Hydroperoxide isomerase: a new enzyme of lipid metabolism

An enzyme has been isolated from flaxseed (Linum usitatissimum) which utilizes the product of lipoxidase for its substrate. The enzyme, termed hydroperoxide isomerase, converts the conjugated diene hydroperoxide of linoleic acid to the corresponding monoenoic ketohydroxy fatty acid. The structure of the latter has been determined by ultraviolet, infrared, and nuclear magnetic resonance spectroscopy; periodate and permangate oxidation; gas chromatography; and thin layer chromatography. Hydroperoxide isomerase activity has also been demonstrated in crude extracts from barley (Hordeum vulgare), wheat germ (Triticum aestivum), mung beans (Phaseolus aureus), and corn (Zea mays) and from partially purified extracts of soybean (Glycine max).     

Allene oxide cyclase is essential for theobroxide-induced jasmonic acid biosynthesis in Pharbitis nil

Theobroxide, a natural product, strongly stimulates the biosynthesis of jasmonic acid (JA) in Pharbitis nil. In this study, we investigated the accumulation of protein by the immunoblot analysis of lipoxygenase (LOX), allene oxide synthase (AOS), and allene oxide cyclase (AOC), key enzymes in JA biosynthesis, and how the endogenous levels of JA in P. nil are affected by theobroxide. The effect of JA on the accumulations of these proteins was monitored simultaneously. The results show that theobroxide treatment led to a high level accumulation of JA, which is due to high accumulations of LOX, AOS, and AOC proteins induced by theobroxide treatment both under short day (SD) and long day (LD) conditions. However, under SD conditions AOS and AOC proteins are not enhanced by JA treatment. Kinetic analysis of protein levels shows that a biphasic activation of AOC protein by theobroxide is displayed and the first activation of AOC protein together with elevated JA levels is observed within 30min after treatment. Meanwhile, AOS and LOX proteins are activated by theobroxide later than AOC protein, suggesting that AOC plays an essential role in the initial JA formation induced by theobroxide. Since theobroxide-increased JA levels also show a biphasic manner similar to AOC activation and AOS, LOX proteins are activated later than AOC, and thus we propose a positive JA feedback regulation. Interestingly, AOS protein, which is also the enzyme for the biosynthesis of 9,10-ketol-octadecadienoic acid (KODA, a flowering inducing factor), accumulates markedly due to the simultaneous involvement of theobroxide and SD conditions, suggesting that AOS probably plays a role in flower bud formation in P. nil.     

Acyl-CoA oxidase 1 from Arabidopsis thaliana. Structure of a key enzyme in plant lipid metabolism

The peroxisomal acyl-CoA oxidase family plays an essential role in lipid metabolism by catalyzing the conversion of acyl-CoA into trans-2-enoyl-CoA during fatty acid beta-oxidation. Here, we report the X-ray structure of the FAD-containing Arabidopsis thaliana acyl-CoA oxidase 1 (ACX1), the first three-dimensional structure of a plant acyl-CoA oxidase. Like other acyl-CoA oxidases, the enzyme is a dimer and it has a fold resembling that of mammalian acyl-CoA oxidase. A comparative analysis including mammalian acyl-CoA oxidase and the related tetrameric mitochondrial acyl-CoA dehydrogenases reveals a substrate-binding architecture that explains the observed preference for long-chained, mono-unsaturated substrates in ACX1. Two anions are found at the ACX1 dimer interface and for the first time the presence of a disulfide bridge in a peroxisomal protein has been observed. The functional differences between the peroxisomal acyl-CoA oxidases and the mitochondrial acyl-CoA dehydrogenases are attributed to structural differences in the FAD environments.     

Allene oxide synthase: a major control point in Arabidopsis thaliana octadecanoid signalling

The analysis of allene oxide synthase (AOS) mRNA levels, of AOS polypeptide levels and specific enzymatic activities, as well as the quantitative determination of the levels of the octadecanoids cis-12-oxophytodienoic acid (cis-OPDA) and JA following a number of treatments, has shown that AOS is a regulatory site in octadecanoid biosynthesis in A. thaliana. AOS activity, mRNA and polypeptide levels are increased in wounded leaves locally and systemically. The methyl esters of OPDA or JA (OPDAME, JAME) and coronatine, are strong inducers of AOS mRNA, polypeptide and enzymatic activity. Ethephon also induces AOS activity. Salicylic acid (SA) was an inducer of AOS activity while abscisic acid (ABA) had no effect. At the level of the octadecanoids, the consequences of induction of AOS by the different inducers were distinctly different, depending on the nature of the inducer. Wounding led to a strong, bi-phasic accumulation of JA in wounded leaves and to a less pronounced increase in JA-levels in systemic leaves. Levels of OPDA changed very little in wounded leaves and remained constant or even declined in systemic leaves. Ethephon treatment resulted in a strong, transient increase in JA-levels kinetically coinciding with the second, more pronounced peak in wound-induced JA. In SA-treated leaves, the level of cis-OPDA increased throughout the experimental period while there was no effect on JA levels during the first 24 h following treatment and only a slight accumulation after 48 h. Clearly, mechanisms in addition to regulating substrate (LA) availability and the regulation of AOS accumulation control the output of the octadecanoid pathway.     

