Glyoxylate Cycle Enzymes Research Articles

Sputum Mycobacterium tuberculosis mRNA as a Marker of Bacteriologic Clearance in Response to Antituberculosis Therapy

mRNA is a marker of cell viability. Quantifying Mycobacterium tuberculosis mRNA in sputum is a promising tool for monitoring response to antituberculosis therapy and evaluating the efficacy of individual drugs. mRNA levels were measured in sputum specimens from patients with tuberculosis (TB) receiving monotherapy in an early bactericidal activity study of fluoroquinolones and in those receiving a standard rifampin-based regimen in an interleukin-2 (IL-2) trial. In the early bactericidal activity study, sputum for quantitative culture and mRNA analysis was collected for 2 days before and daily during 7 days of study drug administration. In the IL-2 trial, sputum was collected for quantitative culture, Bactec 460 liquid culture, and mRNA analysis throughout the intensive treatment phase. RNA was isolated from digested sputum and tested in quantitative reverse transcription-PCR assays for several gene targets. mRNA for the glyoxylate cycle enzyme isocitrate lyase declined at similar rates in patients receiving isoniazid, gatifloxicin, levofloxacin, and moxifloxacin monotherapy. Isocitrate lyase mRNA correlated highly with CFU in sputum prior to therapy and during 7 days of monotherapy in all treatment arms. Isocitrate lyase mRNA was detectable in sputum of culture-positive TB patients receiving a rifampin-based regimen for 1 month. At 2 months, sputum for isocitrate mRNA correlated more closely with growth in liquid culture than did growth on solid culture medium. Data suggest that isocitrate lyase mRNA is a reliable marker of M. tuberculosis viability.

An involvement of yeast peroxisomal channels in transmembrane transfer of glyoxylate cycle intermediates

The separate localization of glyoxylate cycle enzymes in the peroxisomes and the cytosol of the yeast Saccharomyces cerevisiae indicates that the peroxisomal membrane must permit the flow of metabolites between the two compartments. The transfer of these metabolites may require peroxisomal membrane channel(s). We used an electrophysiological approach (reconstitution assay in lipid bilayers) to assess the ability of peroxisomal membrane channels to conduct different solutes including metabolites of the glyoxylate cycle. At least two distinct channel-forming activities were detected in peroxisomal preparations. One of these activities was highly inducible by dithiothreitol and showed large-amplitude current increments when 1 M KCl was used as a bath solution. Single-channel analysis revealed that the inducible channel is anion-selective ( P C l - / P K + = 2.6 ; P citrate / P K + = 1.6 ) and displays flickering at holding potentials over ±30 mV directed upward or downward relative to the main open state of the channel. The channel inducible by DTT facilitates the transfer of solutes with a molecular mass up to 400 Da, sufficient to allow the transmembrane trafficking of glyoxylate cycle intermediates between the peroxisomal lumen and the cytoplasm.

Oil Bodies and Oleosins in Physcomitrella Possess Characteristics Representative of Early Trends in Evolution

Searches of sequenced genomes of diverse organisms revealed that the moss Physcomitrella patens is the most primitive organism possessing oleosin genes. Microscopy examination of Physcomitrella revealed that oil bodies (OBs) were abundant in the photosynthetic vegetative gametophyte and the reproductive spore. Chromatography illustrated the neutral lipids in OBs isolated from the gametophyte to be largely steryl esters and triacylglycerols, and SDS-PAGE showed the major proteins to be oleosins. Reverse transcription-PCR revealed the expression of all three oleosin genes to be tissue specific. This tissue specificity was greatly altered via alternative splicing, a control mechanism of oleosin gene expression unknown in higher plants. During the production of sex organs at the tips of gametophyte branches, the number of OBs in the top gametophyte tissue decreased concomitant with increases in the number of peroxisomes and level of transcripts encoding the glyoxylate cycle enzymes; thus, the OBs are food reserves for gluconeogenesis. In spores during germination, peroxisomes adjacent to OBs, along with transcripts encoding the glyoxylate cycle enzymes, appeared; thus, the spore OBs are food reserves for gluconeogenesis and equivalent to seed OBs. The one-cell-layer gametophyte could be observed easily with confocal microscopy for the subcellular OBs and other structures. Transient expression of various gene constructs transformed into gametophyte cells revealed that all OBs were linked to the endoplasmic reticulum (ER), that oleosins were synthesized in extended regions of the ER, and that two different oleosins were colocated in all OBs.

