Effect of regulatory mutations of sulphur metabolism on the levels of cysteine- and homocysteine-synthesizing enzymes in Neurospora crassa

1. Regulation of four enzymes involved in cysteine and homocysteine synthesis, i.e. cysteine synthase (EC 4.2.99.8), homocysteine synthase (EC 4.1.99.10), cystathionine beta-synthase (EC 2.1.22) and gamma-cystathionase (EC 4.4.1.1) was studied in the wild type and sulphur regulatory mutants of Neurospora crassa. 2. Homocysteine synthase and cystathionine beta-synthase were found to be regulatory enzymes but only the former is under control of the cys-3 - scon system regulating several enzymes of sulphur metabolism, including gamma-cystathionase. 3. The results obtained with the mutants strongly suggest that homocysteine synthase plays a physiological role as an enzyme of the alternative pathway of methionine synthesis. Cysteine synthase activity was similar in all strains examined irrespective of growth conditions. 4. The sconc strain with derepressed enzymes of sulphur metabolism showed an increased pool of sulphur amino acids, except for methionine. Particularly characteristic for this pool is a high content of hypotaurine, a product of cysteine catabolism.     

Energy-Metabolising Enzymes in Brain Regions of Adult and Aging Rats

Abstract: The regional enzyme activities of glucose metabolism in the rat brain were investigated. Hexokinase (EC 2.7.1.1) and pyruvate dehydrogenase (EC 1.2.4.1), key enzymes for glucose metabolism, showed no changes in activity in all the regions studied of the aging brain as compared with the adult brain. However, the activity of d -3-hydroxybutyrate dehydrogenase (EC 1.1.1.30) is low throughout the adult brain and, in contrast with hexokinase and pyruvate dehydrogenase, its activity decreases significantly during aging. Other enzymes that showed significant decreases during aging are aldolase (EC 4.1.2.13), lactate dehydrogenase (EC 1.1.1.27), citrate synthase (EC 4.1.3.7), and NAD⁺-linked isocitrate dehydrogenase (EC 1.1.1.41). The catabolic enzyme in cholinergic metabolism, acetylcholinesterase (EC 3.1.1.7), selected as an example of a non-energy-metabolising enzyme, also showed significant decreases in all regions of the brain in aging, although its highest activity remained in the striatum. These results are discussed with respect to the energy metabolism in various brain regions and their status with aging.     

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.     

Control of nitrogen and carbon metabolism in root nodules

Because legume root nodules have high rates of carbon and nitrogen metabolism, they are ideal for the study of plant physiology, biochemistry and molecular biology. Many plant enzymes involved in carbon and nitrogen assimilation have enhanced activity and enzyme protein in nodules as compared to other plant organs. For all intents and purposes the interior of the root nodule is O₂ limited. Both plant and bacterial components of effective root nodules have unique adaptive features for maximizing carbon and nitrogen metabolism in an O₂-limited environment. Plant glycolysis appears to be shunted to malic acid synthesis with further reductive synthesis to fumarate and succinate. Nodule bacteroids utilize these organic acids for the energy to fuel nitrogenase activity. Activities of the plant enzymes phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), malate dehydrogenase (MDH, EC 1.1.1.37) and aspartate aminotransferase (AAT, EC 2.6.1.1), which are very high in nodules, may mediate the flux of carbon between organic and amino acid pools. Dark CO₂ fixation via nodule PEPC can provide up to 25% of the carbon needed for malate and aspartate synthesis. At least three of the plant proteins showing enhanced expression in root nodules are O₂ regulated. Isolation of alfalfa cDNAs encoding PEPC, AAT, NADH-glutamate synthase (NADH-GOGAT, EC 1.4.1.14) and aldolase (EC 4.1.2.13) will offer new tools to assess molecular events controlling nodule carbon and nitrogen metabolism.     

Rhodothermus marinus: a thermophilic bacterium producing dimeric and hexameric citrate synthase isoenzymes

Two separate citrate synthases from the extremely thermophilic bacterium Rhodothermus marinus have been identified and purified. One of the enzymes is a hexameric protein and is the first thermostable, hexameric citrate synthase to be isolated. The other is a dimeric enzyme, which is also thermostable but possesses both citrate synthase and 2-methyl citrate synthase activities. 2-Methyl citrate synthase uses propionyl-coenzyme A as one of its substrates and in Escherichia coli, for example, it has been implicated in the metabolism of propionate. However, no growth of R. marinus was observed using minimal medium with propionate as the sole carbon source, and both hexameric and dimeric enzymes were produced irrespective of whether propionate was included in the growth medium. The data are discussed with respect to the evolutionary relationships between the known hexameric and dimeric citrate synthases and 2-methyl citrate synthase.     

