

     

Interaction of Bovine β-Lactoglobulin and Other Bovine and Human Whey Proteins with Retinol and Fatty Acids

The interaction of bovine and human whey proteins with retinol and palmitic acid has been studied. Using gel filtration it was found that bovine β-lactoglobulin and α-lactalbumin and serum albumin from both species bind retinol in vitro while the ability to bind palmitic acid is restricted to bovine β-lactoglobulin and bovine and human serum albumin. Using equilibrium dialysis, β-lactoglobulin was found to display two binding sites for retinol per dimeric molecule with an association constant of 1.5 × 10⁴m^-1. Competition experiments showed that when the concentration ratio between total fatty acids and retinol is similar to that found in milk, palmitic acid competes with the binding of retinol to β-lactoglobulin.     

Distinct Effects of Fatty Acids on Translocation of γ- and ε-Subspecies of Protein Kinase C

Effects of fatty acids on translocation of the γ- and ε-subspecies of protein kinase C (PKC) in living cells were investigated using their proteins fused with green fluorescent protein (GFP). γ-PKC–GFP and ε-PKC–GFP predominated in the cytoplasm, but only a small amount of γ-PKC–GFP was found in the nucleus. Except at a high concentration of linoleic acid, all the fatty acids examined induced the translocation of γ-PKC–GFP from the cytoplasm to the plasma membrane within 30 s with a return to the cytoplasm in 3 min, but they had no effect on γ-PKC–GFP in the nucleus. Arachidonic and linoleic acids induced slow translocation of ε-PKC–GFP from the cytoplasm to the perinuclear region, whereas the other fatty acids (except for palmitic acid) induced rapid translocation to the plasma membrane. The target site of the slower translocation of ε-PKC–GFP by arachidonic acid was identified as the Golgi network. The critical concentration of fatty acid that induced translocation varied among the 11 fatty acids tested. In general, a higher concentration was required to induce the translocation of ε-PKC–GFP than that of γ-PKC–GFP, the exceptions being tridecanoic acid, linoleic acid, and arachidonic acid. Furthermore, arachidonic acid and the diacylglycerol analogue (DiC8) had synergistic effects on the translocation of γ-PKC–GFP. Simultaneous application of arachidonic acid (25 μM) and DiC8 (10 μM) elicited a slow, irreversible translocation of γ-PKC– GFP from the cytoplasm to the plasma membrane after rapid, reversible translocation, but a single application of arachidonic acid or DiC8 at the same concentration induced no translocation.These findings confirm the involvement of fatty acids in the translocation of γ- and ε-PKC, and they also indicate that each subspecies has a specific targeting mechanism that depends on the extracellular signals and that a combination of intracellular activators alters the target site of PKCs.     

Structure and Stability Analysis of Cytotoxic Complex of Camel α-Lactalbumin and Unsaturated Fatty Acids Produced at High Temperature

Abstract

α-Lactalbumin (α-La), together with oleic acid can be converted to a complex, which kills tumor cells selectively. Cytotoxic α-La -oleic acid and a-La -linoleic acid complexes were generated by adding fatty acid to camel holo a-La at 60°C (referred to as La-OA-60 and La-LA-60 state, respectively). Structural properties of these complexes were studied and compared to the camel α-La. The experimental results show that linoleic acid induces a-La partial unfolding but oleic acid does not change the protein structure significantly. Also the stability of La-OA-60 and La- LA-60 toward thermal denaturation was measured. The order of temperature at the transition midpoint is as follows: La-LA-60 < La-0A-60 < α-La. La-0A-60 complex inhibited tubulin polymerization in vitro. Although the structures of La-0A-60 and La-LA-60 were different, these two complexes had similar cytotoxic effect to DU145 human prostate cancer cells. Samples of La-0A-60 that have been renatured after denaturation lost the specific biological activity toward tumor cells.     

