The structural basis for specificity in lipoxygenase catalysis

Many intriguing facets of lipoxygenase (LOX) catalysis are open to a detailed structural analysis. Polyunsaturated fatty acids with two to six double bonds are oxygenated precisely on a particular carbon, typically forming a single chiral fatty acid hydroperoxide product. Molecular oxygen is not bound or liganded during catalysis, yet it is directed precisely to one position and one stereo configuration on the reacting fatty acid. The transformations proceed upon exposure of substrate to enzyme in the presence of O₂ (RH + O₂ → ROOH), so it has proved challenging to capture the precise mode of substrate binding in the LOX active site. Beginning with crystal structures with bound inhibitors or surrogate substrates, and most recently arachidonic acid bound under anaerobic conditions, a picture is consolidating of catalysis in a U‐shaped fatty acid binding channel in which individual LOX enzymes use distinct amino acids to control the head‐to‐tail orientation of the fatty acid and register of the selected pentadiene opposite the non‐heme iron, suitably positioned for the initial stereoselective hydrogen abstraction and subsequent reaction with O₂. Drawing on the crystal structures available currently, this review features the roles of the N‐terminal β‐barrel (C2‐like, or PLAT domain) in substrate acquisition and sensitivity to cellular calcium, and the α‐helical catalytic domain in fatty acid binding and reactions with O₂ that produce hydroperoxide products with regio and stereospecificity. LOX structures combine to explain how similar enzymes with conserved catalytic machinery differ in product, but not substrate, specificities.     

Characterization of antioxidant enzymes and peroxisomes of olive (Olea europaea L.) fruits

The presence of peroxisomes in olive (Olea europaea L.) fruits and different antioxidant enzymes occurring in this plant tissue is reported for the first time. Ultrastructural analysis showed that olive cells were characterized by the presence of large vacuoles and lipid drops. Plastids, mitochondria and peroxisomes were placed near the cell wall, showing some type of association with it. Olive fruit peroxisomes were purified by sucrose density-gradient centrifugation, and catalase, glutathione reductase and ascorbate peroxidase were found in peroxisomes. In olive fruit tissue the presence of a battery of antioxidant enzymes was demonstrated, including catalase, four superoxide dismutase isozymes (mainly an Fe-SOD plus 2 Cu,Zn-SOD and a Mn-SOD), all the enzymes of the ascorbate–glutathione cycle, reduced and oxidized glutathione, ascorbate, and four NADPH-recycling dehydrogenases. The knowledge of the full composition of antioxidants (enzymatic and non-enzymatic) in olive fruits is crucial to be able to understand the processes regulating the antioxidant composition of olive oil.     

Fatty-acid metabolism is involved in stress-resistance mechanisms of Caenorhabditis elegans

Fatty acids are the major components of the phospholipid bilayer and are involved in several functions of cell membrane. We previously reported that fatty-acid metabolism is involved in the regulation of DAF-2/insulin signal in Caenorhabditis elegans. In this study, we investigate the role of fatty-acid metabolism in stress resistance with respect to daf-16 in nematode. We found that fatty-acid metabolism regulates heat, osmotic, and oxidative-stress resistance in C. elegans. RNA interference (RNAi) of fat-6, fat-7, and elo-2 enhanced heat resistance but decreased oxidative-stress tolerance. RNAi of fat-2 strongly increased osmotic-stress resistance, whereas nhr-49-RNAi remarkably reduced osmotic and oxidative-stress tolerance. In daf-16 mutants (mgDf50), RNAi of fat-2 and fat-7 increased viability under osmotic stress, while RNAi of fat-6, fat-7, and elo-2 enhanced heat resistance. Exposure of saturated fatty acids to RNAi worms of fat-1-, fat-7-, and nhr-49 increased osmotic resistance. On the other hand, polyunsaturated fatty acids (PUFAs) reduced osmotic-stress tolerance in fat-2-RNAi worms, whereas PUFAs enhanced it in nhr-49-RNAi worms. Heat-stress resistance in fat-6- and fat-7-RNAi worms was suppressed by oleic acid.These results suggest that stress-resistance mechanisms are regulated by fatty-acid metabolism with or without DAF-16 activity.     

