Dark metabolism of acetate in Rhodospirillum rubrum cells, grown under photoheterotropic conditions

The mechanism of the dark assimilation of acetate in the photoheterotrophically grown nonsulfur bacterium Rhodospirillum rubrum was studied. Both in the light and in the dark, acetate assimilation in Rsp. rubrum cells, which lack the glyoxylate pathway, was accompanied by the excretion of glyoxylate into the growth medium. The assimilation of propionate was accompanied by the excretion of pyruvate. Acetate assimilation was found to be stimulated by bicarbonate, pyruvate, the C4-dicarboxylic acids of the Krebs cycle, and glyoxylate, but not by propionate. These data implied that the citramalate (CM) cycle in Rsp. rubrum cells grown aerobically in the dark can function as an anaplerotic pathway. This supposition was confirmed by respiration measurements. The respiration of cells oxidizing acetate depended on the presence of CO2 in the medium. The fact that the intermediates of the CM cycle (citramalate and mesaconate) markedly inhibited acetate assimilation but had almost no effect on cell respiration indicative that citramalate and mesaconate are intermediates of the acetate assimilation pathway. The inhibition of acetate assimilation and cell respiration by itaconate was due to its inhibitory effect on propionyl-CoA carboxylase, an enzyme of the CM cycle. The addition of 5 mM itaconate to extracts of Rsp. rubrum cells inhibited the activity of this enzyme by 85%. The data obtained suggest that the CM cycle continues to function in Rsp. rubrum cells that have been grown anaerobically in the light and then transferred to the dark and incubated aerobically.     

Photosynthesis in Rhodospirillum rubrum. II. Photoheterotrophic Carbon Dioxide Fixation

The contribution of the reductive pentose phosphate cycle to the photometabolism of carbon dioxide and to carbon metabolism in Rhodospirillum rubrum grown photoheterotrophically with l-malate as the carbon source is nil, unlike autotrophically grown R. rubrum. Glycolic acid appears to be the first stable product of CO(2) fixation in R. rubrum cultured photoheterotrophically on l-malate. The results obtained in (14)CO(2) fixation experiments suggest that the photometabolism of CO(2) through glycolate into malate is a major pathway of CO(2) fixation in such cells. However, l-malate was a much more efficient precursor of phosphate esters, and of glutamic acid, than was carbon dioxide; l-malate is therefore, in this case, a far more important source of cell carbon than is carbon dioxide.The products of the light-dependent incorporation of CO(2) and of acetate were investigated in R. rubrum grown photoheterotrophically on acetate. Carboxylation reactions and the reductive pentose phosphate cycle are apparently of greater significance in the photometabolism of acetate heterotrophs than in malate heterotrophs; the photometabolism of the acetate photoheterotrophs seems to be intermediate between the photoheterotrophy of malate heterotrophs and strict autotrophy.     

The mechanism of acetate assimilation in purple nonsulfur bacteria lacking the glyoxylate pathway: enzymes of the citramalate cycle in Rhodobacter sphaeroides

The mechanism of acetate assimilation by the purple nonsulfur bacterium Rhodobacter sphaeroides, which lacks the glyoxylate shortcut, has been studied. In a previous work, proceeding from data on acetate assimilation by Rba. sphaeroides cell suspensions, a suggestion was made regarding the operation, in this bacterium, of the citramalate cycle. This cycle was earlier found in Rhodospirillum rubrum in the form of an anaplerotic reaction sequence that operates during growth on acetate instead of the glyoxylate shortcut, which is not present in the latter bacterium. The present work considers the enzymes responsible for acetate assimilation in Rba. sphaeroides. It is shown that this bacterium possesses the key enzymes of the citramalate cycle: citramalate synthase, which catalyzes condensation of acetyl-CoA and pyruvate and, as a result, forms citramalate, and 3-methylmalyl-CoA lyase, which catalyzes the cleavage of 3-methylmalyl-CoA to glyoxylate and propionyl-CoA. The regeneration of pyruvate, which is the acetyl-CoA acceptor in the citramalate cycle, involves propionyl-CoA and occurs via the following reaction sequence: propionyl-CoA (+ CO2) --> methylmalonyl-CoA --> succinyl-CoA --> succinate --> fumarate --> malate --> oxalacetate (- CO2) --> phosphoenolpyruvate --> pyruvate. The independence of the cell growth and the acetate assimilation of CO2 is due to the accumulation of CO2/HCO3- (released during acetate assimilation) in cells to a level sufficient for the effective operation of propionyl-CoA carboxylase.     