Substrate Specificity and Regioselectivity of Δ12 and ω3 Fatty Acid Desaturases from Saccharomyces kluyveri

Δ12 and ω3 fatty acid desaturases are key enzymes in the synthesis of polyunsaturated fatty acids (PUFAs), which are important constituents of membrane glycerolipids and also precursors to signaling molecules in many organisms. In this study, we determined the substrate specificity and regioselectivity of the Δ12 and ω3 fatty acid desaturases from Saccharomyces kluyveri (Sk-FAD2 and Sk-FAD3). Based on heterologous expression in Saccharomyces cerevisiae, it was found that Sk-FAD2 converted C16–20 monounsaturated fatty acids to diunsaturated fatty acids by the introduction of a second double bond at the ν+3 position, while Sk-FAD3 recognized the ω3 position of C18 and C20. Furthermore, fatty acid analysis of major phospholipids suggested that Sk-FAD2 and Sk-FAD3 have no strong substrate specificity toward the lipid polar head group or the sn-positions of fatty acyl groups in phospholipids.     

Fatty acid–genotype interactions and cardiovascular risk

Cardiovascular disease (CVD) risk and rate of progression is determined by genetic, environmental and behavioural factors. Majority of genotype–diet–CVD phenotype research till date has focussed on the interactive impact of single nucleotide polymorphisms (SNP) and dietary fat composition, on blood lipids levels, with strong evidence of the existence of hypo- and hyper-responders. However, a recognised concern in the field of nutrigenetics is a lack of consistency between findings of different studies. This apparent lack of consistency is likely to be attributable to the impact of factors such as ethnicity and gender on the ‘size’ of nutrigenetic interactions, a clear understanding of which needs to be gained. Although not yet ready for widespread use, in the future a greater use of genetic profiling is likely to enhance current strategies of CVD prediction, and improve the design of more personalised approaches to minimise risk in the individual.     

Fatty acid composition of odonate’s eyes

We have studied the fatty acid composition of eyes of amphibiotic insects, namely, the odonate Sympetrum flaveolum. The main polyunsaturated fatty acid of odonate’s eyes has been found to be 20:5n-3 (eicosapentaenoic fatty acid, EPA) rather than 18:2n-6 and 18:3n-3, which usually dominate in eyes of terrestrial insects, or 22:6n-3, which dominates in eyes of vertebrates. The prevalence of EPA in odonate’s eyes probably provides a more effective transmission of light signal in this animal compared to terrestrial insects. It is important for odonates because vision plays a decisive role in finding and catching prey.     

Inflammation and Resolution Are Associated with Upregulation of Fatty Acid β-Oxidation in Zymosan-Induced Peritonitis

Inflammation is a fundamental defensive response to harmful stimuli. However, it can cause damage if it does not subside. To avoid such damage, organisms have developed a mechanism called resolution of inflammation. Here we applied an untargeted metabolomics approach to a sterile and self-resolving animal model of acute inflammation, namely zymosan-induced peritonitis in mice, to examine the effect of inflammation and resolution on the metabolomic profiles. Significant and time-dependent changes in metabolite profiles after zymosan administration were observed in both peritoneal wash fluid (PWF) and plasma. These metabolomic changes correlated well with inflammatory chemokine or cytokine production. In PWF, most of metabolites that could detected increased in zymosan-treated mice, which is suggestive of inflammation, oxidative stress and increased energy demands. In plasma, most metabolites in the central metabolic pathway (glycolysis and TCA cycle) were significantly downregulated after zymosan administration. The concentration of the ketone body 3-hydroxybutyric acid (3-HB) in plasma and PWF increased in zymosan-injected animals indicating upregulation of fatty acid β-oxidation. Increased 3-HB level was observed in the cells that infiltrated into the peritoneal cavity and these infiltrated cells might contribute, at least in part, to the production of 3-HB in the peritoneal cavity.     

