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.     

Studies on the Respiratory Physiology of Euglena gracilis Cultured on Acetate or Glucose

SYNOPSIS. The glyoxylate cycle operates at a high level in Euglena gracilis when acetate is the only carbon source, and at a low level when glucose is the only carbon source, as indicated by activities of malate synthase. Altho glucose causes a moderate repression of some of the enzymes of the glyoxylate cycle, it neither represses nor inhibits malate synthase. The specific activity of the malic enzyme was about 5-fold greater in acetate-grown Euglena than in glucose-grown cells, but the absolute rate of CO₂ fixation was about twice as great in cells grown on glucose. The respiratory quotient was unity regardless of substrate.     

A multienzyme complex for CO2 fixation.

Acetyl-coenzyme A carboxylase from Euglena gracilis strain Z was isolated as a component of a multienzyme complex which includes phosphoenolpyruvate carboxylase and malate dehydrogenase. The multienzyme complex was shown to exist in crude extracts and was purified to a homogeneous protein with a molecular weight of 360,000 by gel filtration. The ratio of the activities of the constituent enzymes was acetyl-CoA carboxylase:phosphoenolpyruvate carboxylase:malate dehydrogenase, 1:25:500. The complex is proposed to operate in conjunction with malic enzyme, which is present in Euglena, to facilitate the formation of substrates, malonyl-CoA, and NADPH, for fatty acid biosynthesis. The interaction of the enzymes may represent a means of control of acetyl-CoA carboxylase activity in organisms which do not possess an enzyme subject to allosteric regulation. The acetyl-CoA carboxylase activity from Euglena is unaffected by citrate and isocitrate.     

Soybean (Glycine max. L.) and bacteroid glyoxylate cycle activities during nodular senescence

Soybean (Glycine max. L.) nodular senescence results in the dismantling of the peribacteroid membrane (PBM) and in an increase of soybean isocitrate lyase (ICL; EC 4.1.3.1) and malate synthase (MS; EC 4.1.3.2) mRNA and protein levels. This suggests that in senescing soybean nodular cells, the specific glyoxylate cycle enzyme activities might be induced to reallocate carbon obtained from the PBM degradation. In order to evaluate as well the carbon metabolism of the nitrogen-fixing Bradyrhizobium japonicum endosymbiotic bacteroids during nodular senescence, their glyoxylate cycle activities were also investigated. To this end, partial DNA sequences were isolated from their icl and ms genes, but the corresponding mRNAs were not detected in the microorganisms. It was also observed that the bacteroid ICL and MS activities were negligible during nodular senescence. This suggests that glyoxylate cycle activities are not reinitiated in the bacteroids under these physiological conditions. In case the microorganisms nevertheless feed on the PBM degradation products, this might occur via the citric acid cycle exclusively.     

Properties of Partially Purified Malate Synthase from Euglena gracilis

SYNOPSIS. Properties of partially purified malate synthase from Euglena gracilis were characterized. The pH optimum is 7.0 and the temperature optimum about 30 C; the activation energy is 12,000 calories. A Km of 4 × 10⁻⁵ M was found for both reactants, glyoxylate and acetyl-CoA. The reaction is partially inhibited by a number of normal metabolites, suggesting allosteric control; glycolate is severely inhibitory. The enzyme is not active in cells grown with phototrophic nutrition, but is found in all heterotrophic cells grown on a wide range of carbon sources; the specific activity is greatly dependent on carbon source. High rates of oxygen consumption are usually, but not always, correlated with high enzyme levels.     

Kinetic studies of the fatty acid synthetase multienzyme complex from Euglena gracilis variety bacillaris.