Allantoin catabolism influences the production of antibiotics in Streptomyces coelicolor

Purines are a primary source of carbon and nitrogen in soil; however, their metabolism is poorly understood in Streptomyces. Using a combination of proteomics, metabolomics, and metabolic engineering, we characterized the allantoin pathway in Streptomyces coelicolor. When cells grew in glucose minimal medium with allantoin as the sole nitrogen source, quantitative proteomics identified 38 enzymes upregulated and 28 downregulated. This allowed identifying six new functional enzymes involved in allantoin metabolism in S. coelicolor. From those, using a combination of biochemical and genetic engineering tools, it was found that allantoinase (EC 3.5.2.5) and allantoicase (EC 3.5.3.4) are essential for allantoin metabolism in S. coelicolor. Metabolomics showed that under these growth conditions, there is a significant intracellular accumulation of urea and amino acids, which eventually results in urea and ammonium release into the culture medium. Antibiotic production of a urease mutant strain showed that the catabolism of allantoin, and the subsequent release of ammonium, inhibits antibiotic production. These observations link the antibiotic production impairment with an imbalance in nitrogen metabolism and provide the first evidence of an interaction between purine metabolism and antibiotic biosynthesis.     

Acceleration of gluconeogenesis from propionate by dl-carnitine in the rat kidney cortex

1. The rate of gluconeogenesis from propionate in rat kidney-cortex slices was stimulated up to 3.5-fold by dl-carnitine and by bicarbonate, and was inhibited by inorganic phosphate or high concentrations of propionate (above 3mm). 2. The stimulatory effect of carnitine was dependent on the bicarbonate concentration and could be replaced at low propionate concentration by addition of 25mm-bicarbonate-carbon dioxide buffer. At low bicarbonate concentration the carnitine concentration can be rate-limiting. 3. All observations are in accordance with the view that the action of carnitine is in principle the same as that established for other fatty acids in other tissues, namely that carnitine promotes the appearance of propionyl-CoA within the mitochondrion by acting as a carrier. 4. The accelerating effects of carnitine and bicarbonate and the inhibitory effect of phosphate can be explained on the basis of the known properties of key enzymes of propionate metabolism, i.e. the reversibility of the reactions leading to the formation of methylmalonyl-CoA from propionyl-CoA. 5. 5mm-Propionate caused a five- to ten-fold fall in the free CoA content of the tissue. This fall can account for the inhibition of respiration and gluconeogenesis caused by high propionate concentration. 6. Relatively large quantities of propionyl-l-carnitine (15% of the propionate removed) were formed when dl-carnitine was present; thus the ;activation' of propionate proceeded at a faster rate than the carboxylation of propionyl-CoA. The metabolism of added propionyl-l-carnitine was accompanied by glucose synthesis. 7. The appearance of radioactivity from [2-(14)C]propionate in both glucose and carbon dioxide was as expected on account of the randomization of C-2 and C-3 of propionate, i.e. the formation of succinate as an intermediate. 8. The maximum rate of glucose synthesis from propionate (93.3+/-3.3mumoles/g. dry wt./hr.) was not affected by dietary changes aimed at varying the rate of caecal volatile fatty acid formation in the rat. 9. Inhibition of gluconeogenesis by high propionate concentration was not found in those species where the rate of caecal or ruminal propionate production is high under normal conditions (rabbit, sheep and cow).     

The Role of Chloride in Acetylcholine Metabolism

Abstract: The chloride dependence of acetylcholine (ACh) synthesis and release and of choline uptake was studied in synaptosomal preparations from rat brain. The substitution of propionate for chloride, in the presence of 35 m m -potassium, lowered the ACh content of the synaptosomes. However, in the presence of 5 m m -potassium, the ACh level in synaptosomes was reduced, but significantly less so. Propionate had no effect on choline acetyltransferase (EC 2.3.1.6) activity when measured in a standard chloride-containing medium. In the presence of propionate, the spontaneous release of ACh was unchanged, but potassium-stimulated release of ACh was markedly reduced as compared with a chloride-containing medium. The synthesis of ACh, as measured by the net increase in the amount of ACh in the synaptosomes and that released to the medium, was reduced with propionate at 5 m m -potassium and was totally inhibited when the potassium concentration was increased to 35 m m . Choline uptake studies revealed that with propionate only a low-affinity component of the choline transport system existed. Further, the V _max was markedly reduced when the potassium concentration was increased to 35 m m . The results suggest that under certain conditions choline transported by a low-affinity system might provide a substantial source of choline for ACh synthesis.     

