Hormone-responsive myofibrillar protease activity in cultured rat myoblasts

The phosphorylation of NADP-specific isocitrate dehydrogenase in an isocitrate lyase and in a malate synthase mutant of Escherichia coli has been investigated. The results clearly demonstrate that isocitrate dehydrogenase may undergo an acetate-induced phosphorylation in organisms which do not have a functional glyoxylate cycle. This observation, together with those reported in Salmonella typhimurium, suggest that the current notion concerning the interrelationship between the glyoxylate cycle and the reversible phosphorylation of NADP-isocitrate dehydrogenase in microbial physiology should be reevaluated, and that phosphoenolpyruvate may be a key factor in the regulation of the reversible covalent modification of this enzyme in vivo.     

L-Malyl-coenzyme A lyase/beta-methylmalyl-coenzyme A lyase from Chloroflexus aurantiacus,a bifunctional enzyme involved in autotrophic CO(2) fixation

The 3-hydroxypropionate cycle is a bicyclic autotrophic CO(2) fixation pathway in the phototrophic Chloroflexus aurantiacus (Bacteria), and a similar pathway is operating in autotrophic members of the Sulfolobaceae (Archaea). The proposed pathway involves in a first cycle the conversion of acetyl-coenzyme A (acetyl-CoA) and two bicarbonates to L-malyl-CoA via 3-hydroxypropionate and propionyl-CoA; L-malyl-CoA is cleaved by L-malyl-CoA lyase into acetyl-CoA and glyoxylate. In a second cycle, glyoxylate and another molecule of propionyl-CoA (derived from acetyl-CoA and bicarbonate) are condensed by a putative beta-methylmalyl-CoA lyase to beta-methylmalyl-CoA, which is converted to acetyl-CoA and pyruvate. The putative L-malyl-CoA lyase gene of C. aurantiacus was cloned and expressed in Escherichia coli, and the recombinant enzyme was purified and studied. Beta-methylmalyl-CoA lyase was purified from cell extracts of C. aurantiacus and characterized. We show that these two enzymes are identical and that both enzymatic reactions are catalyzed by one single bifunctional enzyme, L-malyl-CoA lyase/beta-methylmalyl-CoA lyase. Interestingly, this enzyme works with two different substrates in two different directions: in the first cycle of CO(2) fixation, it cleaves L-malyl-CoA into acetyl-CoA and glyoxylate (lyase reaction), and in the second cycle it condenses glyoxylate with propionyl-CoA to beta-methylmalyl-CoA (condensation reaction). The combination of forward and reverse directions of a reversible enzymatic reaction, using two different substrates, is rather uncommon and reduces the number of enzymes required in the pathway. In summary, L-malyl-CoA lyase/beta-methylmalyl-CoA lyase catalyzes the interconversion of L-malyl-CoA plus propionyl-CoA to beta-methylmalyl-CoA plus acetyl-CoA.     

Isocitrate lyase from higher plants

Work on isocitrate lyase, the first enzyme unique to the glyoxylate cycle, is reviewed.     

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.     

Taurine catabolism. III. Evidence for the participation of the glyoxylate cycle

The metabolic fate of acetate, produced during taurine catabolism in Pseudomonas aeruginosa TAU-5, appears to involve the glyoxylate cycle. Organisms grown on taurine have significantly higher levels of malate synthetase and isocritrate lyase than cells grown on nutrient broth, but were comparable to the levels found in acetate-grown organisms. Itaconate, an isocitrate lyase inhibitor, produced a prolonged lag phase and reduced the growth rate of organisms when it was present in the taurine or acetate growth medium. Ethylmethanesulfonate treatment of TAU-5 yielded mutant strains unable to grow on taurine or acetate as sole carbon sources, due to a lack of either malate synthetase or isocitrate lyase. Spontaneous revertants derived from these mutant strains regained the missing enzyme activity and the ability to grow on taurine or acetate.     

Taurine catabolism: III. Evidence for the participation of the glyoxylate cycle

The metabolic fate of acetate, produced during taurine catabolism in Pseudomonas aeruginosa TAU-5, appear to involve the glyoxylate cycle. Organisms grown on taurine have significantly higher levels of malate synthetase and isocitrate lyase than cells grown on nutrient broth, but were comparable to the levels found in acetate-grown organisms. Itaconate, an isocitrate lyase inhibitor, produced a prolonged lag phase and reduced the growth rate of organisms when it was present in the taurine or acetate growth medium. Ethylmethanesulfonate treatment of TAU-5 yielded mutant strains unable to grow on taurine or acetate as sole carbon sources, due to a lack of either malate synthetase or isocitrate lyase. Spontaneous revertants derived from these mutant strains regained the missing enzyme activity and the ability to grow on taurine or acetate.     

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.     

Vanadate-dependent photomodification of serine 319 and 321 in the active site of isocitrate lyase from Escherichia coli.