Peroxisome-associated matrix protein degradation in Arabidopsis

Peroxisomes are ubiquitous eukaryotic organelles housing diverse enzymatic reactions, including several that produce toxic reactive oxygen species. Although understanding of the mechanisms whereby enzymes enter peroxisomes with the help of peroxin (PEX) proteins is increasing, mechanisms by which damaged or obsolete peroxisomal proteins are degraded are not understood. We have exploited unique aspects of plant development to characterize peroxisome-associated protein degradation (PexAD) in Arabidopsis. Oilseed seedlings undergo a developmentally regulated remodeling of peroxisomal matrix protein composition in which the glyoxylate cycle enzymes isocitrate lyase (ICL) and malate synthase (MLS) are replaced by photorespiration enzymes. We found that mutations expected to increase or decrease peroxisomal H(2)O(2) levels accelerated or delayed ICL and MLS disappearance, respectively, suggesting that oxidative damage promotes peroxisomal protein degradation. ICL, MLS, and the beta-oxidation enzyme thiolase were stabilized in the pex4-1 pex22-1 double mutant, which is defective in a peroxisome-associated ubiquitin-conjugating enzyme and its membrane tether. Moreover, the stabilized ICL, thiolase, and an ICL-GFP reporter remained peroxisome associated in pex4-1 pex22-1. ICL also was stabilized and peroxisome associated in pex6-1, a mutant defective in a peroxisome-tethered ATPase. ICL and thiolase were mislocalized to the cytosol but only ICL was stabilized in pex5-10, a mutant defective in a matrix protein import receptor, suggesting that peroxisome entry is necessary for degradation of certain matrix proteins. Together, our data reveal new roles for PEX4, PEX5, PEX6, and PEX22 in PexAD of damaged or obsolete matrix proteins in addition to their canonical roles in peroxisome biogenesis.

Fat-to-glucose interconversion by hydrodynamic transfer of two glyoxylate cycle enzyme genes

The glyoxylate cycle, which is well characterized in higher plants and some microorganisms but not in vertebrates, is able to bypass the citric acid cycle to achieve fat-to-carbohydrate interconversion. In this context, the hydrodynamic transfer of two glyoxylate cycle enzymes, such as isocytrate lyase (ICL) and malate synthase (MS), could accomplish the shift of using fat for the synthesis of glucose. Therefore, 20 mice weighing 23.37 ± 0.96 g were hydrodinamically gene transferred by administering into the tail vein a bolus with ICL and MS. After 36 hours, body weight, plasma glucose, respiratory quotient and energy expenditure were measured. The respiratory quotient was increased by gene transfer, which suggests that a higher carbohydrate/lipid ratio is oxidized in such animals. This application could help, if adequate protocols are designed, to induce fat utilization for glucose synthesis, which might be eventually useful to reduce body fat depots in situations of obesity and diabetes.

Drought, salinity and heat differently affect seed germination of Pinus pinea

Drought, salinity and forest fires are adverse abiotic factors responsible for the failure of plant regeneration in the Mediterranean regions. The objective of this study was to evaluate Pinus pinea seed germination in response to heat, osmotic and salinity stress. Separate experiments were conducted, using polyethylene glycol (PEG) as an osmotic agent to reproduce drought stress, and sodium chloride (NaCl) and diluted seawater as salinizing agents to simulate salinity stress. Different temperatures were used to reproduce the effects of different fire regimes. Seed germination and the activities of the main enzymes involved in Pinus seed reserve utilization (glyoxylate cycle enzymes) decreased with increasing PEG, NaCl and seawater concentrations. Seawater appears to be the least toxic salinizing agent on Pinus pinea seed germination. The present study indicates that the germination of Pinus pinea seeds is influenced by the concentrations and even more by the nature and interactions of the ions present in the solutions. As regard heat treatments, the maximum germination percentage (80%) was obtained at 80°C and short exposure time (3 min). Increasing exposure time and temperature led to the germination percentage progressively decreasing.