Fatty acid α-hydroxylation and its relation to myelination

Summary -Hydroxylation is an enzymatic reaction by which long-chain fatty acids are converted to their -hydroxy derivatives. This reaction, in animals, can be detected only in developing brain and is the rate-determining step in the synthesis of hydroxycerebroside, which is an indispensable and abundant myelin lipid. In addition to a particulate fraction from brain, two cytoplasmic factors, one heat-stable and the other heat-labile, are required for -hydroxylation. During the past eight years we have been investigating -hydroxylation. Our progress is summarized and discussed here.     

Diversity of Yeasts from Puddles in the Vicinity of Midre Lovénbreen Glacier, Arctic and Bioprospecting for Enzymes and Fatty Acids

A total of 132 yeast strains were characterised from 4 sediment samples collected from small puddles in the vicinity of Midre Lovénbreen glacier, Arctic. Based on the D1/D2 domain sequence similarity, the isolates could be categorised into 6 groups. The nearest phylogenetic neighbour of groups I to VI were identified as Cryptococcus gastricus, Cryptococcus terricolus, Rhodotorula muscorum, Mrakia psychrophila, Mrakia gelida and Rhodotorula glacialis, respectively. Strains representative of the six groups were psychrophilic and salt tolerant but varied in their ability to produce cold-active extracellular enzymes such as lipase, protease, pectinase, cellulase and amylase. C_{18:1 (w9C)} and C_{18:2 (w9,12C)} were the only two fatty acids common to all the yeasts and branched and (or) unsaturated fatty acids increased in yeasts growing at 8°C compared to 22°C, probably as an adaptation to low temperature. The present study establishes that psychrophilic yeasts are predominant in Arctic and could be used as work horses to produce cold-active enzymes and poly unsaturated fatty acids which have been implicated in low temperature adaptation and also for their use in biotechnology.     

Interaction of Sesamin and Eicosapentaenoic Acid against Δ5 Desaturation and n-6/ n-3 Ratio of Essential Fatty Acids in Rat

Sesamin is a specific inhibitor of Δ5 desaturation, the conversion from dihomo-γ-linolenic acid (20: 3, n-6) to arachidonic acid (AA, 20: 4, n-6). Previously, we reported that sesamin inhibited Δ5 desaturation of n-6 fatty acids in rat hepatocytes but not that of n-3 fatty acids, from 20: 4 (n-3) to eicosapentaenoic acid (EPA, 20: 5, n-3). In this study, we investigated the interaction of sesamin and EPA on Δ5 desaturation of both series and the n-6/n-3 fatty acids ratio by measuring actural fatty acid contents in vivo. Rats were fed three types of dietary oils; 1) linoleic acid (LA, 18: 2, n-6): linolenic acid (LLA, 18: 3, n-3) = 3: 1, n-6/n-3 ratio of 3: 1 (LA group), 2) LA: LLA =1: 3, n-6/n-3 ratio of 1: 3 (LLA group), 3) LA: LLA: EPA =1: 0.5: 3, n-6/n-3 ratio of 1: 3.5 (EPA group) with or without sesamin (0.5% w/w) for 4 weeks. In all groups, sesamin administration increased the content of dihomo-γ-linolenic acid (20: 3, n-6) in the liver and decreased the Δ5 desaturation index of n-6 fatty acid, the ratio of 20: 4/20: 3 (n-6). On the contrary, the Δ5 desaturation index of n-3 fatty acid, the ratio of 20: 5 + 22: 5 + 22: 6/20: 4 (n-3), was increased by the administration of sesamin. These results suggest that sesamin inhibits the A5 desaturation of n-6 fatty acid, but not that of n-3 fatty acid in rat livers. Sesamin administration decreased incorporation of EPA (n-3) and simultaneously increased the AA (n-6) content in the liver. The n-6/n-3 ratio in the liver was increased by administering sesamin under n-3 rich conditions, i.e., the LLA and EPA groups.     