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.     

Acetate metabolism in purple non-sulfur bacteria

Abstract The photosynthetic non-sulfur purple bacterium Rhodobacter capsulatus E1F1 can grow on acetate or dl -malate photoheterotrophically under anerobic conditions or chemoheterotrophically in the dark in the presence of dioxygen. Bacterial cells grown under both anaerobic and aerobic conditions exhibited high amounts of the tricarboxylic acid cycle enzymes especially in dark-aerobic cultures. A high activity of isocitrate lyase was found in cells of R. capsulatus E1F1 and, to a lesser extent, in those of R. capsulatus IP2, Rhodobacter sphaeroides and Rhodospirillum rubrum grown photoheterotrophically on acetate under anaerobic conditions. The second enzyme of the glyoxylate shunt, malate synthase, appears to be constitutive. Itaconate, a powerful inhibitor of isocitrate lyase, severely inhibited growth of R. capsulatus, R. rubrum and R. sphaeroides on acetate, thus corroborating a physiological role of the enzyme in acetate metabolism by Rhodospirillaceae.     

An energy-conserving pyruvate-to-acetate pathway in Entamoeba histolytica. Pyruvate synthase and a new acetate thiokinase.

Under anaerobic conditions, cells of Entamoeba histolytica grown with bacteria produce H2 and acetate while cells grown axenically produce neither. Aerobically, acetate is produced and O2 is consumed by amebae from either type of cells. Centrifuged extracts, 2.4 x 106 x g x min, from both types of cells contain pyruvate synthase (EC 1.2.7.1) and an acetate thiokinase which, together, form a system capable of converting pyruvate to acetate. Pyruvate synthase catalyzes the reaction: pyruvate + CoA leads to CO2 + acetyl-CoA + 2E. Electron acceptors which function with this enzyme are FAD, FMN, riboflavin, ferredoxin, and methyl viologen, but not NAD or NADP. The amebal acetate thiokinase catalyzes the reaction acetyl-CoA + ADP + Pi leads to acetate + ATP + CoA. For this apparently new enzyme we suggest the trivial name acetyl-CoA-synthetase (ADP-forming). Extracts from axenic amebae do not contain hydrogenase, but extracts from cells grown with bacteria do. It is postulated that in bacteria-grown amebae electrons generated at the pyruvate synthase step are utilized anaerobically to produce H2 via the hydrogenase and that the acetyl-CoA is converted to acetate in an energy-conserving step catalyzed by amebal acetyl-CoA synthetase. Aerobically, cells grown under either regimen may utilize the energy-conserving pyruvate-to-acetate pathway since O2 then serves as the ultimate electron acceptor.     

Isolation, characterization, and crystallization of ribulosebisphosphate carboxylase from autotrophically grown Rhodospirillum rubrum.

Serial culture of Rhodospirillum rubrum with 2% CO2 in H2 as the exclusive carbon source resulted in a rather large fraction of the soluble protein (greater than 40%) being comprised of ribulosebisphosphate carboxylase (about sixfold higher than the highest value previously reported). Isolation of the enzyme from these cells revealed that it has physical and kinetic properties similar to those previously described for the enzyme derived from cells grown on butyrate. Notably, the small subunit (which is a constituent of the carboxylase from eucaryotes and most procaryotes) was absent in the enzyme from autotrophically grown R. rubrum. Edman degradation of the purified enzyme revealed that the NH2 terminus is free (in contrast to the catalytic subunit of the carboxylase from eucaryotes) and that the NH2-terminal sequence is Met-Asp-Gln-Ser-Ser-Arg-Tyr-Val-Asn-Leu-Ala-Leu-Lys-Glu-Glu-Asp-Leu-Ile-Ala-Gly-Gly-Glx-His-Val-Leu-. Crystals of the enzyme were readily obtained by dialysis against distilled water.     

Derepression of the synthesis of D-ribulose 1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum.