Ethanol physiology in the warehouse-staining fungus, Baudoinia compniacensis

The fungus Baudoinia compniacensis colonizes the exterior surfaces of a range of materials, such as buildings, outdoor furnishings, fences, signs, and vegetation, in regions subject to periodic exposure to low levels of ethanol vapour, such as those in the vicinity of distillery aging warehouses and commercial bakeries. Here we investigated the basis of ethanol metabolism in Baudoinia and investigate the role of ethanol in cell germination and growth. Germination of mycelia of Baudoinia was enhanced by up to roughly 1 d exposure to low ethanol concentrations, optimally 10 ppm when delivered in vapour form and 5 m m in liquid form. However, growth was strongly inhibited following exposure to higher ethanol concentrations for shorter durations (e.g., 1.7 m for 6 h). We found that ethanol was catabolized into central metabolism via alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ACDH). Isocitrate dehydrogenases (IDHs) were active in cells grown on glucose, but these enzymes were not expressed when ethanol was provided as a sole or companion carbon source. The glyoxylate cycle enzymes isocitrate lyase (ICL) and malate synthase (MS) activities observed in cells grown on acetate were comparable to those reported for other microorganisms. By replenishing tricarboxylic acid (TCA) cycle intermediates, it is likely that the functionality of the glyoxylate cycle is important in the establishment of luxuriant growth of Baudoinia compniacensis on ethanol-exposed, nutrient-deprived, exposed surfaces. In other fungi, such as Saccharomyces cerevisiae, ADH II catalyses the conversion of ethanol to acetaldehyde, which then can be metabolized via the TCA cycle. ADH II is known to be strongly repressed in the presence of glucose.

Visceral adipose tissue specific persistence of Mycobacterium tuberculosis may be reason for the metabolic syndrome

Mycobacterium tuberculosis (Mtb) is highly successful intracellular pathogen. Infection is maintained in spite of acquired immunity and resists eradication by antimicrobials. Following bacillaemia, small numbers of bacteria are disseminated to the extrapulmonary organs most likely including visceral adipose tissue by a mechanism that may involve the migration of M. tuberculosis within dendritic cells. In this lipid rich environment, Mtb can metabolize the fatty acids in a glyoxylate cycle dependent manner, and a state of chronic persistence may ensue. The persistent bacilli primarily use fatty acids as their carbon source. Expression of isocitrate lyase (ICL), gating enzyme of glyoxylate cycle, is upregulated during infection. ICL is important for survival during the persistent phase of infection. Expression of adipokines, particularly monocyte chemoattractant protein-1 (MCP-1), which is a potent proinflammatory cytokine, may be increased. MCP-1 contributes both to the recruitment of macrophages to adipose tissue and to the development of insulin resistance in humans. In addition, prolonged low level immune stimulation induces local adipolipogenesis, increasing visceral fat. Increased delivery of free fatty acid to the liver may stimulate the glyoxylate cycle-induced gluconeogenesis, raising hepatic glucose output. Hence, inhibition of the triggering enzyme ICL, which initiates all the pathologies related to persistent Mtb infection, may block the growth of the bacteria and may resolve the systemic metabolic complications.

Functional Characterization of a Vitamin B 12 -Dependent Methylmalonyl Pathway in Mycobacterium tuberculosis : Implications for Propionate Metabolism during Growth on Fatty Acids