Effects of n–3 Polyunsaturated Fatty Acids and Lectins on Immunoglobulin Production by Spleen Lymphocytes of Sprague-Dawley Rats

We examined the effects of n-3 polyunsaturated fatty acid (PUFA), such as α-linolenic (α -LA), eicosapentaenoic (EPA), and docosahexaenoic acid (DHA) on immunoglobulin (Ig) production by spleen lymphocytes of Sprague-Dawley rats, n-3 polyunsaturated fatty acid (PUFA) strongly inhibited the production of IgA and IgM and that of IgG weakly at 100 μΜ. When the lymphocytes were treated with n-3 PUFA in the presence of other inhibitory biomaterials such as lectins, some PUFA attenuated their inhibitory effect on Ig production. In the presence of concanavalin A (ConA), all n-3 PUFA attenuated the inhibitory effect of ConA on the production of IgM or IgG but increased its inhibition of IgA synthesis. Thus, the interaction of n-3 polyunsaturated fatty acid and lectins in spleen interfere with each other or the expression of Ig production regulating activity.     

Effect of Animal and Industrial Trans Fatty Acids on HDL and LDL Cholesterol Levels in Humans – A Quantitative Review

Background

Trans fatty acids are produced either by industrial hydrogenation or by biohydrogenation in the rumens of cows and sheep. Industrial trans fatty acids lower HDL cholesterol, raise LDL cholesterol, and increase the risk of coronary heart disease. The effects of conjugated linoleic acid and trans fatty acids from ruminant animals are less clear. We reviewed the literature, estimated the effects trans fatty acids from ruminant sources and of conjugated trans linoleic acid (CLA) on blood lipoproteins, and compared these with industrial trans fatty acids.

Methodology/Principal Findings

We searched Medline and scanned reference lists for intervention trials that reported effects of industrial trans fatty acids, ruminant trans fatty acids or conjugated linoleic acid on LDL and HDL cholesterol in humans. The 39 studies that met our criteria provided results of 29 treatments with industrial trans fatty acids, 6 with ruminant trans fatty acids and 17 with CLA. Control treatments differed between studies; to enable comparison between studies we recalculated for each study what the effect of trans fatty acids on lipoprotein would be if they isocalorically replaced cis mono unsaturated fatty acids. In linear regression analysis the plasma LDL to HDL cholesterol ratio increased by 0.055 (95%CI 0.044–0.066) for each % of dietary energy from industrial trans fatty acids replacing cis monounsaturated fatty acids The increase in the LDL to HDL ratio for each % of energy was 0.038 (95%CI 0.012–0.065) for ruminant trans fatty acids, and 0.043 (95% CI 0.012–0.074) for conjugated linoleic acid (p = 0.99 for difference between CLA and industrial trans fatty acids; p = 0.37 for ruminant versus industrial trans fatty acids).

Conclusions/Significance

Published data suggest that all fatty acids with a double bond in the trans configuration raise the ratio of plasma LDL to HDL cholesterol.     

Fatty acid β-oxidation and glyoxylate cycle enzyme activities of induced glyoxysomes from anise suspension cultures

Homogenates of dedifferentiated anise (Pimpinella anisum L.) suspension cultures grown in B-5 medium with sucrose as source of carbon show all but 3 glyoxysomal enzyme activities: NAD-dependent oxidation of palmitoyl-CoA, isocitrate lyase, and malate synthase are lacking. Substitution of 20 mmol/l acetate for sucrose leads to the appearance of these enzyme activities. Only then glyoxysomes with a buoyant density of 1.23 kg/l in sucrose gradients are formed showing the enzyme activities for both ß-oxidation of fatty acids and glyoxylate cycle. Quantitatively and qualitatively they resemble glyoxysomes isolated from endosperm of 4 d old anise seedlings. Therefore, the suspension cultures constitute a valuable system for the study of both mechanisms and regulation of glyoxysome formation in anise.     

The Family of Peanut Fatty Acid Desaturase Genes and a Functional Analysis of Four ω-3 AhFAD3 Members

Plant Molecular Biology Reporter - The synthesis of α-linolenic acid (ALA) requires the activity of ω-3 fatty acid desaturases (ω-3 FADs). The quality of peanut oil would be much...