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Steven M. Smith - One of the best experts on this subject based on the ideXlab platform.
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Arabidopsis peroxisomal malate dehydrogenase functions in β-oxidation but not in the Glyoxylate Cycle
The Plant Journal, 2007Co-Authors: Itsara Pracharoenwattana, Johanna E. Cornah, Steven M. SmithAbstract:The aim was to determine the function of peroxisomal NAD(+)-malate dehydrogenase (PMDH) in fatty acid beta-oxidation and the Glyoxylate Cycle in Arabidopsis. Seeds in which both PMDH genes are disrupted by T-DNA insertions germinate, but seedling establishment is dependent on exogenous sugar. Mutant seedlings mobilize their triacylglycerol very slowly and growth is insensitive to 2,4-dichlorophenoxybutyric acid. Thus mutant seedlings are severely impaired in beta-oxidation, even though microarray analysis shows that beta-oxidation genes are expressed normally. The mutant phenotype was complemented by expression of a cDNA encoding PMDH with either its native peroxisome targeting signal-2 (PTS2) targeting sequence or a heterologous PTS1 sequence. In contrast to the block in beta-oxidation in mutant seedlings, [(14)C]acetate is readily metabolized into sugars and organic acids, thereby demonstrating normal activity of the Glyoxylate Cycle. We conclude that PMDH serves to reoxidize NADH produced from fatty acid beta-oxidation and does not participate directly in the Glyoxylate Cycle.
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A central role for the peroxisomal membrane in Glyoxylate Cycle function
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2006Co-Authors: Markus Kunze, Itsara Pracharoenwattana, Steven M. Smith, Andreas HartigAbstract:The Glyoxylate Cycle provides the means to convert C2-units to C4-precursors for biosynthesis, allowing growth on fatty acids and C2-compounds. The conventional view that the Glyoxylate Cycle is contained within peroxisomes in fungi and plants is no longer valid. Glyoxylate Cycle enzymes are located both inside and outside the peroxisome. Thus, the operation of the Glyoxylate Cycle requires transport of several intermediates across the peroxisomal membrane. Glyoxylate Cycle progression is also dependent upon mitochondrial metabolism. An understanding of the operation and regulation of the Glyoxylate Cycle, and its integration with cellular metabolism, will require further investigation of the participating metabolite transporters in the peroxisomal membrane.
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lipid utilization gluconeogenesis and seedling growth in arabidopsis mutants lacking the Glyoxylate Cycle enzyme malate synthase
Journal of Biological Chemistry, 2004Co-Authors: Johanna E. Cornah, Véronique Germain, Jane L Ward, Michael H Beale, Steven M. SmithAbstract:Abstract The aim of this research was to test the role of the Glyoxylate Cycle enzyme malate synthase (MLS) in lipid utilization, gluconeogenesis, and seedling growth in Arabidopsis. We hypothesized that in the absence of MLS, succinate produced by isocitrate lyase (ICL) could still feed into the tricarboxylic acid Cycle, whereas Glyoxylate could be converted to sugars using enzymes of the photorespiratory pathway. To test this hypothesis we isolated knock-out mls mutants and studied their growth and metabolism in comparison to wild type and icl mutant seedlings. In contrast to icl seedlings, which grow slowly and are unable to convert lipid into sugars (Eastmond, P. J., Germain, V., Lange, P. R., Bryce, J. H., Smith, S. M. & Graham, I. A. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 5669–5674), mls seedlings grow faster, use their lipid more rapidly, and are better able to establish as plantlets. Transcriptome and metabolome analyses show that icl seedlings exhibit many features characteristic of carbohydrate starvation, whereas mls seedlings differ relatively little from wild type. In the light mls seedlings generate more sugars than icl seedlings, and when fed with [14C]acetate, 14C-labeling of sugars is three times greater than in icl seedlings and more than half that in wild type seedlings. The mls seedlings also accumulate more glycine and serine than icl or wild type seedlings, consistent with a diversion of Glyoxylate into these intermediates of the photorespiratory pathway. We conclude that, in contrast to bacteria and fungi in which MLS is essential for gluconeogenesis from acetate or fatty acids, MLS is partially dispensable for lipid utilization and gluconeogenesis in Arabidopsis seedlings.