Enzyme Activities in Regions of the Hypothalamus

Abstract: The activities of certain key enzymes have been measured in the ventral medial and ventral lateral areas of the hypothalamus, which are implicated in feeding behaviour, and compared with enzyme activities in the cortex and brainstem. The enzymes measured are concerned with glucose metabolism [hexokinase (EC 2.7.1.1) and glucosesphosphate dehydrogenase (EC 1.1.1.49)], ketone body metabolism [3-hydroxybutyrate dehydrogenase (EC 1.1.1.30)], fatty acid utilisation [carnitine palmitoyl transferase (EC 2.3.1.7)], citric acid cycle activity [pyruvate dehydrogenase (EC 1.2.4.2) and citrate synthase (EC 4.1.3.7)] and neurotransmitter synthesis [glutamate dehydrogenase (EC 1.4.1.3)].     

Gluconate accumulation and enzyme activities with extremely nitrogen-limited surface cultures of Aspergillus niger

Batch cultures of Aspergillus niger grown from conidia on a medium with high C/N ratio accumulated gluconate from glucose with a yield of 57%. During almost the whole time of accumulation there was no net synthesis of total protein in the mycelium but the activity per flask and the specific activity of glucose oxidase (EC 1.1.3.4) in mycelial extracts increased whereas both values decreased for glucose dehydrogenase (EC 1.1.99.10) gluconate 6-phosphatase (cf. EC 3.1.3.1, 3.1.3.2), gluconokinase (EC 2.7.1.12), glucose 6-phosphate and phosphogluconate dehydrogenases (EC 1.1.1.49, EC 1.1.1.44), phosphoglucomutase (EC 2.7.5.1), and most enzymes of the Embden-Meyerhof pathway and the tricarboxylic acid cycle. Gluconate dehydratase (EC 4.2.1.39), gluconate dehydrogenase (EC 1.1.99.3) and enzymes of the Entner-Doudoroff pathway could not be detected. By cycloheximide the increase of glucose oxidase activity was inhibited. It is concluded that the high yield of gluconate was due mainly to the net (de novo) synthesis of glucose oxidase which occurred during protein turnover after the exhaustion of the nitrogen source, and which was not accompanied by a net synthesis of the other enzymes investigated. Some gluconate may also have been formed by hydrolytic cleavage of gluconate 6-phosphate.Abbreviations GOD glucose oxidase - GD glucose dehydrogenase - PP pentose phosphate - EM Embden-Meyerhof - TCA tricarboxylic acid     

Enzyme des Stärkeumsatzes in den Wurzelhaubenzellen von Zea mays L.

Summary In connection with the problem of the well-known stability of statolith starch, some enzymes of starch metabolism have been investigated qualitatively in the root cap cells of Zea mays L. No activity of granule-bound UDPG- and ADPG-transglucosylase (EC 2.4.1.21) could be found. In the soluble enzyme fraction of the root cap cells, on the other hand, activities of phosphorylase (EC 2.4.1.1), sucrose synthetase (EC 2.4.1.13), UDPG-pyrophosphorylase (EC 2.7.7.9), -Amylase (EC 3.2.11), Maltase (EC 3.2.1.20), and D-enzyme (EC 2.4.1.25) were clearly shown to be present. However, no measurable activities of ADPG-pyrophosphorylase, sucrose-6-phosphate-synthetase (EC 2.4.1.14) and UDPG-dehydrogenase (EC 1.1.1.22) could be found. It is concluded that the stability of statolith starch in the root cap cells is not caused by the lack of enzymes of starch metabolism, but perhaps by a dynamic equilibrium between the degradation and the synthesis of starch. The later could proceed by the activity of phosphorylase working in the direction of starch synthesis because of removal of the inorganic phosphate by phosphorylating mitochondria accumulating in the neighbourhood of the statolith amyloplasts.     

Effect of Anoxia on Carbohydrate Metabolism in Rice Seedlings

The metabolism of carbohydrates was investigated in rice (Oryza sativa L.) seedlings grown under anoxia. Two phases can be recognized in the utilization of carbohydrates: during the first days of germination under anoxia, the metabolism of sugars is mainly degradative, whereas after the induction of [alpha]-amylase (EC 3.2.1.1) has taken place, the increased presence of glucose and sucrose indicates that both starch degradation and sucrose synthesis operate. The analysis of the enzymes involved in carbohydrate metabolism indicates that anoxic rice seedlings possess a set of enzymes that allow the efficient metabolism of starch and sucrose to fructose-6-phosphate. We propose that cytosolic sucrose metabolism in anoxic rice seedlings takes place mainly through a sucrose synthase (EC 2.4.1.13) pathway with nucleoside diphosphate kinase (EC 2.7.4.6), allowing the cycling of urydilates needed for the operation of this pathway.     