Observation of eight ancient olive trees (Olea europaea L.) growing in the Garden of Gethsemane

For thousands of years, olive trees (Olea europaea L.) have been a significant presence and a symbol in the Garden of Gethsemane, a place located at the foot of the Mount of Olives, Jerusalem, remembered for the agony of Jesus Christ before his arrest. This investigation comprises the first morphological and genetic characterization of eight olive trees in the Garden of Gethsemane. Pomological traits, morphometric, and ultrastructural observations as well as SSR (Simple Sequence Repeat) analysis were performed to identify the olive trees. Statistical analyses were conducted to evaluate their morphological variability. The study revealed a low morphological variability and minimal dissimilarity among the olive trees. According to molecular analysis, these trees showed the same allelic profile at all microsatellite loci analyzed. Combining the results of the different analyses carried out in the frame of the present work, we could conclude that the eight olive trees of the Gethsemane Garden have been propagated from a single genotype.     

Developmental events in differentiating floral buds of four olive (Olea europaea L.) cultivars during late winter to early spring

The olive tree (Olea europaea L.) is a very important evergreen fruit tree because of the high interest of olive oil and table olives in the human diet. Differentiation of olive floral buds during winter is strictly related to flowering during spring and finally to fruit production during autumn–winter. In order to determine which are the developmental events in differentiating olive floral buds we studied in four olive cultivars “Amfissis”, “Kalamon”, “Manzanillo” and “Chalkidikis” the anatomical progress of the ontogeny using median longitudinal sections of floral buds (cutting odd nodes), in weekly intervals from early February (initiation of differentiation) to mid-April (one week after floral bud burst) keeping the cultivars under the same environmental conditions. At the same time, we determined the changes in their water extractable proteins, as an index of the meristematic activity. At the beginning of differentiation, floral buds consisted of the apical and one pair of developed (odd) axillary meristems which were covered with the corresponding bracts. Floral buds showed a slow gradual enlargement in all cultivars. The sequence of the developmental events (initiation of intense mitotic activity in the apical and the first axillary meristems, initiation of mitotic activity in the second axillary meristem, development of the third pair of bracts (except from cv. Chalkidikis), appearance of mitotic activity in the third axillary meristem, swelling of apical and axillary meristems and appearance of primordial whorls) are presented comparatively. All cultivars showed similar ontogenetic trends but there were temporal differences in the initiation, the progress and the completion of the process. Cv. Amfissis initiated differentiation one week later compared to the other cultivars, also delaying the development of the second and the third axillary meristems. On the other hand, cvs. Kalamon and Chalkidikis delayed one week to complete the process when compared to cvs. Amfissis and Manzanillo.     

Lauroylethanolamide is a potent competitive inhibitor of lipoxygenase activity

The lipoxygenase (LOX) pathway was proposed to compete with hydrolysis and be partly responsible for the metabolism of polyunsaturated N-acylethanolamines (PU-NAEs). Treatment of Arabidopsis seedlings with lauroylethanolamide (NAE 12:0) resulted in elevated levels of PU-NAE species, and this was most pronounced in plants with reduced NAE hydrolase activity. Enzyme activity assays revealed that NAE 12:0 inhibited LOX-mediated oxidation of PU lipid substrates in a dose-dependent and competitive manner. NAE 12:0 was 10-20 times more potent an inhibitor of LOX activities than lauric acid (FFA 12:0). Furthermore, treatment of intact Arabidopsis seedlings with NAE 12:0 (but not FFA 12:0) substantially blocked the wound-induced formation of jasmonic acid (JA), suggesting that NAE 12:0 may be used in planta to manipulate oxylipin metabolism.     