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Synthesis and Function of Glyoxylate Cycle Enzymes
Plant Peroxisomes, 2002Co-Authors: Johanna E. Cornah, Steven M. SmithAbstract:The Glyoxylate Cycle in plants has been the subject of much research for several decades, and many hundreds of primary research papers have been published. There have been numerous reviews covering different aspects of the synthesis and function of Glyoxylate Cycle enzymes (Beevers, 1979; Trelease, 1984; Escher and Widmer 1997; Eastmond and Graham, 2001). Here we choose firstly to summarize the main features of the Glyoxylate Cycle as understood from the many studies of its role in lipid metabolism in seedlings of oilseed species. Then we focus on aspects that have been discovered since the previous treatise on plant peroxisomes (Huang et al, 1983) and highlight those aspects which are still to be understood. Among these topics are the discovery that aconitase is a cytosolic enzyme while other key enzymes of the Cycle are peroxisomal, and that the metabolic pathway of the Cycle is still unknown, principally because we do not know how reducing equivalents are exported from the peroxisome. We review the role of the Glyoxylate Cycle in seed and pollen development, senescence and starvation, highlighting different potential functions. The importance of genetic aproaches for future research is illustrated by studies of knock-out mutants of both isocitrate lyase and malate synthase in Arabidopsis thaliana.
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Postgerminative growth and lipid catabolism in oilseeds lacking the Glyoxylate Cycle
Proceedings of the National Academy of Sciences, 2000Co-Authors: Peter J. Eastmond, Steven M. Smith, Véronique Germain, Peter R. Lange, James H. Bryce, Ian A. GrahamAbstract:The Glyoxylate Cycle is regarded as essential for postgerminative growth and seedling establishment in oilseed plants. We have identified two allelic Arabidopsis mutants, icl-1 and icl-2, which lack the Glyoxylate Cycle because of the absence of the key enzyme isocitrate lyase. These mutants demonstrate that the Glyoxylate Cycle is not essential for germination. Furthermore, photosynthesis can compensate for the absence of the Glyoxylate Cycle during postgerminative growth, and only when light intensity or day length is decreased does seedling establishment become compromised. The provision of exogenous sugars can overcome this growth deficiency. The icl mutants also demonstrate that the Glyoxylate Cycle is important for seedling survival and recovery after prolonged dark conditions that approximate growth in nature. Surprisingly, despite their inability to catalyze the net conversion of acetate to carbohydrate, mutant seedlings are able to break down storage lipids. Results suggest that lipids can be used as a source of carbon for respiration in germinating oilseeds and that products of fatty acid catabolism can pass from the peroxisome to the mitochondrion independently of the Glyoxylate Cycle. However, an additional anaplerotic source of carbon is required for lipid breakdown and seedling establishment. This source can be provided by the Glyoxylate Cycle or, in its absence, by exogenous sucrose or photosynthesis.
Kazutaka Miyatake - One of the best experts on this subject based on the ideXlab platform.
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characterization of a bifunctional Glyoxylate Cycle enzyme malate synthase isocitrate lyase of euglena gracilis
Journal of Eukaryotic Microbiology, 2011Co-Authors: Masami Nakazawa, Hiroshi Inui, Yoshihisa Nakano, Mitsuhiro Ueda, Masaaki Nishimura, Kengo Inoue, Kazutaka MiyatakeAbstract:The Glyoxylate Cycle is a modified form of the tricarboxylic acid Cycle, which enables organisms to synthesize carbohydrates from C2 compounds. In the protozoan Euglena gracilis, the key enzyme activities of the Glyoxylate Cycle, isocitrate lyase (ICL) and malate synthase (MS), are conferred by a single bifunctional protein named Glyoxylate Cycle enzyme (Euglena gracilis Glyoxylate Cycle enzyme [EgGCE]). We analyzed the enzymatic properties of recombinant EgGCE to determine the functions of its different domains. The 62-kDa N-terminal domain of EgGCE was sufficient to provide the MS activity as expected from an analysis of the deduced amino acid sequence. In contrast, expression of the 67-kDa C-terminal domain of EgGCE failed to yield ICL activity even though this domain was structurally similar to ICL family enzymes. Analyses of truncation mutants suggested that the N-terminal residues of EgGCE are critical for both the ICL and MS activities. The ICL activity of EgGCE increased in the presence of micro-molar concentrations of acetyl-coenzyme A (CoA). Acetyl-CoA also increased the activity in a mutant type EgGCE with a mutation at the acetyl-CoA binding site in the MS domain of EgGCE. This suggests that acetyl-CoA regulates the ICL reaction by binding to a site other than the catalytic center of the MS reaction.