A fatty acid synthetase multienzyme complex was purified from Euglena gracilis variety bacillaris. The fatty acid synthetase activity is specifically inhibited by antibodies against Escherichia coli acyl-carrier protein. The Euglena enzyme system requires both NADPH and NADH for maximal activity. An analysis was done of the steady-state kinetics of the reaction catalysed by the fatty acid synthetase multienzyme complex. Initial-velocity studies were done in which the concentrations of the following pairs of substrates were varied: malonyl-CoA and acetyl-CoA, NADPH and acetyl-CoA, malonyl-CoA and NADPH. In all three cases patterns of the Ping Pong type were obtained. Product-inhibition studies were done with NADP+ and CoA. NADP+ is a competitive inhibitor with respect to NADPH, and uncompetitive with respect to malonyl-CoA and acetyl-CoA. CoA is uncompetitive with respect to NADPH and competitive with respect to malonyl-CoA and acetyl-CoA. When the concentrations of acetyl-CoA and malonyl-CoA were varied over a wide range, mutual competitive substrate inhibition was observed. When the fatty acid synthetase was incubated with radiolabelled acetyl-CoA or malonyl-CoA, labelled acyl-enzyme was isolated. The results are consistent with the idea that fatty acid synthesis proceeds by a multisite substituted-enzyme mechanism involving Ping Pong reactions at the following enzyme sites: acetyl transacylase, malonyl transacylase, beta-oxo acyl-enzyme synthetase and fatty acyl transacylase.     

Structure of isocitrate lyase, a persistence factor of Mycobacterium tuberculosis

Isocitrate lyase (ICL) plays a pivotal role in the persistence of Mycobacterium tuberculosis in mice by sustaining intracellular infection in inflammatory macrophages. The enzyme allows net carbon gain by diverting acetyl-CoA from beta-oxidation of fatty acids into the glyoxylate shunt pathway. Given its potential as a drug target against persistent infections, we solved its structure without ligand and in complex with two inhibitors. Covalent modification of an active site residue, Cys 191, by the inhibitor 3-bromopyruvate traps the enzyme in a catalytic conformation with the active site completely inaccessible to solvent. The structure of a C191S mutant of the enzyme with the inhibitor 3-nitropropionate provides further insight into the reaction mechanism.     

Purification and characterization of a cold-adapted isocitrate lyase and a malate synthase from Colwellia maris, a psychrophilic bacterium.

Isocitrate lyase (ICL) and malate synthase (MS) of a psychrophilic marine bacterium, Colwellia maris, were purified to electrophoretically homogeneous state. The molecular mass of the ICL was found to be 240 kDa, composed of four identical subunits of 64.7 kDa. MS was a dimeric enzyme composed of 76.3 kDa subunits. N-Terminal amino acid sequences of the ICL and MS were analyzed. Purified ICL had its maximum activity at 20 degrees C and was rapidly inactivated at the temperatures above 30 degrees C, but the optimum temperature for the activity of MS was 45 degrees C. NaCl was found to protect ICL from heat inactivation above 30 degrees C, but the salt did not stabilize MS. Effects of temperatures on the kinetic parameters of both the enzymes were examined. The Km for the substrate (isocitrate) of ICL was decreased with decreasing temperature. On the other hand, the Km for the substrate (glyoxylate) of MS was increased with decreasing temperature. The calculated value of free energy of activation of ICL was on the same level as that of MS.     

Purification and Characterization of a Cold-adapted Isocitrate Lyase and a Malate Synthase from Colwellia maris,a Psychrophilic Bacterium

Isocitrate lyase (ICL) and malate synthase (MS) of a psychrophilic marine bacterium, Colwellia maris, were purified to electrophoretically homogeneous state. The molecular mass of the ICL was found to be 240 kDa, composed of four identical subunits of 64.7 kDa. MS was a dimeric enzyme composed of 76.3 kDa subunits. N-Terminal amino acid sequences of the ICL and MS were analyzed. Purified ICL had its maximum activity at 20°C and was rapidly inactivated at the temperatures above 30°C, but the optimum temperature for the activity of MS was 45°C. NaCl was found to protect ICL from heat inactivation above 30°C, but the salt did not stabilize MS. Effects of temperatures on the kinetic parameters of both the enzymes were examined. The K_m for the substrate (isocitrate) of ICL was decreased with decreasing temperature. On the other hand, the K_m for the substrate (glyoxylate) of MS was increased with decreasing temperature. The calculated value of free energy of activation of ICL was on the same level as that of MS.     