Butyrylcholinesterase in lipid metabolism: A new outlook

Cholinesterase enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are traditionally associated with the termination of acetylcholine mediated neural signaling. The fact that these ubiquitous enzymes are also found in tissues not involved in neurotransmission has led to search for alternative functions for these enzymes. Cholinesterases are reported to be involved in many lipid related disease states. Taking into view that lipases and cholinesterases belong to the same enzyme class and by comparing the catalytic sites, we propose a new outlook on the link between BChE and lipid metabolism. The lipogenic substrates of BChE that have recently emerged in contrast to traditional cholinesterase substrates are explained through the hydrolytic capacity of BChE for ghrelin, 4-methyumbelliferyl (4-mu) palmitate, and arachidonoylcholine and through endogenous lipid mediators such as cannabinoids like anandamide and essential fatty acids. The abundance of BChE in brain, intestine, liver, and plasma, tissues with active lipid metabolism, supports the idea that BChE may be involved in lipid hydrolysis. BChE is also regulated by various lipids such as linoleic acid, alpha-linolenic acid or dioctanoylglycerol, whereas AChE is inhibited. The finding that BChE is able to hydrolyze 4-mu palmitate at a pH where lipases are less efficient points to its role as a backup in lipolysis. In diseases such as Alzheimer, in which elevated BChE and impaired lipid levels are observed, the lipolytic activity of BChE might be involved. It is possible to suggest that fatty acids such as 4-mu palmitate, ghrelin, arachidonoylcholine, essential fatty acids, and other related lipid mediators regulate cholinesterases, which could lead to some sort of compensatory mechanism at high lipid concentrations.

     

Malate metabolism by NADP-malic enzyme in plant defense

Malate is involved in various metabolic pathways, and there are several enzymes that metabolize it. One important malate metabolizing enzyme is NADP-malic enzyme (NADP-ME). NADP-ME functions in many different pathways in plants, having an important role in C₄ photosynthesis where it releases the CO₂ to be used in carbon fixation by Rubisco. Apart from this specialized role, NADP-ME is thought to fulfill diverse housekeeping functions because of its universal presence in different plant tissues. NADP-ME is induced after wounding or exposure to UV-B radiation. In this way, the enzyme is implicated in defense-related deposition of lignin by providing NADPH for the two NADPH-dependent reductive steps in monolignol biosynthesis. On the other hand, it can supply NADPH for flavonoid biosynthesis as many steps in the flavonoid biosynthesis pathway require reductive power. Pyruvate, another product of NADP-ME reaction, can be used for obtaining ATP through respiration in the mitochondria; and may serve as a precursor for synthesis of phosphoenolpyruvate (PEP). PEP is utilized in the shikimate pathway, leading to the synthesis of aromatic amino acids including phenylalanine, the common substrate for lignin and flavonoid synthesis. Moreover, NADP-ME can be involved in mechanisms producing NADPH for synthesis of activated oxygen species that are produced in order to kill or damage pathogens. In conclusion, an increase in the levels of NADP-ME could provide building blocks and energy for biosynthesis of defense compounds, suggesting a role of malate metabolism in plant defense.     

Nitric oxide: a new player in plant signalling and defence responses

There is increasing evidence that nitric oxide (NO), which was first identified as a unique diffusible molecular messenger in animals, plays important roles in diverse (patho)physiological processes in plants. NO functions include the modulation of hormonal, wounding and defence responses, as well as the regulation of cell death. Enzymes that catalyse NO synthesis and signalling cascades that mediate NO effects have recently been discovered, providing a better understanding of the mechanisms by which NO influences plant responses to various stimuli. Additionally, growing evidence suggests that NO signalling interacts with the salicylic acid and jasmonic acid signalling pathways.     

