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.     

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.     

Methylamine metabolism in a pseudomonas species

The mechanism by which a nonphotosynthetic bacterium Pseudomonas sp. (Shaw Strain MA) grows on the one-carbon source, methylamine, was investigated by comparing enzyme levels of cells grown on methylamine, to cells grown on acetate or succinate. Cells grown on methylamine have elevated levels of the enzymes serine hydroxymethyl transferase, serine dehydratase, malic enzyme, glycerate dehydrogenase and malate lyase (CoA acetylating ATP-cleaving). These enzymes, in conjunction with a constitutive glyoxylate transaminase, can account for the net conversion of two one-carbon units into acetyl CoA. Cells grown on acetate or methylamine, but not succinate, contain the enzyme isocitrate lyase; while cells grown on acetate or succinate, but not methylamine, contain significant levels of malate synthetase. These findings suggest that the acetyl CoA derived from one-carbon units in methylamine grown cells, condenses with oxalacetate to yield citrate and then isocitrate, followed by cleavage to succinate and glyoxylate. Thus, growth on methylamine is accomplished by the net synthesis of succinate from two molecules of methyamine and two molecules of CO₂.     

Microbial metabolism of C1 and C2 compounds. The role of glyoxylate, glycollate and acetate in the growth of Pseudomonas AM1 on ethanol and on C1 compounds

Succinate (or a product of succinate metabolism) is a catabolite repressor of some enzymes of the serine pathway (hydroxypyruvate reductase, serine-glyoxylate aminotransferase and glycerate kinase) but not of methanol dehydrogenase nor methylamine dehydrogenase. A mutant (PCT64) of Pseudomonas AM1, which is unable to grow on C(1) compounds, lacks glycerate kinase, showing that this enzyme is essential for the operation of the serine pathway. Mutant PCT48, unable to convert acetate into glycollate, has lost the ability to grow both on C(1) compounds and on ethanol. The properties of a third mutant (PCT57) show that Pseudomonas AM1 contains enzymes catalysing the conversion of acetate into glyoxylate. Evidence is presented that hydroxypyruvate reductase is involved in the oxidation of glycollate to glyoxylate during growth on ethanol. A scheme is proposed for the conversion of ethanol and of C(1) compounds into glyoxylate in which acetate (or a derivative) and glycollate are intermediates.     

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.     

Metabolism of C2 compounds inAcetobacter aceti

The presence of isocitrate lyase and malate synthase was detected in cell-free extracts ofAcetobacter aceti, grown in a mineral medium with acetate as sole carbon source. The presence of these enzymes explains the ability of this strain to grow with ethanol or acetate as sole carbon source, which is an important characteristic in Frateur's classification system forAcetobacter. In addition to isocitrate lyase and malate synthase, these cell-free extracts were found to contain glyoxylate carboligase, tartronicsemialdehyde reductase and glycerate kinase. The induction of these enzymes during growth on acetate is thought to be caused by the very high activity of isocitrate lyase, which may lead to an accumulation of glyoxylate. The importance of this pathway in cells growing with acetate as sole carbon source for the synthesis of their carbohydrate components is discussed. The presence of the enzymes from the pathway from glyoxylate to 3-phosphoglycerate explains the ability of this strain to grow with ethyleneglycol and glycollate as sole carbon source.     

Study of an alternate glyoxylate cycle for acetate assimilation by Rhodobacter sphaeroides

Organisms, which grow on organic substrates that are metabolized via acetyl-CoA, are faced with the problem to form all cell constituents from this C(2)-unit. The problem was solved by the seminal work of Kornberg and is known as the glyoxylate cycle. However, many bacteria are known to not contain isocitrate lyase, the key enzyme of this pathway. This problem was addressed in acetate-grown Rhodobacter sphaeroides. An acetate-minus mutant identified by transposon mutagenesis was affected in the gene for beta-ketothiolase forming acetoacetyl-CoA from two molecules of acetyl-CoA. This enzyme activity was missing in this mutant, which grew on acetoacetate and on acetate plus glyoxylate. A second acetate/acetoacetate-minus mutant was affected in the gene for a putative mesaconyl-CoA hydratase, an enzyme which catalyses the hydration of mesaconyl-CoA to beta-methylmalyl-CoA. Beta-methylmalyl-CoA is further cleaved into glyoxylate and propionyl-CoA. These results as well as identification of acetate-upregulated proteins by two-dimensional gel electrophoresis lead to the proposal of a new pathway for acetate assimilation. In a first part, affected by the mutations, two molecules of acetyl-CoA and one molecule CO(2) are converted via acetoacetyl-CoA and mesaconyl-CoA to glyoxylate and propionyl-CoA. In a second part glyoxylate and propionyl-CoA are converted with another molecule of acetyl-CoA and CO(2) to l-malyl-CoA and succinyl-CoA.     

Role and control of isocitrate lyase in Candida lipolytica.

Mutants of Candida lipolytica that were unable to grow on acetate but able to utilize succinate or glycerol as a sole carbon source were isolated. Amongst the mutants isolated, one strain (Icl-) was specifically deficient in isocitrate lyase activity, whereas another strain (Acos-) was deficient in acetyl coenzyme A synthetase activity. Since the Icl- mutant could not grow either on n-alkane or its derivatives, such as fatty acid and long-chain dicarboxylic acid, any anaplerotic route other than the glyoxylate pathway was inconceivable as far as growth on these carbon sources was concerned. Acetyl coenzyme A is most likely a metabolic inducer of isocitrate lyase and malate synthase, because the Acos- mutant was characterized by the least susceptibility to induction of these enzymes by acetate. The structural gene for isocitrate lyase was most probably impaired in the Icl- mutant, since revertants (Icl-) produced thermolabile isocitrate lyase. The production of isocitrate from n-alkane by the revertants was enhanced in comparison with the parental strain.     