Mycobacterium tuberculosis is predicted to subsist on alternative carbon sources during persistence within the human host. Catabolism of odd- and branched-chain fatty acids, branched-chain amino acids, and cholesterol generates propionyl-coenzyme A (CoA) as a terminal, three-carbon (C(3)) product. Propionate constitutes a key precursor in lipid biosynthesis but is toxic if accumulated, potentially implicating its metabolism in M. tuberculosis pathogenesis. In addition to the well-characterized methylcitrate cycle, the M. tuberculosis genome contains a complete methylmalonyl pathway, including a mutAB-encoded methylmalonyl-CoA mutase (MCM) that requires a vitamin B(12)-derived cofactor for activity. Here, we demonstrate the ability of M. tuberculosis to utilize propionate as the sole carbon source in the absence of a functional methylcitrate cycle, provided that vitamin B(12) is supplied exogenously. We show that this ability is dependent on mutAB and, furthermore, that an active methylmalonyl pathway allows the bypass of the glyoxylate cycle during growth on propionate in vitro. Importantly, although the glyoxylate and methylcitrate cycles supported robust growth of M. tuberculosis on the C(17) fatty acid heptadecanoate, growth on valerate (C(5)) was significantly enhanced through vitamin B(12) supplementation. Moreover, both wild-type and methylcitrate cycle mutant strains grew on B(12)-supplemented valerate in the presence of 3-nitropropionate, an inhibitor of the glyoxylate cycle enzyme isocitrate lyase, indicating an anaplerotic role for the methylmalonyl pathway. The demonstrated functionality of MCM reinforces the potential relevance of vitamin B(12) to mycobacterial pathogenesis and suggests that vitamin B(12) availability in vivo might resolve the paradoxical dispensability of the methylcitrate cycle for the growth and persistence of M. tuberculosis in mice.

Changes in germination and glyoxylate and respiratory enzymes of Pinus pinea seeds under various abiotic stresses

This study examined Pinus pinea seeds for their tolerance to osmotic potentials of −0.30MPa (10% polyethylene glycol [PEG]), −0.58MPa (18% PEG), −0.80MPa (21% PEG), −1.05MPa (24% PEG), pH values of 4, 5, 6, 7, 8, 9, 10, and different calcareous solutions (5, 10, 20 and 40% CaCO3). The main enzymes of glyoxylate cycle and respiratory pathway were tested. Pinus pinea seeds under no stressful condition (Control) and 5% CaCO3 reached 100% of germination. Higher concentrations of CaCO3 (20, 40%) and lower pH (4–5) adversely affected seed germination percentage, glyoxylic and respiratory enzyme activities. PEG caused the most detrimental effects on Pinus seeds; increasing the osmotic potential the germination was completely inhibited. These results suggest that Pinus pinea is able to germinate in calcareous and alkaline soils rather than in soils with lower water availability and acidic conditions.

Role of the methylcitrate cycle in propionate metabolism and detoxification in Mycobacterium smegmatis

Catabolism of odd-chain-length fatty acids yields acetyl-CoA and propionyl-CoA. A common pathway of propionyl-CoA metabolism in micro-organisms is the methylcitrate cycle, which includes the dedicated enzymes methylcitrate synthase (MCS), methylcitrate dehydratase (MCD) and methylisocitrate lyase (MCL). The methylcitrate cycle is essential for propionate metabolism in Mycobacterium tuberculosis. Unusually, M. tuberculosis lacks an MCL orthologue and this activity is provided instead by two isoforms of the glyoxylate cycle enzyme isocitrate lyase (ICL1 and ICL2). These bifunctional (ICL/MCL) enzymes are jointly required for propionate metabolism and for growth and survival in mice. In contrast, the non-pathogenic species Mycobacterium smegmatis encodes a canonical MCL enzyme in addition to ICL1 and ICL2. The M. smegmatis gene encoding MCL (prpB) is clustered with genes encoding MCS (prpC) and MCD (prpD). Here we show that deletion of the M. smegmatis prpDBC locus reduced but did not eliminate MCL activity in cell-free extracts. The residual MCL activity was abolished by deletion of icl1 and icl2 in the DeltaprpDBC background, suggesting that these genes encode bifunctional ICL/MCL enzymes. A DeltaprpB Deltaicl1 Deltaicl2 mutant was unable to grow on propionate or mixtures of propionate and glucose. We hypothesize that incomplete propionyl-CoA metabolism might cause toxic metabolites to accumulate. Consistent with this idea, deletion of prpC and prpD in the DeltaprpB Deltaicl1 Deltaicl2 background paradoxically restored growth on propionate-containing media. These observations suggest that the marked attenuation of ICL1/ICL2-deficient M. tuberculosis in mice could be due to the accumulation of toxic propionyl-CoA metabolites, rather than inability to utilize fatty acids per se.