RIFL aims to be a new player in lipid metabolism

RIFL (refeeding induced in fat and liver) is highly expressed in brown and white fat as well as in liver. In white adipose tissue and liver, RIFL expression is induced by refeeding and is also elevated in ob/ob mice. The function of RIFL is unknown, and there is some evidence to suggest it may be secreted. RIFL expression is induced during adipogenesis in rodent and human model systems, and cellular knockdown and mouse knockout studies demonstrate that RIFL expression correlates with lipid levels. Overall, these studies indicate that RIFL is a new important player in lipid metabolism.     

S-adenosyl-L-methionine:thioether S-methyltransferase, a new enzyme in sulfur and selenium metabolism

The final urinary excretion product of selenium detoxification is trimethylselenonium ion. An assay has been developed for the enzyme, S-adenosylmethionine:thioether S-methyltransferase, responsible for this final methylation reaction. This assay employed high pressure liquid chromatography separation and quantitation of the trimethylselenonium ion produced by thioether methyltransferase acting on S-adenosylmethionine and dimethyl selenide. The enzyme was shown to reside primarily in the cytosol of mouse lung (30 pmol/mg protein/min) and liver (7 pmol/mg protein/min). Purification from mouse lung to a preparation that exhibited a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was achieved by DEAE, gel filtration, and chromatofocusing chromatographies. Thioether methyltransferase is monomeric with a molecular weight of 28,000 and has a pI of 5.3. The pH optimum was 6.3, and Km values for dimethyl selenide and S-adenosylmethionine were 0.4 and 1.0 microM, respectively. The enzyme was inhibited 50% by 25 microM sinefungin, an analog of S-adenosylmethionine, or 40 microM S-adenosylhomocysteine, the reaction product. Pure thioether methyltransferase methylated selenium in dimethyl selenide, tellurium in dimethyl telluride, and S in dimethyl sulfide and many other thioethers. These data suggest a general role for this novel enzyme in the synthesis of onium compounds with increased aqueous solubility helpful in their excretion.     

Allene oxide cyclase dependence of the wound response and vascular bundle-specific generation of jasmonates in tomato - amplification in wound signalling

The allene oxide cyclase (AOC)-catalyzed step in jasmonate (JA) biosynthesis is important in the wound response of tomato. As shown by treatments with systemin and its inactive analog, and by analysis of 35S::prosysteminsense and 35S::prosysteminantisense plants, the AOC seems to be activated by systemin (and JA) leading to elevated formation of JA. Data are presented on the local wound response following activation of AOC and generation of JA, both in vascular bundles. The tissue-specific occurrence of AOC protein and generation of JA is kept upon wounding or other stresses, but is compromised in 35S::AOCsense plants, whereas 35S::AOCantisense plants exhibited residual AOC expression, a less than 10% rise in JA, and no detectable expression of wound response genes. The (i). activation of systemin-dependent AOC and JA biosynthesis occurring only upon substrate generation, (ii). the tissue-specific occurrence of AOC in vascular bundles, where the prosystemin gene is expressed, and (iii). the tissue-specific generation of JA suggest an amplification in the wound response of tomato leaves allowing local and rapid defense responses.     

Allene oxide and aldehyde biosynthesis in starfish oocytes.

Allene oxides are a very unusual type of epoxide that, in biological systems, are formed by the enzymic dehydration of fatty acid hydroperoxides (lipoxygenase products). This reaction occurs widely in plants, in which allene oxide synthesis is a key step in the conversion of linolenic acid to jasmonic acid, the plant growth regulator. We report biosynthesis of the allene oxide (8R)-8,9-epoxyeicosa-(5Z,9,11Z,14Z)-tetraenoic acid via the (8R)-lipoxygenase metabolism of arachidonic acid in starfish oocytes. Formation of the allene oxide was deduced from high pressure liquid chromatography, UV, gas chromatography-mass spectrometry and 1H-NMR analyses of the precise structure and mechanism of biosynthesis of its major hydrolysis product, the alpha-ketol 8-hydroxy-9-ketoeicosa-(5Z,11Z,14Z)-trienoic acid. A second enzymic activity detected in the oocytes (hydroperoxide lyase) cleaves specifically the (8R)-hydroperoxy substrate into C7 and C13 fragments, identified as the hydroxyacid, (5Z)-7-hydroxyheptenoic acid, and two aldehydes, (2E,4Z,7Z)-tridecenal and its 4E isomer. Discovery of the allene oxide synthase and hydroperoxide lyase marks the first definitive localization of these enzymic activities to an animal cell. It was established previously that the (8R)-lipoxygenase metabolite (8R)-HETE will activate the maturation (re-initiation of meiosis) of starfish oocytes. The individual 8-lipoxygenase products may be involved at distinct stages of cell development.     