The synthesis of ribulose 1,5-bisphosphate carboxylase/oxygenase in Rhodospirillum rubrum was greatly influenced by the conditions of culture. When grown photolithotrophically in an atmosphere containing low levels of CO2 (1.5 to 2%), enzyme synthesis was derepressed, with the result that the enzyme comprised up to 50% of the soluble protein of the cells as determined by immunological quantitation. This response was not observed when R. rubrum was grown photolithotrophically in an atmosphere of 5% CO2 in hydrogen. Similarly, the derepression of ribulose 1,5-bisphosphate carboxylase/oxygenase was observed in photoheterotrophically (butyrate)-grown cultures only after the HCO3- supply was nearly exhausted. The increase in enzyme activity observed in derepressed cultures was not paralleled by an increase in the in vivo CO2 fixation rate. Apparently, R. rubrum derepresses the synthesis of ribulose 1,5-bisphosphate carboxylase/oxygenase when exposed to low CO2 concentrations to scavenge the limited CO2 available to such cultures.     

Carbon monoxide dehydrogenase from Rhodospirillum rubrum

The carbon monoxide dehydrogenase from the photosynthetic bacterium Rhodospirillum rubrum was purified over 600-fold by DEAE-cellulose chromatography, heat treatment, hydroxylapatite chromatography, and preparative scale gel electrophoresis. In vitro, this enzyme catalyzed a two-electron oxidation of CO to form CO2 as the product. The reaction was dependent on the addition of an electron acceptor. The enzyme was oxygen labile, heat stable, and resistant to tryptic and chymotryptic digestion. Optimum in vitro activity occurred at pH 10.0. A sensitive, hemoglobin-based assay for measuring dissolved CO levels is presented. The in vitro Km for CO was determined to be 110 microM. CO, through an unknown mechanism, stimulated hydrogen evolution in whole cells, suggesting the presence of a reversible hydrogenase in R. rubrum which is CO insensitive in vivo.     

Properties of R-citramalyl-coenzyme A lyase and its role in the autotrophic 3-hydroxypropionate cycle of Chloroflexus aurantiacus

The autotrophic CO(2) fixation pathway (3-hydroxypropionate cycle) in Chloroflexus aurantiacus results in the fixation of two molecules of bicarbonate into one molecule of glyoxylate. Glyoxylate conversion to the CO(2) acceptor molecule acetyl-coenzyme A (CoA) requires condensation with propionyl-CoA (derived from one molecule of acetyl-CoA and one molecule of CO(2)) to beta-methylmalyl-CoA, which is converted to citramalyl-CoA. Extracts of autotrophically grown cells contained both S- and R-citramalyl-CoA lyase activities, which formed acetyl-CoA and pyruvate. Pyruvate is taken out of the cycle and used for cellular carbon biosynthesis. Both the S- and R-citramalyl-CoA lyases were up-regulated severalfold during autotrophic growth. S-Citramalyl-CoA lyase activity was found to be due to l-malyl-CoA lyase/beta-methylmalyl-CoA lyase. This promiscuous enzyme is involved in the CO(2) fixation pathway, forms acetyl-CoA and glyoxylate from l-malyl-CoA, and condenses glyoxylate with propionyl-CoA to beta-methylmalyl-CoA. R-Citramalyl-CoA lyase was further studied. Its putative gene was expressed and the recombinant protein was purified. This new enzyme belongs to the 3-hydroxy-3-methylglutaryl-CoA lyase family and is a homodimer with 34-kDa subunits that was 10-fold stimulated by adding Mg(2) or Mn(2+) ions and dithioerythritol. The up-regulation under autotrophic conditions suggests that the enzyme functions in the ultimate step of the acetyl-CoA regeneration route in C. aurantiacus. Genes similar to those involved in CO(2) fixation in C. aurantiacus, including an R-citramalyl-CoA lyase gene, were found in Roseiflexus sp., suggesting the operation of the 3-hydroxypropionate cycle in this bacterium. Incomplete sets of genes were found in aerobic phototrophic bacteria and in the gamma-proteobacterium Congregibacter litoralis. This may indicate that part of the reactions may be involved in a different metabolic process.     