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molecular characterization of a bifunctional Glyoxylate Cycle enzyme malate synthase isocitrate lyase in euglena gracilis
Comparative Biochemistry and Physiology B, 2005Co-Authors: Masami Nakazawa, Hiroshi Inui, Yoshihisa Nakano, Tomomi Minami, Koji Teramura, Shohei Kumamoto, Sayaka Hanato, Shigeo Takenaka, Mitsuhiro Ueda, Kazutaka MiyatakeAbstract:Euglena gracilis induced Glyoxylate Cycle enzymes when ethanol was fed as a sole carbon source. We purified, cloned and characterized a bifunctional Glyoxylate Cycle enzyme from E. gracilis (EgGCE). This enzyme consists of an N-terminal malate synthase (MS) domain fused to a C-terminal isocitrate lyase (ICL) domain in a single polypeptide chain. This domain order is inverted compared to the bifunctional Glyoxylate Cycle enzyme in Caenorhabditis elegans, an N-terminal ICL domain fused to a C-terminal MS domain. Purified EgGCE catalyzed the sequential ICL and MS reactions. ICL activity of purified EgGCE increased in the existence of acetyl-CoA at a concentration of micro-molar order. We discussed the physiological roles of the bifunctional Glyoxylate Cycle enzyme in these organisms as well as its molecular evolution.
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Presence of Glyoxylate Cycle enzymes in the mitochondria of Euglena gracilis.
The Journal of Eukaryotic Microbiology, 2003Co-Authors: Kouki Ono, Maki Kondo, Tetsuaki Osafune, Kazutaka Miyatake, Hiroshi Inui, Shozaburo Kitaoka, Yoshihisa NakanoAbstract:Abstract Isocitrate lyase and malate synthase are specific enzymes of the Glyoxylate Cycle, used here as glyoxysomal markers. Both enzymes were found in the mitochondrial fraction after organelle fractionation by isopycnic centrifugation. Electron microscopy of this fraction indicated that mitochondria were the only recognizable organelles. Using an immunogold labeling method with anti- (malate synthase) antiserum, the only organelles stained in cells were the mitochondria. These results show that the Glyoxylate Cycle is present in mitochondria in Euglena.
Donald A Bryant - One of the best experts on this subject based on the ideXlab platform.
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biochemical validation of the Glyoxylate Cycle in the cyanobacterium chlorogloeopsis fritschii strain pcc 9212
Journal of Biological Chemistry, 2015Co-Authors: Shuyi Zhang, Donald A BryantAbstract:Abstract Cyanobacteria are important photoautotrophic bacteria with extensive but variable metabolic capacities. The existence of the Glyoxylate Cycle, a variant of the TCA Cycle, is still poorly documented in cyanobacteria. Previous studies reported the activities of isocitrate lyase and malate synthase, the key enzymes of the Glyoxylate Cycle in some cyanobacteria, but other studies concluded that these enzymes are missing. In this study the genes encoding isocitrate lyase and malate synthase from Chlorogloeopsis fritschii PCC 9212 were identified, and the recombinant enzymes were biochemically characterized. Consistent with the presence of the enzymes of the Glyoxylate Cycle, C. fritschii could assimilate acetate under both light and dark growth conditions. Transcript abundances for isocitrate lyase and malate synthase increased, and C. fritschii grew faster, when the growth medium was supplemented with acetate. Adding acetate to the growth medium also increased the yield of poly-3-hydroxybutyrate. When the genes encoding isocitrate lyase and malate synthase were expressed in Synechococcus sp. PCC 7002, the acetate assimilation capacity of the resulting strain was greater than that of wild type. Database searches showed that the genes for the Glyoxylate Cycle exist in only a few other cyanobacteria, all of which are able to fix nitrogen. This study demonstrates that the Glyoxylate Cycle exists in a few cyanobacteria, and that this pathway plays an important role in the assimilation of acetate for growth in one of those organisms. The Glyoxylate Cycle might play a role in coordinating carbon and nitrogen metabolism under conditions of nitrogen fixation.