On the singular, dual, and multiple positional specificity of manganese lipoxygenase and its G316A mutant

Abstract manganese lipoxygenase (Mn-LO) oxygenates 18:3n-3 and 18:2n-6 to bis-allylic 11S-hydroperoxy fatty acids, which are converted to 13R-hydroperoxy fatty acids. Other unsaturated C(16)-C(22) fatty acids, except 17:3n-3, are poor substrates, possibly because of ineffective enzyme activation (Mn(II)-->Mn(III)) by the produced hydroperoxides. Our aim was to determine whether unsaturated C(16)-C(22) fatty acids were oxidized by Mn(III)-LO. Mn(III)-LO oxidized C(16), C(19), C(20), and C(22) n-3 and n-6 fatty acids. The carbon chain length influenced the position of hydrogen abstraction (n-8, n-5) and oxygen insertion at the terminal or the penultimate 1Z,4Z-pentadienes. Dilinoleoyl-glycerophosphatidylcholine was oxidized by Mn-LO, in agreement with a "tail-first" model. 16:3n-3 was oxidized at the bis-allylic n-5 carbon and at positions n-3, n-7, and n-6. Long fatty acids, 19:3n-3, 20:3n-3, 20:4n-6, 22:5n-3, and 22:5n-6, were oxidized mainly at the n-6 and the bis-allylic n-8 positions (in ratios of approximately 3:2). The bis-allylic hydroperoxides accumulated with one exception, 13-hydroperoxyeicosatetraenoic acid (13-HPETE). Mn(III)-LO oxidized 20:4n-6 to 15R-HPETE ( approximately 60%) and 13-HPETE ( approximately 37%) and converted 13-HPETE to 15R-HPETE. Mn(III)-LO G316A oxygenated mainly 16:3n-3 at positions n-7 and n-6, 19:3n-3 at n-10, n-8, and n-6, and 20:3n-3 at n-10 and n-8. We conclude that Mn-LO likely binds fatty acids tail-first and oxygenates many C(16), C(18), C(20), and C(22) fatty acids to significant amounts of bis-allylic hydroperoxides.     

Molecular cloning and functional analysis of two FAD2 genes from American grape (Vitis labrusca L.)

The synthesis of polyunsaturated fatty acids (PUFAs), the most abundant fatty acids in plants, begins with a reaction catalyzed by fatty acid desaturase 2 (FAD2; EC 1.3.1.35), also called microsomal oleate Δ12-desaturase. Since the FAD2 gene was first identified in Arabidopsis thaliana, FAD2 research has gained wide interest as the essential enzyme for synthesizing PUFA. Grapes are one of the most frequently cultivated fruits in the world, with most commercial growers cultivating Vitis vinifera and V. labrusca. Grapeseed oil contains a high proportion, 60–70% of linoleic acid (18:2). We cloned two putative FAD2 genes from V. labrusca cv. Campbell Early based on V. vinifera genome sequences. Deduced amino acid sequences of two putative genes showed that VlFAD2s show high similarity to Arabidopsis FAD2 and commonly contain six transmembrane domain, three histidine boxes and endoplasmic reticulum (ER) retrieval motif representing the characteristics of fatty acid desaturase. Phylogenetic analyses of various plant FAD2s showed that VlFAD2-1 and VlFAD2-2 are separately grouped with constitutive and seed-type FAD2s, respectively. Southern blot showed that one or two bands are found in each lane. Because Campbell Early is a hybrid cultivar, FAD2-1 and FAD2-2 genes may exist as one copy in V. labrusca. Expression analysis in different tissues indicated that VlFAD2-1 is a constitutive gene but VlFAD2-2 is a seed-type gene. Complementation experiments of fad2-1 mutant Arabidopsis with VlFAD2-1 or VlFAD2-2 demonstrated that VlFAD2-1 and VlFAD2-2 can restore low PUFA proportion of fad2 to normal PUFA proportion.     

