Molecular characterization of a bifunctional glyoxylate cycle enzyme, malate synthase/isocitrate lyase, in Euglena gracilis

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.     

Characterization of a bifunctional glyoxylate cycle enzyme, malate synthase/isocitrate lyase, of Euglena gracilis

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.     

Sucrose controls storage lipid breakdown on gene expression level in germinating yellow lupine (Lupinus luteus L.) seeds

This study revealed that cytosolic aconitase (ACO, EC 4.2.1.3) and isocitrate lyase (ICL, EC 4.1.3.1, marker of the glyoxylate cycle) are active in germinating protein seeds of yellow lupine. The glyoxylate cycle seems to function not only in the storage tissues of food-storage organs, but also in embryonic tissue of growing embryo axes. Sucrose (60 mM) added to the medium of in vitro culture of embryo axes and cotyledons decreased activity of lipase (LIP, EC 3.1.1.3) and activity of glutamate dehydrogenase (NADH-GDH, EC 1.4.1.2). The opposite effect was caused by sucrose on activity of cytosolic ACO, ICL as well as NADP⁺-dependent (EC 1.1.1.42) and NAD⁺-dependent (EC 1.1.1.41) isocitrate dehydrogenase (NADP-IDH and NAD-IDH, respectively); activity of these enzymes was clearly stimulated by sucrose. Changes in the activity of LIP, ACO, NADP-IDH, and NAD-IDH caused by sucrose were based on modifications in gene expression because corresponding changes in the enzyme activities and in the mRNA levels were observed. The significance of cytosolic ACO and NADP-IDH in carbon flow from storage lipid to amino acids, as well as the peculiar features of storage lipid breakdown during germination of lupine seeds are discussed.     

Enzyme profiles of Thiobacillus versutus after aerobic and denitrifying growth: Regulation of isocitrate lyase

The specific activities of the tricarboxylic acid (TCA) cycle enzymes in Thiobacillus versutus were invariably lower after aerobic growth as compared to denitrifying growth in acetate- or succinate-limited chemostat cultures. Of the glyoxylate cycle enzymes, isocitrate lyase (ICL) activity was nil during aerobic and 76 nmol·min^-1·mg^-1 protein during denitrifying growth on acetate whereas malate synthase (MS) did not change. In succinate-grown cells ICL was always near nil. The change in ICL and MS was followed after pulse additions of acetate and nitrate to an aerobic acetate-limited chemostat culture made anaerobic prior to the first pulse. ICL remained nil during denitrifying growth after the first pulse but increased to 47 and 81 nmol ·min^-1·mg^-1 protein after the second and third pulse, respectively. MS remained unaltered. The appearance of ICL was dependent upon de novo protein synthesis. During transition in a steady state culture on acetate from oxygen to nitrate as terminal electron acceptor, denitrifying growth started after 0.6 volume replacements. The resumption of growth was concomitant with an increase in TCA cycle enzyme activities. ICL was observed only after two volume replacements. During the reverse transition, ICL disappeared at a rate twice the dilution rate. SDS polyacrylamide gelectrophoresis of cell-free extracts containing ICL showed a major protein band with a R_f value identical to purified ICL and a mol·wt. of 60,000. ICL from T. versutus was inhibited by 1.5 mM itaconate but not by 10 mM phosphoenolpyruvate. Its activity was dependent upon the presence of Mg²⁺ and cysteine.Abbreviations TCA Tricarboxylic acid - ICL isocitrate lyase - MS malate synthase - FPLC fast protein liquid chromatography - maximum specific oxygen consumption rate     

Adaptation of <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> to Al-Citrate: Involvement of Tricarboxylic Acid and Glyoxylate Cycle Enzymes and the Influence of Phosphate

The degradation of Aluminum-citrate by Pseudomonas fluorescens necessitated a major restructuring of the various enzymatic activities involved in the TCA and glyoxylate cycles. While a six-fold increase in fumarase (FUM EC 4.2.1.2) activity was observed in cells subjected to Al-citrate compared to control cells, citrate synthase (CS EC 4.1.3.7) activity experienced a two-fold increase. On the other hand, in the Al-stressed cells malate synthase (MS EC 4.1.3.2) activity underwent a five-fold decrease in activity. This modulation of enzymatic activities appeared to be evoked by Al stress, as the incubation of Al-stressed cells in control media led to the complete reversal of these enzymatic profiles. These observations were further confirmed by ¹H NMR and ¹³C NMR spectroscopy. No significant variations were observed in the activities of other glyoxylate and TCA cycle enzymes, like isocitrate lyase (ICL EC 4.1.3.1), malate dehydrogenase (MDH EC 1.1.1.37), and succinate dehydrogenase (SDH EC 1.3.99.1). This reconfiguration of the metabolic pathway appears to favour the production of a citrate-rich aluminophore that is involved in the sequestration of Al.     