Nitrogen Fixation by Rhodospirillum rubrum Grown in Nitrogen-limited Continuous Culture

Cell-free extracts of the photosynthetic bacterium Rhodospirillum rubrum were inconsistent in reducing N(2). An internally illuminated fermentor, designed for the continuous culture of this organism on N(2) under nitrogen-limited conditions, produced cells which yielded cell extracts with consistent activity for cell-free N(2) fixation. A nitrogen-limited continuous culture, supplied ammonia rather than N(2), gave cell-free extracts with even more active N(2) fixation. Extracts of cells grown in the fermentor with glutamate nitrogen as the limiting nutrient in continuous culture did not reduce N(2), but whole cells fixed (15)N-enriched N(2). The discovery that cells from ammonia and glutamate nitrogen-limited continuous cultures are capable of N(2) reduction suggests that R. rubrum cells produce the N(2)-reducing enzymes in response to conditions of nitrogen deficiency rather than in response to the presence of N(2). Examination of the effect of the pN(2) on N(2) reduction by cell-free preparations of R. rubrum indicated that the K(N(2)) is approximately 0.071 atm. Cell-free extracts from R. rubrum were tested for their ability to reduce substrates other than N(2).     

Ribulose diphosphate carboxylase from autotrophic microorganisms

Thiobacillus denitrificans was grown anaerobically with nitrate as an acceptor in both sterile and nonsterile media. Ribulose diphosphate carboxylase was stable throughout the exponential growth phase and declined slowly only after cells reached the stationary phase. Reversible inactivation of the carboxylase occurred in extracts as a result of bicarbonate omission. The enzyme was purified 32-fold with excellent recovery of a preparation which was 50 to 60% pure by the criterion of polyacrylamide gel electrophoresis. This purified preparation catalyzed the fixation of 1.25 mumoles of CO(2) per min per mg of protein at pH 8.1 and 30 C, and the molecular weight of ribulose diphosphate carboxylase was approximately 350,000 daltons. A striking biphasic time course of CO(2) fixation that was independent of protein and ribulose diphosphate concentration was observed. The optimal pH of the enzyme assay was fairly broad, ranging from 7 to 8.2. Kinetic dependence upon bicarbonate, ribulose diphosphate, and Mg(2+) was characterized and indicated that bicarbonate and Mg(2+) must combine with enzyme prior to addition of ribulose diphosphate. Antiserum to ribulose diphosphate carboxylase from Hydrogenomonas eutropha was only slightly inhibitory when added to the enzyme from T. denitrificans, and the mixture did not precipitate. Cyanide (4 x 10(-5)m) gave 61% inhibition of the enzyme from T. denitrificans. Ribulose diphosphate carboxylase in extracts of H. eutropha, H. facilis, Chromatium D, Rhodospirillum rubrum, and Chlorella pyrenoidosa were also inhibited to varying extents by cyanide and antiserum to the H. eutropha enzyme.     

Propionyl-coenzyme A synthase from Chloroflexus aurantiacus, a key enzyme of the 3-hydroxypropionate cycle for autotrophic CO2 fixation

The 3-hydroxypropionate cycle has been proposed as a new autotrophic CO(2) fixation pathway for the phototrophic green non-sulfur eubacterium Chloroflexus aurantiacus and for some chemotrophic archaebacteria. The cycle requires the reductive conversion of the characteristic intermediate 3-hydroxypropionate to propionyl-CoA. The specific activity of the 3-hydroxypropionate-, CoA-, K(+)-, and MgATP-dependent oxidation of NADPH in autotrophically grown cells was 0.09 micromol min(-1) mg(-1) protein, which was 2-fold down-regulated in heterotrophically grown cells. Unexpectedly, a single enzyme catalyzes the entire reaction sequence: 3-hydroxypropionate + MgATP + CoA + NADPH + H(+) --> propionyl-CoA + MgAMP + PP(i) + NADP(+) + H(2)O. The enzyme was purified 30-fold to near homogeneity and has a very large native molecular mass between 500 and 800 kDa, with subunits of about 185 kDa as judged by SDS-PAGE, suggesting a homotrimeric or homotetrameric structure. Upon incubation of this new enzyme, termed propionyl-CoA synthase, with the proteinase trypsin, the NADPH oxidation function of the enzyme was lost, whereas the enzyme still activated 3-hydroxypropionate to its CoA-thioester and dehydrated it to acrylyl-CoA. SDS-PAGE revealed that the subunits of propionyl-CoA synthase had been cleaved once and the N-terminal amino acid sequences of the two trypsin digestion products were determined. Two parts of the gene encoding propionyl-CoA synthase (pcs) were identified on two contigs of an incomplete genome data base of C. aurantiacus, and the sequence of the pcs gene was completed. Propionyl-CoA synthase is a natural fusion protein of 201 kDa consisting of a CoA ligase, an enoyl-CoA hydratase, and an enoyl-CoA reductase, the reductase domain containing the trypsin cleavage site. Similar polyfunctional large enzymes are common in secondary metabolism (e.g. polyketide synthases) but rare in primary metabolism (e.g. eukaryotic type I fatty acid synthase). These results lend strong support to the operation of the proposed pathway in autotrophic CO(2) fixation.     