Ability to grow on lipids accounts for the fully virulent phenotype in neutropenic mice of Aspergillus fumigatus null mutants in the key glyoxylate cycle enzymes

Incidence and mortality rates of invasive aspergillosis clearly indicate the need of novel antifungals to treat patients suffering from this disease. Fungal proteins playing a crucial role in pathogenesis and with no orthologue in human cells are considered as primary therapeutic targets for the development of new antifungals with a high therapeutic index, one of the major drawbacks of the standard antifungal therapy, so far. In this work, we have analyzed the role in pathogenesis of the key enzymes of the Aspergillus fumigatus glyxoxylate cycle, isocitrate lyase and malate synthase, two possible candidates to primary therapeutic targets in this fungus. Deletion strains lacking isocitrate lyase (Δ acuD strains) or malate synthase (Δ acuE mutants) were constructed in this work. The Neurospora crassa pyr-4 gene was used as the replacing marker in gene deletion experiments. The pathogenicities of Δ acuD and Δ acuE mutants were tested in neutropenic mice and compared with those of two reference wild-type isolates A. fumigatus 237 and A. fumigatus 293. Interestingly, virulence and cytological studies clearly indicated the dispensability of the A. fumigatus glyoxylate cycle for pathogenicity. In addition, these results suggested the suitability of the pyr-4 gene as a valuable replacing marker for virulence studies in this fungus, a fact that was further confirmed by gene expression analyses. Finally, growth tests were performed to investigate the germination and growth of the Δ acuD and ΔacuE strains in nutrient deprivation environments, resembling the conditions that A. fumigatus conidia face after phagocytosis. Results obtained in this work strongly suggest that the ability to grow on lipids (triglycerides) of A. fumigatus isocitrate lyase and malate synthase deletion strains accounts for their fully virulent phenotype.

Developmental regulation of the glyoxylate cycle in the human pathogen Penicillium marneffei

Penicillium marneffei is a thermally dimorphic opportunistic human pathogen with a saprophytic filamentous hyphal form at 25 degrees C and a pathogenic unicellular yeast form at 37 degrees C. During infection. P. marneffei yeast cells exist intracellularly in macrophages. To cope with nutrient deprivation during the infection process, a number of pathogens employ the glyoxylate cycle to utilize fatty acids as carbon sources. The genes which constitute this pathway have been implicated in pathogenesis. To investigate acetate and fatty acid utilization, the acuD gene encoding a key glyoxylate cycle enzyme (isocitrate lyase) was cloned. The acuD gene is regulated by both carbon source and temperature in P. marneffei, being strongly induced at 37 degrees C even in the presence of a repressing carbon source such as glucose. When introduced into the non-pathogenic monomorphic fungus Aspergillus nidulans, the P. marneffei acuD promoter only responds to carbon source. Similarly, when the A. nidulans acuD promoter is introduced into P. marneffei it only responds to carbon source suggesting that P. marneffei possesses both cis elements and trans-acting factors to control acuD by temperature. The Zn(II)2Cys6 DNA binding motif transcriptional activator FacB was cloned and is responsible for carbon source-, but not temperature-, dependent induction of acuD. The expression of acuD at 37 degrees C is induced by AbaA, a key regulator of morphogenesis in P. marneffei, but deletion of abaA does not completely eliminate temperature-dependent induction, suggesting that acuD and the glyoxylate cycle are regulated by a complex network of factors in P. marneffei which may contribute to its pathogenicity.

Revisiting the glyoxylate cycle: alternate pathways for microbial acetate assimilation

The glyoxylate cycle, identified by Kornberg et al. in 1957, provides a simple and efficient strategy for converting acetyl-CoA into anapleurotic and gluconeogenic compounds. Studies of a number of bacteria capable of growth with C2 compounds as the sole carbon source have revealed that they lack the key glyoxylate cycle enzyme isocitrate lyase, suggesting that alternative pathway(s) for acetate assimilation exist in these bacteria. Recent studies of acetate assimilation in methylotrophs and purple phototrophs have revealed remarkable and complex new pathways for assimilation of acetate in the absence of isocitrate lyase. The details of these new pathways are the subject of this MicroCommentary.

Antioxidative enzymes from chloroplasts, mitochondria, and peroxisomes during leaf senescence of nodulated pea plants.