Regiospecificity of a novel bacterial lipoxygenase from Myxococcus xanthus for polyunsaturated fatty acids

Lipoxygenase (LOX) is the key enzyme involved in the synthesis of oxylipins as signaling compounds that are important for cell growth and development, inflammation, and pathogenesis in various organisms. The regiospecificity of LOX from Myxococcus xanthus, a gram-negative bacterium, was investigated. The enzyme catalyzed oxygenation at the n-9 position in C20 and C22 polyunsaturated fatty acids (PUFAs) to form 12S- and 14S-hydroxy fatty acids (HFAs), respectively, and oxygenation at the n-6 position in C18 PUFAs to form 13-HFAs. The 12S-form products of C20 and C22 PUFAs by M. xanthus LOX is the first report of bacterial LOXs. The residues involved in regiospecificity were determined to be Thr397, Ala461, and Ile664 by analyzing amino acid alignment and a homology model based on human arachidonate 15-LOX with a sequence identity of 25%. Among these variants, the regiospecificity of the T397Y variant for C20 and C22 PUFAs was changed. This may be because of the reduced size of the substrate-binding pocket by substitution of the smaller Thr to the larger Tyr residue. The T397Y variant catalyzed oxygenation at the n-6 position in C20 and C22 PUFAs to form 15- and 17-hydroperoxy fatty acids, respectively. However, the oxygenation position of T397Y for C18 PUFAs was not changed. The discovery of bacterial LOX with novel regiospecificity will facilitate the biosynthesis of regiospecific?oxygenated signaling compounds.     

Hydroperoxide specificity of plant and human tissue lipoxygenase: an in vitro evaluation using N-demethylation of phenothiazines

Since hydroperoxide specificity of lipoxygenase (LO) is poorly understood at present, we investigated the ability of cumene hydroperoxide (CHP) and tert-butyl hydroperoxide (TBHP) to support cooxidase activity of the enzyme toward the selected xenobiotics. Considering the fact that in the past, studies of xenobiotic N-demethylation have focused on heme-proteins such as P450 and peroxidases, in this study, we investigated the ability of non-heme iron proteins, namely soybean LO (SLO) and human term placental LO (HTPLO) to mediate N-demethylation of phenothiazines. In addition to being dependent on peroxide concentration, the reaction was dependent on enzyme concentration, substrate concentration, incubation time, and pH of the medium. Using Nash reagent to estimate formaldehyde production, the specific activity under optimal assay conditions for the SLO mediated N-demethylation of chlorpromazine (CPZ), a prototypic phenothiazine, in the presence of TBHP, was determined to be 117±12 nmol HCHO/min/mg protein, while that of HTPLO was 3.9±0.40 nmol HCHO/min/mg protein. Similar experiments in the presence of CHP yielded specific activities of 106±11 nmol HCHO/min/mg SLO, and 3.2±0.35 nmol HCHO/min/mg HTPLO. As expected, nordihydroguaiaretic acid and gossypol, the classical inhibitors of LOs, as well as antioxidants and free radical reducing agents, caused a marked reduction in the rate of formaldehyde production from CPZ by SLO in the reaction media fortified with either CHP or TBHP. Besides chlorpromazine, both SLO and HTPLO also mediated the N-demethylation of other phenothiazines in the presence of these organic hydroperoxides.     

Lipid and oxylipin profiles during aging and sprout development in potato tubers (Solanum tuberosum L.)

Potato tubers (Solanum tuberosum L. cv Bintje) were stored at 20 °C for 210 days without desprouting to study the lipoxygenase pathway during aging. After 15 days of storage, potato tubers sprouted, while after 45–60 days, apical dominance was lost and multiple sprouts developed. Analysis of the fatty acid hydroperoxides (HPOs) revealed that 9-S-hydroperoxide of linoleic acid (9-HPOD) was the main oxylipin formed. Between 45 and 60 days of storage, increases in the levels of 9-HPOD and colneleic acid were observed. Analysis of phospholipids and galactolipids by electrospray ionisation tandem mass spectrometry (ESI-MS/MS) showed that a decrease in the levels of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), digalactosyldiacylglycerol (DGDG), and monogalactosyldiacylglycerol (MGDG) occurred between 0 and 45 days of aging. The decrease in the amount of linoleic acid in complex lipids correlates well with the amount of 9-HPOD and colneleic acid produced.     

Functional characterization of two microsomal fatty acid desaturases from Jatropha curcas L.