Cytochemical immuno-localization of allene oxide cyclase, a jasmonic acid biosynthetic enzyme, in developing potato stolons

The involvement of jasmonates in the tuber development has been proved by the presence of many of these compounds in potato stolons, modification of their levels during the transition of the stolon into tuber, and induction of cell expansion upon exogenous jasmonates treatment. However, to date there is only little evidence of the presence of the jasmonic acid-biosynthetic enzymes in stolons or young tubers. As allene oxide cyclase represents the major control point for jasmonic acid biosynthesis, we studied the occurrence of allene oxide cyclase by immunological approaches in the early stages of tuber formation. In developing stolons, allene oxide cyclase as well as lipoxygenase were clearly detectable, but their levels did not change during development. Jasmonic acid treatment for 24h, however, increased lipoxygenase and allene oxide cyclase protein levels in both developmental stages analyzed. In longitudinal sections of stolons of stages 1 and 2, allene oxide cyclase and lipoxygenase occurred in the apex and along the stolon axis. Allene oxide cyclase was clearly detectable in epidermal, cortical and pith parenchymatic cells, showing the highest levels in vascular tissues surrounding cells. Lipoxygenase was mainly located in the parenchymatic cortex cells. The occurrence of allene oxide cyclase in stolons together with the previous identification of jasmonates from developing stolons reveals that these organs are capable to synthesize and metabolize jasmonates.     

Cloning, expression, and sequencing of squalene-hopene cyclase, a key enzyme in triterpenoid metabolism.

The pentacyclic hopanoids, a class of eubacterial lipids, are synthesized by squalene-hopene cyclase and side chain-elongating enzymes. With the aid of DNA probes based on the amino-terminal sequence of purified squalene-hopene cyclase from Bacillus acidocaldarius, clones of Escherichia coli that express this enzyme in the cytoplasmic membrane were isolated. According to the DNA sequence, the cyclase contained 627 amino acids with a molecular mass of 69,473 Da. A high percentage of the amino acids were basic. No significant similarity to existing sequenced proteins was found.     

The effect of hypolipidemic drugs on plant lipid metabolism

The effect of hypolipidemic drugs, WY14643 and DH990, on plant lipid metabolism has been studied. The total incorporation of [14C]acetate into lipids was inhibited by addition of both drugs to aged potato (Solanum tuberosum) tuber discs, spinach (Spinacia oleracea) leaves, and spinach chloroplasts, while the incorporation in Chlorella vulgaris cells was affected only by DH990. Moreover, DH990 inhibited the incorporation of 14C-labeled fatty acids into phosphatidylcholine and phosphatidylethanolamine of potato discs, and decreased the incorporation into phosphatidylglycerol of Chlorella cells. DH990 inhibited the formation of polyunsaturated fatty acids in potato discs, Chlorella cells, and spinach leaves, whereas WY14643 had no effect on the formation of these fatty acids. Stearoyl-ACP desaturase from safflower (Carthamus tinctorius) seeds was very sensitive to both drugs, especially DH990, which completely blocked the activity at 2 mM levels. When safflower lysophospholipid acyltransferases were solubilized by detergent treatment, only DH990 inhibited the incorporation of [14C]oleoyl-CoA into lysophosphatidylcholine or lysophosphatidylethanolamine. Both drugs inhibited fatty acid synthesis from [14C]malonyl-CoA in the microsomal fraction from safflower seeds, but only DH990 inhibited FAS activity in the soluble fraction; both drugs inhibited severely the formation of stearic acid. Both acetyl-CoA carboxylase and acetyl-CoA synthetase were sensitive to both drugs.     

Towards model-driven characterization and manipulation of plant lipid metabolism

Plant lipids have versatile applications and provide essential fatty acids in human diet. Therefore, there has been a growing interest to better characterize the genetic basis, regulatory networks, and metabolic pathways that shape lipid quantity and composition. Addressing these issues is challenging due to context-specificity of lipid metabolism integrating environmental, developmental, and tissue-specific cues. Here we systematically review the known metabolic pathways and regulatory interactions that modulate the levels of storage lipids in oilseeds. We argue that the current understanding of lipid metabolism provides the basis for its study in the context of genome-wide plant metabolic networks with the help of approaches from constraint-based modeling and metabolic flux analysis. The focus is on providing a comprehensive summary of the state-of-the-art of modeling plant lipid metabolic pathways, which we then contrast with the existing modeling efforts in yeast and microalgae. We then point out the gaps in knowledge of lipid metabolism, and enumerate the recent advances of using genome-wide association and quantitative trait loci mapping studies to unravel the genetic regulations of lipid metabolism. Finally, we offer a perspective on how advances in the constraint-based modeling framework can propel further characterization of plant lipid metabolism and its rational manipulation.