L-malyl-coenzyme A/beta-methylmalyl-coenzyme A lyase is involved in acetate assimilation of the isocitrate lyase-negative bacterium Rhodobacter capsulatus

Cell extracts of Rhodobacter capsulatus grown on acetate contained an apparent malate synthase activity but lacked isocitrate lyase activity. Therefore, R. capsulatus cannot use the glyoxylate cycle for acetate assimilation, and a different pathway must exist. It is shown that the apparent malate synthase activity is due to the combination of a malyl-coenzyme A (CoA) lyase and a malyl-CoA-hydrolyzing enzyme. Malyl-CoA lyase activity was 20-fold up-regulated in acetate-grown cells versus glucose-grown cells. Malyl-CoA lyase was purified 250-fold with a recovery of 6%. The enzyme catalyzed not only the reversible condensation of glyoxylate and acetyl-CoA to L-malyl-CoA but also the reversible condensation of glyoxylate and propionyl-CoA to beta-methylmalyl-CoA. Enzyme activity was stimulated by divalent ions with preference for Mn(2+) and was inhibited by EDTA. The N-terminal amino acid sequence was determined, and a corresponding gene coding for a 34.2-kDa protein was identified and designated mcl1. The native molecular mass of the purified protein was 195 +/- 20 kDa, indicating a homohexameric composition. A homologous mcl1 gene was found in the genomes of the isocitrate lyase-negative bacteria Rhodobacter sphaeroides and Rhodospirillum rubrum in similar genomic environments. For Streptomyces coelicolor and Methylobacterium extorquens, mcl1 homologs are located within gene clusters implicated in acetate metabolism. We therefore propose that L-malyl-CoA/beta-methylmalyl-CoA lyase encoded by mcl1 is involved in acetate assimilation by R. capsulatus and possibly other glyoxylate cycle-negative bacteria.     

Functional characterization of D-galacturonic acid reductase, a key enzyme of the ascorbate biosynthesis pathway, from Euglena gracilis

D-Galacturonic acid reductase, a key enzyme in ascorbate biosynthesis, was purified to homogeneity from Euglena gracilis. The enzyme was a monomer with a molecular mass of 38-39 kDa, as judged by SDS-PAGE and gel filtration. Apparently it utilized NADPH with a Km value of 62.5+/-4.5 microM and uronic acids, such as D-galacturonic acid (Km=3.79+/-0.5 mM) and D-glucuronic acid (Km=4.67+/-0.6 mM). It failed to catalyze the reverse reaction with L-galactonic acid and NADP(+). The optimal pH for the reduction of D-galacturonic acid was 7.2. The enzyme was activated 45.6% by 0.1 mM H(2)O(2), suggesting that enzyme activity is regulated by cellular redox status. No feedback regulation of the enzyme activity by L-galactono-1,4-lactone or ascorbate was observed. N-terminal amino acid sequence analysis revealed that the enzyme is closely related to the malate dehydrogenase families.     

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     

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     

Modulation of TCA cycle enzymes and aluminum stress in Pseudomonas fluorescens.