Linoleic acid (LA, C18:2) and α-linolenic acid (ALA, C18:3) are polyunsaturated fatty acids (PUFAs) and major storage compounds in plant seed oils. Microsomal ω-6 and ω-3 fatty acid (FA) desaturases catalyze the synthesis of seed oil LA and ALA, respectively. Jatropha curcas L. seed oils contain large proportions of LA, but very little ALA. In this study, two microsomal desaturase genes, named JcFAD2 and JcFAD3, were isolated from J. curcas. Both deduced amino acid sequences possessed eight histidines shown to be essential for desaturases activity, and contained motif in the C-terminal for endoplasmic reticulum localization. Heterologous expression in Saccharomyces cerevisiae and Arabidopsis thaliana confirmed that the isolated JcFAD2 and JcFAD3 proteins could catalyze LA and ALA synthesis, respectively. The results indicate that JcFAD2 and JcFAD3 are functional in controlling PUFA contents of seed oils and could be exploited in the genetic engineering of J. curcas, and potentially other plants.     

Olive (Olea europaea) leaf extract effective in patients with stage-1 hypertension: Comparison with Captopril

A double-blind, randomized, parallel and active-controlled clinical study was conducted to evaluate the anti-hypertensive effect as well as the tolerability of Olive leaf extract in comparison with Captopril in patients with stage-1 hypertension. Additionally, this study also investigated the hypolipidemic effects of Olive leaf extract in such patients. It consisted of a run-in period of 4 weeks continued subsequently by an 8-week treatment period. Olive (Olea europaea L.) leaf extract (EFLA^®943) was given orally at the dose of 500 mg twice daily in a flat-dose manner throughout the 8 weeks. Captopril was given at the dosage regimen of 12.5 mg twice daily at start. After 2 weeks, if necessary, the dose of Captopril would be titrated to 25 mg twice daily, based on subject's response to treatment. The primary efficacy endpoint was reduction in systolic blood pressure (SBP) from baseline to week-8 of treatment. The secondary efficacy endpoints were SBP as well as diastolic blood pressure (DBP) changes at every time-point evaluation and lipid profile improvement. Evaluation of BP was performed every week for 8 weeks of treatment; while of lipid profile at a 4-week interval. Mean SBP at baseline was 149.3 ± 5.58 mm Hg in Olive group and 148.4 ± 5.56 mm Hg in Captopril group; and mean DBPs were 93.9 ± 4.51 and 93.8 ± 4.88 mm Hg, respectively. After 8 weeks of treatment, both groups experienced a significant reduction of SBP as well as DBP from baseline; while such reductions were not significantly different between groups. Means of SBP reduction from baseline to the end of study were −11.5 ± 8.5 and −13.7 ± 7.6 mm Hg in Olive and Captopril groups, respectively; and those of DBP were −4.8 ± 5.5 and −6.4 ± 5.2 mm Hg, respectively. A significant reduction of triglyceride level was observed in Olive group, but not in Captopril group. In conclusion, Olive (Olea europaea) leaf extract, at the dosage regimen of 500 mg twice daily, was similarly effective in lowering systolic and diastolic blood pressures in subjects with stage-1 hypertension as Captopril, given at its effective dose of 12.5-25 mg twice daily.     

Defense peptides from barnyard grass (Echinochloa crusgalli L.) seeds

A number of defense polypeptides from latent seeds of weed cereal barnyard grass (Echinochloa crusgalli L.) has been isolated and characterized using an acidic extraction and high performance liquid chromatography methods in combination with MALDI-TOF mass spectrometry and Edman sequencing. Members of three antimicrobial peptide families and two protease inhibitor families were found to be localized in barnyard grass seeds. Their biological activity concerning to Gram-Positive and Gram-Negative phytopathogenic bacteria, as well as oomycete Phytophthora infestans, has been investigated. Diversity of barnyard grass defense peptides is a significant factor that provides a resistance of E. crusgalli seeds to germination and latent phases.     

Studies on the group effect in Acheta domesticus (L.) using artificial diets

Larvae of Acheta domesticus were reared on artificial diets both singly and in groups of six. The growth of group-reared larvae was significantly faster, and survival of singly reared larvae was greatly reduced. Survival of singly reared larvae was raised to that of grouped larvae when 1% wheat-germ oil was added to the diet. Wheat-germ oil also improved the growth of grouped larvae and of singly reared female larvae. The fatty acid composition of body lipid was similar, except for linolenic acid, whether the larvae were singly or group-reared, or whether wheat-germ oil was fed or not. These results are discussed from the point of view of the mechanism of the group effect.     