Molecular cloning of cucumber phosphoenolpyruvate carboxykinase and developmental regulation of gene expression

A cDNA library from RNA of senescing cucumber cotyledons was screened for sequences also expressed in cotyledons during post-germinative growth. One clone encodes ATP-dependent phosphoenolpyruvate carboxykinase (PCK; EC 4.1.1.49), an enzyme of the gluconeogenic pathway. The sequence of a fulllength cDNA predicts a polypeptide of 74397 Da which is 43%, 49% and 57% identical to bacterial, trypanosome and yeast enzymes, respectively. The cDNA was expressed in Escherichia coli and antibodies raised against the resultant protein. The antibody recognises a single polypeptide of ca. 74 kDa, in extracts of cotyledons, leaves and roots. The cucumber genome contains a single pck gene. In the seven-day period after seed imbibition, PCK mRNA and protein steady-state levels increase in amount in cotyledons, peaking at days 2 and 3 respectively, and then decrease. Both accumulate again to a low level in senescing cotyledons. This pattern of gene expression is similar to that of isocitrate lyase (ICL) and malate synthase (MS). When green cotyledons are detached from seedlings and incubated in the dark, ICL and MS mRNAs increase rapidly in amount but PCK mRNA does not. Therefore it seems unlikely that the glyoxylate cycle serves primarily a gluconeogenic role in starved (detached) cotyledons, in contrast to post-germinative and senescing cotyledons where PCK, ICL and MS are coordinately synthesised. While exogenous sucrose greatly represses expression of icl and ms genes in dark-incubated cotyledons, it has a smaller effect on the level of PCK mRNA.     

Expression of a single gene encoding microbody NAD-malate dehydrogenase during glyoxysome and peroxisome development in cucumber

A full-length cDNA clone encoding microbody NAD⁺-dependent malate dehydrogenase (MDH) of cucumber has been isolated. The deduced amino acid sequence is 97% identical to glyoxysomal MDH (gMDH) of watermelon, including the amino terminal putative transit peptide. The cucumber genome contains only a single copy of this gene. Expression of this mdh gene increases dramatically in cotyledons during the few days immediately following seed imbibition, in parallel with genes encoding isocitrate lyase (ICL) and malate synthase (MS), two glyoxylate cycle enzymes. The level of MDH, ICL and MS mRNAs then declines, but then MDH mRNA increases again together with that of peroxisomal NAD⁺-dependent hydroxypyruvate reductase (HPR). The mdh gene is also expressed during cotyledon senescence, together with hpr, icl and ms genes. These results indicate that a single gene encodes MDH which functions in both glyoxysomes and peroxisomes. In contrast to icl and ms genes, expression of the mdh gene is not activated by incubating detached green cotyledons in the dark, nor is it affected by exogenous sucrose in the incubation medium. The function of this microbody MDH and the regulation of its synthesis are discussed.     

An activated-oxygen-mediated role for peroxisomes in the mechanism of senescence of Pisum sativum L. leaves

The possible involvement of peroxisomes and their activated-oxygen metabolism in the mechanism of leaf senescence was investigated in detached pea (Pisum sativum L.) leaves which were induced to senesce by incubation in complete darkness for up to 11 d. At days 0, 3, 8, and 11 of senescence, peroxisomes were purified from leaves and the activities of different peroxisomal and glyoxysomal enzymes were measured. Xanthine-oxidoreductase activity increased with senescence, especially the O ₂ ^{. -} -producing xanthine oxidase (EC 1.1.3.22). The activities of H₂O₂-generating Mn-superoxide dismutase (EC 1.15.1.1) and urate oxidase (EC 1.7.3.3) were also enhanced by senescence, whereas catalase (EC 1.11.1.6) activity was severely depressed. Hydrogen peroxide concentrations increased significantly in senescent leaf peroxisomes. During the progress of senescence, glycollate oxidase (EC 1.1.3.1) and hydroxypyruvate reductase (EC 1.1.1.81), two marker enzymes of photorespiratory metabolism, gradually decreased in activity and disappeared. At the same time, the activities of malate synthase (EC 4.1.3.2) and isocitrate lyase (EC 4.1.3.1), key enzymes of the glyoxylate cycle, which were undetectable in presenescent leaves, increased dramatically upon induction of senescence. Ultrastructural studies of intact leaves showed that the population of peroxisomes and mitochondria increased with senescence. Results indicate that peroxisomes could play a role, mediated by activated oxygen species, in the oxidative mechanism of leaf senescence, and further support the idea, proposed by other authors, that foliar senescence is associated with the transition of leaf peroxisomes into glyoxysomes.Abbreviation Mn-SOD (manganese-containing) superoxide dismutase The authors thank Dr. A.J. Sánchez-Raya (Unidad de Fisiología Vegetal, Estación Experimental del Zaidín, Granada, Spain) for his valuable help in measuring ethylene production, and Dr. G. Barja de Quiroga (Departamento de Biología Animal II, Universidad Complutense, Madrid, Spain) for carrying out the malondialdehyde determinations by HPLC. This work was supported by grant PB87-0404-01 from the DGICYT and the Junta de Andaluc'ia (Research Group # 3315), Spain.     