Acetate metabolism in Rhodopseudomonas gelatinosa and several other Rhodospirillaceae

When Rhodopseudomonas gelatinosa was grown on acetate aerobically in the dark both enzymes of the glyoxylate bypass, isocitrate lyase and malate synthase, could be detected. However, under anaerobic conditions in the light only isocitrate lyase, but not malate synthase, could be found.The reactions, which bypass the malate synthase reaction are those catalyzed by alanine glyoxylate aminotransferase and the enzymes of the serine pathway.Other Rhodospirillaceae were tested for isocitrate lyase and malate synthase activity after growth with acetate; they could be divided into three groups: I. organisms possessing both enzymes; 2. organisms containing malate synthase only; 3. R. gelatinosa containing only isocitrate lyase when grown anaerobically in the light.     

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.     

Regulation of isocitrate lyase in a mutant of Rhodopseudomonas capsulata adapted to growth on acetate

Studies on acetate utilization by Rhodopseudomonas capsulata strain St. Louis indicated that the wild type grew poorly on acetate and made little if any of the glyoxylate cycle enzyme isocitrate lyase. A spontaneous mutant, Ac-l, capable of vigorous and immediate growth on acetate and exhibiting high levels of isocitrate lyase activity, was isolated in the course of those studies.Isocitrate lyase was not formed when the mutant was grown on malate. Addition of malate to cultures of Ac-l growing on acetate resulted in loss of the enzyme by dilution through growth.Starvation of acetate-grown Ac-l for acetate resulted in a rapid and complete loss of isocitrate lyase activity which was shown to be energy dependent. Readdition of acetate to a starved culture previously grown on acetate resulted in a rapid recovery of enzyme activity. The recovery required energy and was sensitive to chloramphenicol inhibition at any time during the recovery phase.     

Metabolic Role, Regulation of Synthesis, Cellular Localization, and Genetic Control of the Glyoxylate Cycle Enzymes in Neurospora crassa

The glyoxylate shunt enzymes, isocitrate lyase and malate synthase, were present at high levels in mycelium grown on acetate as sole source of carbon, compared with mycelium grown on sucrose medium. The glyoxylate shunt activities were also elevated in mycelium grown on glutamate or Casamino Acids as sole source of carbon, and in amino acid-requiring auxotrophic mutants grown in sucrose medium containing limiting amounts of their required amino acid. Under conditions of enhanced catabolite repression in mutants grown in sucrose medium but starved of Krebs cycle intermediates, isocitrate lyase and malate synthase levels were derepressed compared with the levels in wild type grown on sucrose medium. This derepression did not occur in related mutants in which Krebs cycle intermediates were limiting growth but catabolite repression was not enhanced. No Krebs cycle intermediate tested produced an efficient repression of isocitrate lyase activity in acetate medium. Of the two forms of isocitrate lyase in Neurospora, isocitrate lyase-1 constituted over 80% of the isocitrate lyase activity in acetate-grown wild type and also in each of the cases already outlined in which the glyoxylate shunt activities were elevated on sucrose medium. On the basis of these results, it is concluded that the synthesis of isocitrate lyase-1 and malate synthase in Neurospora is regulated by a glycolytic intermediate or derivative. Our data suggest that isocitrate lyase-1 and isocitrate lyase-2 are the products of different structural genes. The metabolic roles of the two forms of isocitrate lyase and of the glyoxylate cycle are discussed on the basis of their metabolic control and intracellular localization.     

Properties of Candida lipolytica mutants with the modified glyoxylate cycle and their ability to produce citric and isocitric acid

Summary Enzyme activities of the tricarboxylic acid (TCA) cycle and the anaplerotic pathways, as well as the cell cytology of two C. lipolytica mutants with the modified glyoxylate cycle and their parent strain were studied during the exponential growth phase on glucose or hexadecane.Among the TCA cycle enzymes, the key enzyme citrate synthase had the highest activity in all three strains grown on both substrates. NAD-dependent isocitrate dehydrogenase had the minimum activity. All strains had well-developed mitochondria.Pyruvate carboxylation was active in the wild strain and mutant 2 grown on glucose, where this reaction is the basic anaplerotic pathway for oxal-acetate synthesis; mutant 1 had actively functioning enzymes for both anaplerotic pathways — pyruvate carboxylase, isocitrate lyase and malate synthase.During hexadecane assimilation, the number of peroxisomes in all strains increased sharply, accompanied by a simultaneous increase in isocitrate lyase activity.The low activities of both isocitrate lyase and pyruvate carboxylase in mutant 2 give reason to believe that this strain has an additional pathway for oxalacetic acid synthesis during the assimilation of n-alkane.     

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.     

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.     

Acetate utilization by a methylotrophic bacterium

The mode of acetate ultilization in the Gram-negative diplococcoid bacterium PAR was investigated. This organism uses the isocitrate lyase-negative serine pathway during growth on C₁-compounds and was found to lack isocitrate lyase on acetate growth also. Enzyme assays revealed the absence of the glycerate and hydroxyaspartate pathways for the metabolism of C₂-compounds. Pulse-labeling experiments indicated the rapid formation of glycine, glutamate, and malate. The rapid formation of malate and the presence of malate synthase suggest that glyoxylate is an intermediate formed from acetate by a means other than the conventional isocitrate cleavage reaction.