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biochemical validation of the Glyoxylate Cycle in the cyanobacterium chlorogloeopsis fritschii strain pcc 9212
Journal of Biological Chemistry, 2015Co-Authors: Shuyi Zhang, Donald A BryantAbstract:Cyanobacteria are important photoautotrophic bacteria with extensive but variable metabolic capacities. The existence of the Glyoxylate Cycle, a variant of the TCA Cycle, is still poorly documented in cyanobacteria. Previous studies reported the activities of isocitrate lyase and malate synthase, the key enzymes of the Glyoxylate Cycle in some cyanobacteria, but other studies concluded that these enzymes are missing. In this study the genes encoding isocitrate lyase and malate synthase from Chlorogloeopsis fritschii PCC 9212 were identified, and the recombinant enzymes were biochemically characterized. Consistent with the presence of the enzymes of the Glyoxylate Cycle, C. fritschii could assimilate acetate under both light and dark growth conditions. Transcript abundances for isocitrate lyase and malate synthase increased, and C. fritschii grew faster, when the growth medium was supplemented with acetate. Adding acetate to the growth medium also increased the yield of poly-3-hydroxybutyrate. When the genes encoding isocitrate lyase and malate synthase were expressed in Synechococcus sp. PCC 7002, the acetate assimilation capacity of the resulting strain was greater than that of wild type. Database searches showed that the genes for the Glyoxylate Cycle exist in only a few other cyanobacteria, all of which are able to fix nitrogen. This study demonstrates that the Glyoxylate Cycle exists in a few cyanobacteria, and that this pathway plays an important role in the assimilation of acetate for growth in one of those organisms. The Glyoxylate Cycle might play a role in coordinating carbon and nitrogen metabolism under conditions of nitrogen fixation. Background: Conflicting claims exist concerning the occurrence of the Glyoxylate Cycle in cyanobacteria. Results: The genes for isocitrate lyase and malate synthase were identified in Chlorogleopsis fritschii PCC 9212 and the purified enzymes were characterized. Conclusion: C. fritschii has a functional Glyoxylate Cycle and can grow in the dark on acetate. Significance: These results clarify the occurrence of the Glyoxylate Cycle in cyanobacteria.
Abir U. Igamberdiev - One of the best experts on this subject based on the ideXlab platform.
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Glyoxylate Cycle and metabolism of organic acids in the scutellum of barley seeds during germination
Plant Science, 2016Co-Authors: Frédéric Marsolais, Mark A. Bernards, Mark W. Sumarah, Natalia V. Bykova, Abir U. IgamberdievAbstract:During the developmental processes from dry seeds to seedling establishment, the Glyoxylate Cycle becomes active in the mobilization of stored oils in the scutellum of barley (Hordeum vulgare L.) seeds, as indicated by the activities of isocitrate lyase and malate synthase. The succinate produced is converted to carbohydrates via phosphoenolpyruvate carboxykinase and to amino acids via aminotransferases, while free organic acids may participate in acidifying the endosperm tissue, releasing stored starch into metabolism. The abundant organic acid in the scutellum was citrate, while malate concentration declined during the first three days of germination, and succinate concentration was low both in scutellum and endosperm. Malate was more abundant in endosperm tissue during the first three days of germination; before citrate became predominant, indicating that malate may be the main acid acidifying the endosperm. The operation of the Glyoxylate Cycle coincided with an increase in the ATP/ADP ratio, a buildup of H2O2 and changes in the redox state of ascorbate and glutathione. It is concluded that operation of the Glyoxylate Cycle in the scutellum of cereals may be important not only for conversion of fatty acids to carbohydrates, but also for the acidification of endosperm and amino acid synthesis.
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Glyoxylate Cycle enzymes are present in liver peroxisomes of alloxan treated rats
FEBS Letters, 1998Co-Authors: Abir U. Igamberdiev, V N Popov, A T Eprintsev, S V VolvenkinAbstract:Abstract Key enzymes of the Glyoxylate Cycle, isocitrate lyase (ICL) and malate synthase (MS), have been detected in the liver of alloxan-treated rats. The activity of ICL in rat liver was 0.040 μmol/min/mg protein and the activity of MS was 0.022 μmol/min/mg protein. These enzymes were associated with the peroxisomal fraction. The activities of citrate synthase, malate synthase and malate dehydrogenase detected in the peroxisomal fraction were also increased by alloxan treatment. Isocitrate lyase was partially purified and displayed catalytic and regulatory properties similar to those of the enzyme isolated from the liver of starved rats (Popov, V.N. et al. (1996) FEBS Lett. 391, 87–90).