Characterization of four variant forms of human propionyl-CoA carboxylase expressed in Escherichia coli

Propionyl-CoA carboxylase (PCC) is a biotin-dependent mitochondrial enzyme that catalyzes the conversion of propionyl-CoA to D-methylmalonyl-CoA. PCC consists of two heterologous subunits, alpha PCC and beta PCC, which are encoded by the nuclear PCCA and PCCB genes, respectively. Deficiency of PCC results in a metabolic disorder, propionic acidemia, which is sufficiently severe to cause neonatal death. We have purified three PCCs containing pathogenic mutations in the beta subunit (R165W, E168K, and R410W) and one PCCB polymorphism (A497V) to homogeneity to elucidate the potential structural and functional effects of these substitutions. We observed no significant difference in Km values for propionyl-CoA between wild-type and the variant enzymes, which indicated that these substitutions had no effect on the affinity of the enzyme for this substrate. Furthermore, the kinetic studies indicated that mutation R410W was not involved in propionyl-CoA binding in contrast to a previous report. The three mutant PCCs had half the catalytic efficiency of wild-type PCC as judged by the kcat/Km ratios. No significant differences have been observed in molecular mass or secondary structure among these enzymes. However, the variant PCCs were less thermostable than the wild-type. Following incubation at 47 degrees C, blue native-PAGE revealed a lower oligomeric form (alpha2beta2) in the three mutants not detectable in wild-type and the polymorphism. Interestingly, the lower oligomeric form was also observed in the corresponding crude Escherichia coli extracts. Our biochemical data and the structural analysis using a beta PCC homology model indicate that the pathogenic nature of these mutations is more likely to be due to a lack of assembly rather than disruption of catalysis. The strong favorable effect of the co-expressed chaperone proteins on PCC folding, assembly, and activity suggest that propionic acidemia may be amenable to chaperone therapy.     

Carbonyl sulfide inhibition of CO dehydrogenase from Rhodospirillum rubrum

Carbonyl sulfide (COS) has been investigated as a rapid-equilibrium inhibitor of CO oxidation by the CO dehydrogenase purified from Rhodospirillum rubrum. The kinetic evidence suggests that the inhibition by COS is largely competitive versus CO (Ki = 2.3 microM) and uncompetitive versus methylviologen as electron acceptor (Ki = 15.8 microM). The data are compatible with a ping-pong mechanism for CO oxidation and COS inhibition. Unlike the substrate CO, COS does not reduce the iron-sulfur centers of dye-oxidized CO dehydrogenase and thus is not an alternative substrate for the enzyme. However, like CO, COS is capable of protecting CO dehydrogenase from slow-binding inhibition by cyanide. A true binding constant (KD) of 2.2 microM for COS has been derived on the basis of the saturable nature of COS protection against cyanide inhibition. The ability of CO, CO2, COS, and related CO/CO2 analogues to reverse cyanide inhibition of CO dehydrogenase is also demonstrated. The kinetic results are interpreted in terms of two binding sites for CO on CO dehydrogenase from R. rubrum.     

Fermentative metabolism of pyruvate by Rhodospirillum rubrum after anaerobic growth in darkness.