Oxalic acid plays a pivotal role in the adaptation of the soil microbe Pseudomonas fluorescens to aluminum (Al) stress. Its production via the oxidation of glyoxylate necessitates a major reconfiguration of the enzymatic reactions involved in the tricarboxylic acid (TCA) cycle. The demand for glyoxylate, the precursor of oxalic acid appears to enhance the activity of isocitrate lyase (ICL). The activity of ICL, an enzyme that participates in the cleavage of isocitrate to glyoxylate and succinate incurred a 4-fold increase in the Al-stressed cells. However, the activity of isocitrate dehydrogenase, a competitor for the substrate isocitrate, appeared to be diminished in cells exposed to Al compared to the control cells. While the demand for oxalate in Al-stressed cells also negatively influenced the activity of the enzyme alpha-ketoglutarate dehydrogenase complex, no apparent change in the activity of malate synthase was recorded. Thus, it appears that the TCA cycle is tailored in order to generate the necessary precursor for oxalate synthesis as a consequence of Al-stress.     

Mono-ADP-Ribosylation of Arginine Residue of Euglena gracilis Z in Synchronous Culture

ABSTRACT. In Euglena gracilis Z, a considerably high activity of mono-ADP-ribosyltransferase occurred and change of it was accompanied by a cell cycle induced by a light-dark cycle. The enzyme activity was strongly inhibited by L-arginine and supported in the presence of poly-L-arginine as a substrate, indicating that ADP-ribosylated amino acid is an arginine residue. Arginine: mono-ADP-ribosyltransferase activity was found in the chloroplasts, mitochondria, microsomes and cytosol as judged from marker enzyme activities and the activity in each organelle fluctuated with the cell cycle.     

Purification and characterization of malate synthase from the glucose-grown wood-rotting basidiomycete Fomitopsis palustris

Malate synthase (EC 4.1.3.2), the key enzyme of the glyoxylate cycle, was purified to a homogeneous protein from the wood-rotting basidiomycete Fomitopsis palustris grown on glucose. The purified enzyme, with a molecular mass of 520 kDa, was found to consist of eight 65-kDa subunits, and to have Km of 45 and 2.2 microM for glyoxylate and acetyl-CoA, respectively. The enzyme activity was competitively inhibited by oxalate (K1, 8.5 microM) and glycolate (Ki, 17 microM), and uncompetitively by coenzyme A (Ki, 100 microM). The potent inhibition of the activity by p-chloromercuribenzoate suggests that the enzyme has a sulfhydryl group at the active center. However, the enzyme was inhibited moderately by adenine nucleotides and weakly by some of the metabolic intermediates of glycolysis and tricarboxylic acid cycle. The enzyme was completely inactive in the absence of metal ions and was maximally activated by Mg2+ (Km, 0.4 microM), which also served to significantly prevent enzyme inactivation during storage.     

Purification and properties of L-alanine:4,5-dioxovalerate aminotransferase from Chlorella regularis

The enzyme L-alanine:4,5-dioxovalerate aminotransferase (EC 2.6.1.43), which catalyzes the synthesis of 5-aminolevulinic acid, was purified 161-fold from Chlorella regularis. The enzyme also showed L-alanine:glyoxylate aminotransferase activity (EC 2.6.1.44). The activity of glyoxylate aminotransferase was 56-fold greater than that of 4,5-dioxovalerate aminotransferase. The ratio of the two activities remained nearly constant during purification, and when the enzyme was subjected to a variety of treatments. 4,5-Dioxovalerate aminotransferase activity was competitively inhibited by glyoxylate, with a Ki value of 0.5 mM. Double-reciprocal plots of velocity versus 4,5-dioxovalerate with varying L-alanine concentrations indicate a ping-pong reaction mechanism. The apparent Km values for 4,5-dioxovalerate and L-alanine were 0.12 and 3.5 mM, respectively. The enzyme is an acidic protein having an isoelectric point of 4.8. The molecular weight of the enzyme was estimated to be 126,000, with two identical subunits. These results suggest that, in Chlorella, as in bovine liver mitochondria and Euglena, both 4,5-dioxovalerate and glyoxylate aminotransferase activities are associated with the same protein. From the activity ratio of transamination and catalytic properties, it is concluded that this enzyme does not function primarily as a part of the 5-carbon pathway to 5-aminolevulinic acid synthesis.