Vanadate was used as a substrate analogue to modify and subsequently localize active site serine residues of isocitrate lyase from Escherichia coli. Irradiation of the enzyme on ice with UV light in the presence of vanadate resulted in inactivation. Inactivation was prevented by the substrates glyoxylate or Ds-isocitrate and to a much lesser extent by succinate. Reduction of photoinactivated isocitrate lyase by NaBH4 partially restored enzyme activity. The photomodified enzyme was labeled by reduction with NaB[3H]4 in the presence and absence of the substrates succinate plus glyoxylate. Highly differential labeling of serine residues 319 and 321 in the absence of substrates suggests their importance in the action of isocitrate lyase. These residues are highly conserved in all five known sequences of this enzyme.     

Biochemical and structural studies of malate synthase from Mycobacterium tuberculosis

Establishment or maintenance of a persistent infection by Mycobacterium tuberculosis requires the glyoxylate pathway. This is a bypass of the tricarboxylic acid cycle in which isocitrate lyase and malate synthase (GlcB) catalyze the net incorporation of carbon during growth of microorganisms on acetate or fatty acids as the primary carbon source. The glcB gene from M. tuberculosis, which encodes malate synthase, was cloned, and GlcB was expressed in Escherichia coli. The influence of media conditions on expression in M. tuberculosis indicated that this enzyme is regulated differentially to isocitrate lyase. Purified GlcB had K(m) values of 57 and 30 microm for its substrates glyoxylate and acetyl coenzyme A, respectively, and was inhibited by bromopyruvate, oxalate, and phosphoenolpyruvate. The GlcB structure was solved to 2.1-A resolution in the presence of glyoxylate and magnesium. We also report the structure of GlcB in complex with the products of the reaction, coenzyme A and malate, solved to 2.7-A resolution. Coenzyme A binds in a bent conformation, and the details of its interactions are described, together with implications on the enzyme mechanism.     

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.     

Itaconic acid: An inhibitor of isocitrate lyase in Tetrahymena pyriformis in vitro and in vivo

The succinate analog itaconic acid was observed to be a competitive inhibitor of the glyoxylate cycle specific enzyme isocitrate lyase (EC 4.1.3.1) in cell-free extracts of Tetrahymena pyriformis. Itaconic acid also inhibited net in vivo glycogen synthesis from glyoxylate cycle-dependent precursors such as acetate but not from glyoxylate cycle-independent precursors such as fructose. The effect of itaconic acid on the incorporation of ¹⁴C into glycogen from various ¹⁴C-labeled precursors was also consistent with inhibition of isocitrate lyase by this compound. Another analog of succinate which shares a common metabolic fate with itaconic acid, mesaconic acid, had no effect on isocitrate lyase activity in vitro or on ¹⁴C-labeled precursor incorporation into glycogen in vivo. In addition, itaconic acid did not affect gluconeogenesis from lactate in isolated perfused rat livers, a system lacking the enzyme isocitrate lyase. These results are taken as evidence that itaconic acid is an inhibitor of glyoxylate cycle-dependent glyconeogenesis Tetrahymena pyriformis via specific competitive inhibition of isocitrate lyase activity.     

The ethylmalonyl-CoA pathway is used in place of the glyoxylate cycle by Methylobacterium extorquens AM1 during growth on acetate

Acetyl-CoA assimilation was extensively studied in organisms harboring the glyoxylate cycle. In this study, we analyzed the metabolism of the facultative methylotroph Methylobacterium extorquens AM1, which lacks isocitrate lyase, the key enzyme in the glyoxylate cycle, during growth on acetate. MS/MS-based proteomic analysis revealed that the protein repertoire of M. extorquens AM1 grown on acetate is similar to that of cells grown on methanol and includes enzymes of the ethylmalonyl-CoA (EMC) pathway that were recently shown to operate during growth on methanol. Dynamic 13C labeling experiments indicate the presence of distinct entry points for acetate: the EMC pathway and the TCA cycle. 13C steady-state metabolic flux analysis showed that oxidation of acetyl-CoA occurs predominantly via the TCA cycle and that assimilation occurs via the EMC pathway. Furthermore, acetyl-CoA condenses with the EMC pathway product glyoxylate, resulting in malate formation. The latter, also formed by the TCA cycle, is converted to phosphoglycerate by a reaction sequence that is reversed with respect to the serine cycle. Thus, the results obtained in this study reveal the utilization of common pathways during the growth of M. extorquens AM1 on C1 and C2 compounds, but with a major redirection of flux within the central metabolism. Furthermore, our results indicate that the metabolic flux distribution is highly complex in this model methylotroph during growth on acetate and is fundamentally different from organisms using the glyoxylate cycle.     

Evidence for an operative glyoxylate cycle in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius

Both key enzymes for the glyoxylate cycle, isocitrate lyase (EC 4.1.3.1) and malate synthase (EC 4.1.3.2), were purified and characterized from the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius. Whereas the former enzyme was copurified with the aconitase, the latter enzyme could be enriched to apparent homogeneity. Amino acid sequencing of three internal peptides of the isocitrate lyase revealed the presence of highly conserved residues. With respect to cofactor requirement and quarternary structure the crenarchaeal malate synthase might represent a novel type of this enzyme family. High activities of both glyoxylate cycle enzymes could already be detected in extracts of glucose grown cells and both increased about two-fold in extracts of acetate grown cells.     