In this work the influence of the nodulation of pea (Pisum sativum L.) plants on the oxidative metabolism of different leaf organelles from young and senescent plants was studied. Chloroplasts, mitochondria, and peroxisomes were purified from leaves of nitrate-fed and Rhizobium leguminosarum-nodulated pea plants at two developmental stages (young and senescent plants). In these cell organelles, the activity of the ascorbate-glutathione cycle enzymes ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), and glutathione reductase (GR), and the ascorbate and glutathione contents were determined. In addition, the total superoxide dismutase (SOD) activity, the pattern of mitochondrial and peroxisomal NADPH-generating dehydrogenases, some of the peroxisomal photorespiratory enzymes, the glyoxylate cycle and oxidative metabolism enzymes were also analysed in these organelles. Results obtained on the metabolism of cell organelles indicate that nodulation with Rhizobium accelerates senescence in pea leaves. A considerable decrease of the ascorbate content of chloroplasts, mitochondria, and peroxisomes was found, and in these conditions a metabolic conversion of leaf peroxisomes into glyoxysomes, characteristic of leaf senescence, took place.

Identification of RamA, a Novel LuxR-Type Transcriptional Regulator of Genes Involved in Acetate Metabolism of Corynebacterium glutamicum

In Corynebacterium glutamicum, the acetate-activating enzymes phosphotransacetylase and acetate kinase and the glyoxylate cycle enzymes isocitrate lyase and malate synthase are coordinately up-regulated in the presence of acetate in the growth medium. This regulation is due to transcriptional control of the respective pta-ack operon and the aceA and aceB genes, brought about at least partly by the action of the negative transcriptional regulator RamB. Using cell extracts of C. glutamicum and employing DNA affinity chromatography, mass spectrometry, and peptide mass fingerprinting, we identified a LuxR-type transcriptional regulator, designated RamA, which binds to the pta-ack and aceA/aceB promoter regions. Inactivation of the ramA gene in the genome of C. glutamicum resulted in mutant RG2. This mutant was unable to grow on acetate as the sole carbon and energy source and, in comparison to the wild type of C. glutamicum, showed very low specific activities of phosphotransacetylase, acetate kinase, isocitrate lyase, and malate synthase, irrespective of the presence of acetate in the medium. Comparative transcriptional cat fusion experiments revealed that this deregulation takes place at the level of transcription. By electrophoretic mobility shift analysis, purified His-tagged RamA protein was shown to bind specifically to the pta-ack and the aceA/aceB promoter regions, and deletion and mutation studies revealed in both regions two binding motifs each consisting of tandem A/C/TG4-6T/C or AC4-5A/G/T stretches separated by four or five arbitrary nucleotides. Our data indicate that RamA represents a novel LuxR-type transcriptional activator of genes involved in acetate metabolism of C. glutamicum.

The glyoxylate cycle enzyme activities in the pathogenic isolates of Candida albicans obtained from HIV/AIDS, diabetic and burn patients

Recently it has been found that Candida albicans harbours enzymes involved in the glyoxylate cycle (GC), which have a role in its virulence, especially the two key enzymes, isocitrate lyase (ICL) and malate synthase (MS). There are however, few studies on the GC enzyme activities isolated in the clinical isolates. Samples were collected from three groups of patients namely, HIV/AIDS, diabetic and burn patients suffering from candidiasis at different body locations. Isolation, identification and the antifungal susceptibility test of all the isolates of C. albicans were followed by the standard techniques. Measurements of all the GC enzyme activities were also carried out by the standard methods. Levels of the principal GC enzymes showed significant changes when calculated and compared taking control strains of C. albicans. The activity of the two key enzymes of the GC, ICL and MS were significantly higher in the isolates from diabetic patients. No significant relationship between the drug susceptibility and the level of enzymes of the GC was observed. As GC activity is absent in mammalian cells, a specific inhibitor for the GC could be developed and these enzymes therefore can be used as a new antifungal target.