Role of energetic coenzyme pools in the production of L-carnitine by Escherichia coli

The aim of this work was to understand the steps controlling the biotransformation of trimethylammonium compounds into L(-)-carnitine by Escherichia coli. The high-cell density reactor steady-state levels of carbon source (glycerol), biotransformation substrate (crotonobetaine), acetate (anaerobiosis product) and fumarate (as an electron acceptor) were pulsed by increasing them fivefold. Following the pulse, the evolution of the enzyme activities involved in the biotransformation process of crotonobetaine into L(-)-carnitine (crotonobetaine hydration), in the synthesis of acetyl-CoA (ACS: acetyl-CoA synthetase and PTA: ATP: acetate phosphotransferase) and in the distribution of metabolites for the tricarboxylic acid (ICDH: isocitrate dehydrogenase) and glyoxylate (ICL: isocitrate lyase) cycles was monitored. In addition, the levels of carnitine, the cell ATP content and the NADH/NAD(+) ratio were measured in order to assess the importance and participation of these energetic coenzymes in the catabolic system. The results provided an experimental demonstration of the important role of the glyoxylate shunt during biotransformation and the need for high levels of ATP to maintain metabolite transport and biotransformation. Moreover, the results obtained for the NADH/NAD(+) pool indicated that it is correlated with the biotransformation process at the NAD(+) regeneration and ATP production level in anaerobiosis. More importantly, a linear correlation between the NADH/NAD(+) ratio and the levels of the ICDH and ICL (carbon and electron flows) and the PTA and ACS (acetate and ATP production and acetyl-CoA synthesis) activity levels was assessed. The main metabolic pathway operating during cell metabolic perturbation with a pulse of glycerol and acetate in the high-cell density membrane reactor was that related to ICDH and ICL, both regulating the carbon metabolism, together with PTA and ACS enzymes (regulating ATP production).     

Glyoxysomal malate dehydrogenase and malate synthase from soybean cotyledons (Glycine max L.): enzyme association,antibody production and cDNA cloning

In order to investigate a possible association between soybean malate synthase (MS; l-malate glyoxylate-lyase, CoA-acetylating, EC 4.1.3.2) and glyoxysomal malate dehydrogenase (gMDH; (S)-malate: NAD⁺ oxidoreductase, EC 1.1.1.37), two consecutive enzymes in the glyoxylate cycle, their elution profiles were analyzed on Superdex 200 HR fast protein liquid chromatography columns equilibrated in low- and high-ionicstrength buffers. Starting with soluble proteins extracted from the cotyledons of 5-d-old soybean seedlings and a 45% ammonium sulfate precipitation, MS and gMDH coeluted on Superdex 200 HR (low-ionic-strength buffer) as a complex with an approximate relative molecular mass (M_r) of 670000. Dissociation was achieved in the presence of 50 mM KCl and 5 mM MgCl₂, with the elution of MS as an octamer of M_r 510000 and of gMDH as a dimer of M_r 73 000. Polyclonal antibodies raised to the native copurified enzymes recognized both denatured MS and gMDH on immunoblots, and their native forms after gel filtration. When these antibodies were used to screen a ZAP II expression library containing cDNA from 3-d-old soybean cotyledons, they identified seven clones encoding gMDH, whereas ten clones encoding MS were identified using an antibody to SDS-PAGE-purified MS. Of these cDNA clones a 1.8 kb clone for MS and a 1.3-kb clone for gMDH were fully sequenced. While 88% identity was found between mature soybean gMDH and watermelon gMDH, the N-terminal transit peptides showed only 37% identity. Despite this low identity, the soybean gMDH transit peptide conserves the consensus R(X₆)HL motif also found in plant and mammalian thiolases.The nucleotide sequence data reported in this paper have been submitted to Genbank and assigned the accession numbers LOI628 for gMDH and L01629 for MS.     