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induction of Glyoxylate Cycle enzymes in rat liver upon food starvation
FEBS Letters, 1996Co-Authors: Abir U. Igamberdiev, V N Popov, S V Volvenkin, Claus SchnarrenbergerAbstract:The key enzymes of the Glyoxylate Cycle, isocitrate lyase and malate synthase, have been detected in liver of foodstarved rats. Activities became measurable 3 days and peaked 5 days after the beginning of starvation. Both enzymes were found in the peroxisomal cell fraction after organelle fractionation by isopycnic centrifugation. Isocitrate lyase was purified 112-fold by ammonium sulfate precipitation, and chromotography on DEAE-cellulose and Toyopearl HW-65. The specific activity of the purified enzyme was 9.0 units per mg protein. The Km(isocitrate) was 68 μM and the pH optimum was at pH 7.4. Malate synthase was enriched 4-fold by ammonium sulfate precipitation. The enzyme had a Km(acetyl-CoA) of 0.2 μM, a Km(Glyoxylate) of 3 mM and a pH optimum of 7.6.
V N Popov - One of the best experts on this subject based on the ideXlab platform.
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comparative analysis of Glyoxylate Cycle key enzyme isocitrate lyase from organisms of different systematic groups
Journal of Evolutionary Biochemistry and Physiology, 2005Co-Authors: V N Popov, E A Moskalev, M U Shevchenko, A T EprintsevAbstract: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.
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subcellular localization and properties of Glyoxylate Cycle enzymes in the liver of rats with alloxan diabetes
Biochemistry, 1999Co-Authors: S V Volvenkin, V N Popov, A T EprintsevAbstract:Key enzymes of the Glyoxylate Cycle (isocitrate lyase and malate synthetase) were found in the liver and kidney of rats suffering from alloxan diabetes. The activities of these enzymes in the liver were 0.080 and 0.0430 U/mg protein, respectively. Isocitrate lyase activity in the kidney was 0.030 U/mg protein, and that of the malate synthetase was 0.018 U/mg protein. Peroxisomal localization of the enzymes was shown. A novel malate dehydrogenase isoform was found in a liver of rats suffering from the alloxan diabetes. The isocitrate lyase was isolated by selective (NH4)2SO4 precipitation and DEAE-Toyopearl chromatography. The resulting enzyme preparation had specific activity 6.1 U/mg protein, corresponding to 76.25-fold purification with 32.6% yield. The isocitrate lyase was found to follow the Michaelis--Menten kinetic scheme (Km for isocitrate, 0.08 mM) and to be competitively inhibited by glucose 1-phosphate (Ki = 1. 25 mM), succinate (Ki = 1.75 mM), and citrate (Ki = 1.0 mM); the pH optimum of the enzyme was 7.5 in Tris-HCl buffer.
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Glyoxylate Cycle enzymes are present in liver peroxisomes of alloxan treated rats
FEBS Letters, 1998Co-Authors: Abir U. Igamberdiev, V N Popov, A T Eprintsev, S V VolvenkinAbstract:Abstract Key enzymes of the Glyoxylate Cycle, isocitrate lyase (ICL) and malate synthase (MS), have been detected in the liver of alloxan-treated rats. The activity of ICL in rat liver was 0.040 μmol/min/mg protein and the activity of MS was 0.022 μmol/min/mg protein. These enzymes were associated with the peroxisomal fraction. The activities of citrate synthase, malate synthase and malate dehydrogenase detected in the peroxisomal fraction were also increased by alloxan treatment. Isocitrate lyase was partially purified and displayed catalytic and regulatory properties similar to those of the enzyme isolated from the liver of starved rats (Popov, V.N. et al. (1996) FEBS Lett. 391, 87–90).
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induction of Glyoxylate Cycle enzymes in rat liver upon food starvation
FEBS Letters, 1996Co-Authors: Abir U. Igamberdiev, V N Popov, S V Volvenkin, Claus SchnarrenbergerAbstract:The key enzymes of the Glyoxylate Cycle, isocitrate lyase and malate synthase, have been detected in liver of foodstarved rats. Activities became measurable 3 days and peaked 5 days after the beginning of starvation. Both enzymes were found in the peroxisomal cell fraction after organelle fractionation by isopycnic centrifugation. Isocitrate lyase was purified 112-fold by ammonium sulfate precipitation, and chromotography on DEAE-cellulose and Toyopearl HW-65. The specific activity of the purified enzyme was 9.0 units per mg protein. The Km(isocitrate) was 68 μM and the pH optimum was at pH 7.4. Malate synthase was enriched 4-fold by ammonium sulfate precipitation. The enzyme had a Km(acetyl-CoA) of 0.2 μM, a Km(Glyoxylate) of 3 mM and a pH optimum of 7.6.