Evolution of glyoxylate cycle enzymes in Metazoa: evidence of multiple horizontal transfer events and pseudogene formation

BackgroundThe glyoxylate cycle is thought to be present in bacteria, protists, plants, fungi, and nematodes, but not in other Metazoa. However, activity of the glyoxylate cycle enzymes, malate synthase (MS) and isocitrate lyase (ICL), in animal tissues has been reported. In order to clarify the status of the MS and ICL genes in animals and get an insight into their evolution, we undertook a comparative-genomic study.ResultsUsing sequence similarity searches, we identified MS genes in arthropods, echinoderms, and vertebrates, including platypus and opossum, but not in the numerous sequenced genomes of placental mammals. The regions of the placental mammals' genomes expected to code for malate synthase, as determined by comparison of the gene orders in vertebrate genomes, show clear similarity to the opossum MS sequence but contain stop codons, indicating that the MS gene became a pseudogene in placental mammals. By contrast, the ICL gene is undetectable in animals other than the nematodes that possess a bifunctional, fused ICL-MS gene. Examination of phylogenetic trees of MS and ICL suggests multiple horizontal gene transfer events that probably went in both directions between several bacterial and eukaryotic lineages. The strongest evidence was obtained for the acquisition of the bifunctional ICL-MS gene from an as yet unknown bacterial source with the corresponding operonic organization by the common ancestor of the nematodes.ConclusionThe distribution of the MS and ICL genes in animals suggests that either they encode alternative enzymes of the glyoxylate cycle that are not orthologous to the known MS and ICL or the animal MS acquired a new function that remains to be characterized. Regardless of the ultimate solution to this conundrum, the genes for the glyoxylate cycle enzymes present a remarkable variety of evolutionary events including unusual horizontal gene transfer from bacteria to animals.ReviewersArcady Mushegian (Stowers Institute for Medical Research), Andrey Osterman (Burnham Institute for Medical Research), Chris Ponting (Oxford University).

Comparative Analysis of Glyoxylate Cycle Key Enzyme Isocitrate Lyase from Organisms of Different Systematic Groups

Isocitrate lyase and malate synthase are the key enzymes of glyoxylate cycle that represents the most important stage on the pathway of conversion of fatty acids to carbohydrates. Until now, induction of enzymes of this metabolic pathway was considered to take place only in cells of prokaryotes, plants, fungi, and nematodes in response to arising demands in carbohydrates. However, the isocitrate lyase activities have been detected in the liver of food-starved rats in our previous work and in pupas of the butterfly Papilio machaon in the present study. The enzymes from both studied objects were purified to homogeneous condition. The main kinetic and physicochemical properties of isocitrate lyase were studied. Organisms of evolutionary distant taxa—mammal, insect, and plant—were chosen for comparative analysis of properties of the studied enzyme. A substantial similarity of kinetic and physicochemical properties of plant and animal isocitrate leases has been found. At the same time, the absence of specific for prokaryotic, plant, and nematode isocitrate lyase nucleotide sequences has been established in mRNA from liver of starved rats and swallowtail pupa. These results are completely confirmed by analysis of the complete genome sequences of the mouse, Drosophila, and human. The obtained data raise the question about the pathway of evolution of genes of the glyoxylate cycle key enzymes.

Hormonal regulation of gluconeogenesis in cereal aleurone is strongly cultivar‐dependent and gibberellin action involves SLENDER1 but not GAMYB

Storage oil is a major constituent in the cereal aleurone layer. The aim of this study was to investigate how gibberellin (GA) and abscisic acid (ABA) regulate conversion of oil to sugar in barley aleurone. The activity of the glyoxylate cycle enzyme isocitrate lyase (ICL) was surveyed in eight barley cultivars. Surprisingly, some cultivars do not require GA for the induction of ICL (e.g. Himalaya), whereas some do (e.g. Golden Promise). Furthermore, in Golden Promise, GA also stimulates triacylglycerol breakdown and enhances the net flux of carbon from acetate to sugar. In contrast, ABA strongly represses ICL activity and the flux of carbon from oil to sugar in both Golden Promise and Himalaya. Biolistics using a promoter reporter showed that GA and ABA regulate ICL at the level of transcription. Studies using barley and rice mutants and pharmacological agents show that GA-dependent induction of ICL activity is mediated by SLENDER1 and requires cGMP, but does not involve the transcription factor GAMYB. Gibberellin and ABA therefore act antagonistically to regulate gluconeogenesis in the aleurone layer as well as controlling the production and secretion of hydrolases into the starchy endosperm. We suggest that the variation between different barley cultivars might be a result of selective breeding to alter seed dormancy.