Ethanol catabolism in Corynebacterium glutamicum

Corynebacterium glutamicum grows on a variety of carbohydrates and organic acids as single or combined sources of carbon and energy. Here we show the ability of C. glutamicum to grow on ethanol with growth rates up to 0.24 h(-1) and biomass yields up to 0.47 g dry weight (g ethanol)(-1). Mutants of C. glutamicum deficient in phosphotransacetylase (PTA), isocitrate lyase (ICL) and malate synthase (MS) were unable to grow on ethanol, indicating that acetate activation and the glyoxylate cycle are essential for utilization of this substrate. In accordance, the expression profile of ethanol-grown C. glutamicum cells compared to that of glucose-grown cells revealed an increased expression of genes encoding acetate kinase (AK), PTA, ICL and MS. Furthermore, the specific activities of these four enzymes as well as those of alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) were found to be high in ethanol-grown and low in glucose-grown cells. Growth of C. glutamicum on a mixture of glucose and ethanol led to a biphasic growth behavior, which was due to the sequential utilization of glucose before ethanol. Accordingly, the specific activities of ADH, ALDH, AK, PTA, ICL and MS in cells grown in medium containing both substrates were as low as in glucose-grown cells in the first growth phase, but increased 5- to 100-fold during the second growth phase. The results indicate that ethanol catabolism in C. glutamicum is subject to carbon source-dependent regulation, i.e., to a carbon catabolite control.     

Immunolocalization of glyoxysomal malate synthase from soybean cotyledons (Glycine max L)

Summary— Malate synthase (MS; EC 4.1.3.2), an enzyme specific to the glyoxylate cycle, was studied in cotyledons of dark-grown soybean (Glycine max L) seedlings with light and electron microscopy techniques. Immunogold localization confirmed biochemical evidence that MS from soybean is a glyoxysomal matrix enzyme.     

A possible role of the key enzymes of the glyoxylate and gluconeogenesis pathways for fruit-body formation of the wood-rotting basidiomycete Flammulina velutipes

 Biochemical roles of the representative enzymes involved in carbon metabolism of glucose were investigated in relation to the fruit-body formation of the basidiomycete Flammulina velutipes. Changes in specific activities of the enzymes of the tricarboxylic acid (TCA) cycle and glyoxylate (GLOX) and gluconeogenesis pathways were measured at different stages of development of the fungus. The enzyme activities of malate synthase (MS) and fructose bisphosphatase (FBP) as the key enzymes for the GLOX-gluconeogenesis pathways increased in mycelia during the fruit-body formation. The activities of isocitrate dehydrogenase (IDH) for the TCA cycle and NADP-linked glutamate dehydrogenase (GLTDH (NADP)) for glutamate synthesis increased more markedly. Moreover, the mycelial mat of the cultures producing fruit bodies yielded greater enzyme activities of isocitrate lyase (ICL), MS, FBP, and IDH than that of the cultures that did not produce fruit bodies. These results suggest that the GLOX-gluconeogenesis pathways as well as the glutamate synthesis have a strong correlation with the fruit-body formation of F. velutipes. Received: January 22, 2002 / Accepted: May 10, 2002     

Effects of growth regulators and light on chloroplasts NAD(P)H dehydrogenase activities of senescent barley leaves

The activities NADH and NADPH dehydrogenases were measured with ferricyanide as electron-acceptor (NADH-FeCN-ox and NADPH-FeCN-ox, respectively) in mitochondria-free chloroplasts of barley leaf segments after receiving various treatments affecting senescence. NADPH-FeCN-ox declined during senescence in the dark, in a way similar to chlorophyll and Hill reaction, and increased when leaf segments were incubated at light. These results suggest that NADPH-FeCN-ox is related to some photosynthetic electron transporter activity (probably ferredoxin-NADP⁺ oxidoreductase). In contrast, NADH-FeCN-ox is notably stable during senescence in the dark and at light. This activity increased during incubation with kinetin or methyl-jasmonate (Me-JA) but decreased when leaf segments were treated with abscisic acid (ABA). The effects of the inhibitors of protein synthesis cycloheximide and chloramphenicol suggest that the changes of NAD(P)H dehydrogenase activities may depend on protein synthesis in chloroplasts. In senescent leaf, chloroplast NADH dehydrogenase might be a way to dissipate NADH produced in the degradation of excess carbon which is released from the degradation of amino acids.Abbreviations ABA abscisic acid - DCPIP 2,6-dichlorophenol-indo-phenol - DOC deoxycholate - Me-JA methyl jasmonate - NADH-FeCN-ox NADH ferricyanide oxidoreductase - NADPH-FeCN-ox NADPH ferricyanide oxidoreductase     

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

Background  

The 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.