Arachidonic and docosahexaenoic acids reduce the growth of A549 human lung-tumor cells increasing lipid peroxidation and PPARs

Polyunsaturated fatty acids (PUFAs) play an important role in both induction and prevention of carcinogenic process. It is well known that several types of neoplastic cells show decreased total PUFA content, contributing to their resistance to chemotherapy and lipid peroxidation. In the light of this, human lung cancer A549 cells, with low PUFA content, were exposed to arachidonic or docosahexaenoic acid to investigate the effect of n-6 and n-3 PUFAs on growth and elucidate underlying mechanisms. The bulk of the results showed that both n-6 PUFAs and n-3 PUFAs decrease human lung-tumor cell growth in a concentration-dependent manner, inducing cell death mainly evident at 100microM concentration. The mechanism underlying the antiproliferative effect of n-6 and n-3 PUFAs appeared to be the same, involving changes in fatty acid composition of biomembranes, production of cytostatic aldehydes derived from lipid peroxidation and able to affect DNA-binding activity of AP-1, and induction of PPAR. From these results it may be hypothesized that n-6 PUFAs, like n-3 PUFAs, are able to inhibit tumor growth.     

A grape berry (Vitis vinifera L.) cation/proton antiporter is associated with berry ripening

We have cloned and characterized VvNHX1, a gene encoding a vacuolar cation/H(+) antiporter from Vitis vinifera cv. Cabernet Sauvignon. VvNHX1 belongs to the vacuolar NHX protein family and showed high similarity to other known vacuolar antiporters. The expression of VvNHX1 partially complements the salt- and hygromycin-sensitive phenotypes of an ena1-4 nhx1 yeast strain. Immunoblots of vacuoles of yeast expressing a VvNHX1, together with the expression of a VvNHX1-GFP (green fluorescent protein) chimera demonstrated that VvNHX1 localized to the vacuoles. VvNHX1 displayed low affinity K(+)/H(+) and Na(+)/H(+) exchange activities (12.8 and 40.2 mM, respectively). The high levels of expression of VvNHX1 during the véraison and post-véraison stages would indicate that the increase in vacuolar K(+) accumulation, mediated by VvNHX1, is needed for vacuolar expansion. This process, together with the rapid accumulation of reducing sugars, would drive water uptake to the berry and the concomitant berry size increase, typical of the post-véraison stage of growth.     

The effect of nitrogen limitation on acetyl-CoA carboxylase expression and fatty acid content in Chromera velia and Isochrysis aff. galbana (TISO)

Lipids from microalgae have become a valuable product with applications ranging from biofuels to human nutrition. While changes in fatty acid (FA) content and composition under nitrogen limitation are well documented, the involved molecular mechanisms are poorly understood. Acetyl-CoA carboxylase (ACCase) is a key enzyme in the FA synthesis and elongation pathway. Plastidial and cytosolic ACCases provide malonyl-CoA for de novo FA synthesis in the plastid and FA elongation in the endoplasmic reticulum, respectively. The present study aimed at investigating the expression of plastidial and cytosolic ACCase in Chromera velia and Isochrysis aff. galbana (TISO) and their impact on FA content and elongation level when grown under nitrogen-deplete conditions. In C. velia, plastidial ACCase was significantly upregulated during nitrogen starvation and with culture age, strongly correlating with increased FA content. Conversely, plastidial ACCase of I. aff. galbana was not differentially expressed in nitrogen-deplete cultures, but upregulated during the logarithmic phase of nitrogen-replete cultures. In contrast to plastidial ACCase, the cytosolic ACCase of C. velia was downregulated with culture age and nitrogen-starvation, strongly correlating with an increase in medium-chain FAs. In conclusion, the expression of plastidial and cytosolic ACCase changed with growth phase and nutrient status in a species-specific manner and nitrogen limitation did not always result in FA accumulation.