Rhodospirillum rubrum grew anaerobically in darkness and fermented sodium pyruvate by a pyruvate formate-lyase reaction. During 30 min of anaerobic dark or light incubation with sodium pyrivate, crude extracts from fermentatively grown cells produced about 6 micronmol of acetylphosphate and formate per mg of protein in reactions performed at pH 8.3. Cell extracts also catalyzed the exchange of sodium [14C]formate into sodium pyruvate at an apparent pH optimum of 7.3 to 7.5, but only about 2.5 micronmol of acetylphosphate was produced at this lower pH value. R. rubrum may also form pyruvate:ferredoxin oxidoreductase activity, as evidenced by low bicarbonate exchange activity. However, its participation in pyruvate metabolism in anaerobic dark-grown cells was not understood. During anaerobic, dark growth with pyruvate, formate was an intermediate in H2 and CO2 gas evolution. In contrast with H2 production by a light-dependent H2-nitrogenase system in photosynthetically grown cells, H2 formation in fermenting R. rubrum occurred through a carbon monoxide-sensitive formic hydrogenlyase reaction not influenced by light.     

Pyruvate:quinone oxidoreductase from Corynebacterium glutamicum: purification and biochemical characterization

Pyruvate:quinone oxidoreductase catalyzes the oxidative decarboxylation of pyruvate to acetate and CO2 with a quinone as the physiological electron acceptor. So far, this enzyme activity has been found only in Escherichia coli. Using 2,6-dichloroindophenol as an artificial electron acceptor, we detected pyruvate:quinone oxidoreductase activity in cell extracts of the amino acid producer Corynebacterium glutamicum. The activity was highest (0.055 +/- 0.005 U/mg of protein) in cells grown on complex medium and about threefold lower when the cells were grown on medium containing glucose, pyruvate, or acetate as the carbon source. From wild-type C. glutamicum, the pyruvate:quinone oxidoreductase was purified about 180-fold to homogeneity in four steps and subjected to biochemical analysis. The enzyme is a flavoprotein, has a molecular mass of about 232 kDa, and consists of four identical subunits of about 62 kDa. It was activated by Triton X-100, phosphatidylglycerol, and dipalmitoyl-phosphatidylglycerol, and the substrates were pyruvate (kcat=37.8 +/- 3 s(-1); Km=30 +/- 3 mM) and 2-oxobutyrate (kcat=33.2 +/- 3 s(-1); Km=90 +/- 8 mM). Thiamine pyrophosphate (Km=1 microM) and certain divalent metal ions such as Mg2+ (Km=29 microM), Mn2+ (Km=2 microM), and Co2+ (Km=11 microM) served as cofactors. In addition to several dyes (2,6-dichloroindophenol, p-iodonitrotetrazolium violet, and nitroblue tetrazolium), menadione (Km=106 microM) was efficiently reduced by the purified pyruvate:quinone oxidoreductase, indicating that a naphthoquinone may be the physiological electron acceptor of this enzyme in C. glutamicum.     

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.     

On the mechanism of action of the antifungal agent propionate.

Propionate is used to protect bread and animal feed from moulds. The mode of action of this short-chain fatty acid was studied using Aspergillus nidulans as a model organism. The filamentous fungus is able to grow slowly on propionate, which is oxidized to acetyl-CoA via propionyl-CoA, methylcitrate and pyruvate. Propionate inhibits growth of A. nidulans on glucose but not on acetate; the latter was shown to inhibit propionate oxidation. When grown on glucose a methylcitrate synthase deletion mutant is much more sensitive towards the presence of propionate in the medium as compared to the wild-type and accumulates 10-fold higher levels of propionyl-CoA, which inhibits CoA-dependent enzymes such as pyruvate dehydrogenase, succinyl-CoA synthetase and ATP citrate lyase. The most important inhibition is that of pyruvate dehydrogenase, as this affects glucose and propionate metabolism directly. In contrast, the blocked succinyl-CoA synthetase can be circumvented by a succinyl-CoA:acetate/propionate CoA-transferase, whereas ATP citrate lyase is required only for biosynthetic purposes. In addition, data are presented that correlate inhibition of fungal polyketide synthesis by propionyl-CoA with the accumulation of this CoA-derivative. A possible toxicity of propionyl-CoA for humans in diseases such as propionic acidaemia and methylmalonic aciduria is also discussed.