Purification and characterization of Acinetobacter calcoaceticus isocitrate lyase.

Acinetobacter calcoaceticus is capable of growing on acetate or compounds that are metabolized to acetate. During adaptation to growth on acetate, A. calcoaceticus B4 exhibits an increase in NADP(+)-isocitrate dehydrogenase and isocitrate lyase activities. In contrast, during adaptation to growth on acetate, Escherichia coli exhibits a decrease in NADP(+)-isocitrate dehydrogenase activity that is caused by reversible phosphorylation of specific serine residues on this enzyme. Also, in E. coli, isocitrate lyase is believed to be active only in the phosphorylated form. This phosphorylation of isocitrate lyase may regulate entry of isocitrate into the glyoxylate bypass. To understand the relationships between these two isocitrate-metabolizing enzymes and the metabolism of acetate in A. calcoaceticus B4 better, we have purified isocitrate lyase to homogeneity. Physical and kinetic characterization of the enzyme as well as the inhibitor specificity and divalent cation requirement have been examined.     

The ICL1 gene from Saccharomyces cerevisiae.

The glyoxylate cycle is essential for the utilization of C2 compounds by the yeast Saccharomyces cerevisiae. Within this cycle, isocitrate lyase catalyzes one of the key reactions. We obtained mutants lacking detectable isocitrate lyase activity, screening for their inability to grow on ethanol. Genetic and biochemical analysis suggested that they carried a defect in the structural gene, ICL1. The mutants were used for the isolation of this gene and it was located on a 3.1-kb BglII-SphI DNA fragment. We then constructed a deletion-substitution mutant in the haploid yeast genome. It did not have any isocitrate lyase activity and lacked the ability to grow on ethanol as the sole carbon source. Both strands of a DNA fragment carrying the gene and its flanking regions were sequenced. An open reading frame of 1671 bp was detected, encoding a protein of 557 amino acids with a calculated molecular mass of 62515 Da. The deduced amino acid sequence shows extensive similarities to genes encoding isocitrate lyases from various organisms. Two putative cAMP-dependent protein-kinase phosphorylation sites may explain the susceptibility of the enzyme to carbon catabolite inactivation.     

Enzymes involved in the assimilation of one-carbon units by Pseudomonas MS.

Pseudomonas MS can grow on methylamine and a number of other compounds containing C1 units as a sole source of carbon and energy. Assimilation of carbon into cell material occurs via the "serine pathway" since enzymes of this pathway are induced after growth on methylamine, but not malate or acetate. A mutant has been isolated which is unable to grow on methylamine or any other related substrate providing C1 units. This mutant is also unable to grow on acetate. Measurment of enzyme activities in cell-free extracts of wild-type cells showed that growth on methylamine caused induction of isocitrate lyase, a key enzyme in the glyoxylate cycle. The mutant organism lacks malate lyase, a key enzyme of the serine pathway, and isocitrate lyase as well. These results suggest that utilization of C1 units by Pseudomonas MS results in the net accumulation of acetate which is then assimilated into cell material via the glyoxylate cycle.     

Phosphorylation of Acinetobacter isocitrate lyase.

During growth on succinate, Acinetobacter calcoaceticus contains two forms of the enzyme isocitrate dehydrogenase. Addition of acetate to a lag-phase culture grown on succinate causes a dramatic increase in activity of form II of isocitrate dehydrogenase and in isocitrate lyase. Form II of isocitrate dehydrogenase may be responsible for the partition of isocitrate between the TCA cycle and the glyoxylate by-pass. This report describes the phosphorylation of the enzyme isocitrate lyase from A. calcoaceticus. This phosphorylation may be a regulatory mechanism for the glyoxylate by-pass.     

Changes in Activity of Glyoxylate Cycle Enzymes During Myxospore Development in Myxococcus xanthus

Activities of the glyoxylate cycle enzymes isocitrate lyase (EC 4.1.3.1) and malate synthase (EC 4.1.3.2) were assayed in extracts prepared at different stages of myxospore formation in liquid cultures of Myxococcus xanthus. Activities of both enzymes attained peak values during conversion of rods to spheres. Isocitrate lyase activity decreased after reaching its peak value. Malate synthase activity also declined but at a much slower rate. The loss of isocitrate lyase activity could be prevented by the addition of chloramphenicol to cultures early in myxospore formation (during the initial rise in enzyme activity), but not by such addition at later stages of myxospore formation. The increase in glyoxylate cycle enzymes was not observed in a mutant unable to form myxospores in liquid culture under conditions suitable for morphological conversion of the wild type, or in wild-type cells incubated in the absence of an inducer for myxospore formation. It is concluded that the changes in the glyoxylate cycle enzymes represent regulatory phenomena associated with the development of the myxospore.