Continuous-culture studies on the regulation of tylosin biosynthesis

The metabolic regulation of tylosin synthesis by Streptomyces fradiae NRRL 2702 was studied in batch and chemostat cultures using a soluble synthetic medium. In batch culture a medium which diminished the trophophase-idiophase kinetic pattern was used to assess the activities of the enzymes involved in tylosin synthesis. The enzymes methylmalonyl-coenzyme A carboxyltransferase (EC 2.1.3.1) and propionyl-coenzyme A carboxylase (EC 6.4.1.3) showed early enzymatic derepression, both enzymes reaching their highest specific activities after 72-96 fermentation. The activity of macrocin 3' -O-methyltransferase, the enzyme catalyzing the conversion of macrocin (tylosin C) to tylosin (tylosin A). also peaked at 72 h. The specific activities of the three enzymes showed close correlation with the q(tylosin) value. In chemostat cultures the activities of the enzymes and the intracellular level of the adenylate pool and energy charge were studied as a function of dilution rate. Under steady-state conditions, increases in the specific growth rate repressed the enzymes activities with a concomitant increase in the intracellular level of the adenylate pool, while the adenylate energy charge remained almost constant and in the range 0.5-0.52. The highest specific activities of the enzymes were observed when D = 0.008 h (-1). The specific rate of tylosin synthesis was inversely proportional to the specific growth rate and the intracellular level of adenylate pool. The pool of adenylate could be a nutritional parameter which had a considerable influence on the biosynthesis of tylosin.     

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.     

Use of pathway analysis and genome context methods for functional genomics of Mycoplasma pneumoniae nucleotide metabolism

Elementary modes analysis allows one to reveal whether a set of known enzymes is sufficient to sustain functionality of the cell. Moreover, it is helpful in detecting missing reactions and predicting which enzymes could fill these gaps. Here, we perform a comprehensive elementary modes analysis and a genomic context analysis of Mycoplasma pneumoniae nucleotide metabolism, and search for new enzyme activities. The purine and pyrimidine networks are reconstructed by assembling enzymes annotated in the genome or found experimentally. We show that these reaction sets are sufficient for enabling synthesis of DNA and RNA in M. pneumoniae. Special focus is on the key modes for growth. Moreover, we make an educated guess on the nutritional requirements of this micro-organism. For the case that M. pneumoniae does not require adenine as a substrate, we suggest adenylosuccinate synthetase (EC 6.3.4.4), adenylosuccinate lyase (EC 4.3.2.2) and GMP reductase (EC 1.7.1.7) to be operative. GMP reductase activity is putatively assigned to the NRDI_MYCPN gene on the basis of the genomic context analysis. For the pyrimidine network, we suggest CTP synthase (EC 6.3.4.2) to be active. Further experiments on the nutritional requirements are needed to make a decision. Pyrimidine metabolism appears to be more appropriate as a drug target than purine metabolism since it shows lower plasticity.     

Carbohydrate metabolism in growing rice seedlings under arsenic toxicity

We studied in the seedlings of two rice cultivars (Malviya-36 and Pant-12) the effect of increasing levels of arsenic in situ on the content of sugars and the activity of several enzymes of starch and sucrose metabolism: alpha-amylase (EC 3.2.1.1), beta-amylase (EC 3.2.1.2), starch phosphorylase (EC 2.4.1.1), acid invertase (EC 3.2.1.26), sucrose synthase (EC 2.4.1.13) and sucrose phosphate synthase (EC 2.4.1.14). During a growth period of 10-20 d As2O3 at 25 and 50 microM in the growth medium caused an increase in reducing, non-reducing and total soluble sugars. An increased conversion of non-reducing to reducing sugars was observed concomitant with As toxicity. The activities of alpha-amylase, beta-amylase and sucrose phosphate synthase declined, whereas starch phosphorylase, acid invertase and sucrose synthase were found to be elevated. Results indicate that in rice seedlings arsenic toxicity causes perturbations in carbohydrate metabolism leading to the accumulation of soluble sugars by altering enzyme activity. Sucrose synthase possibly plays a positive role in synthesis of sucrose under As-toxicity.     

Synthesis of pyrophosphate by chromatophores of Rhodospirillum rubrum in the light and by soluble yeast inorganic pyrophosphatase in water-organic solvent mixtures

Chromatophores of Rhodospirillum rubrum contain a membrane-bound pyrophosphatase that synthesizes pyrophosphate when an electrochemical H+ gradient is formed across the chromatophore membrane upon illumination. In this report it is shown that MgCl2 and Pi have different effects on the synthesis of pyrophosphate in the light depending on whether initial velocities or steady-state levels are examined. When the water activity of the medium is reduced by the addition of organic solvents, soluble yeast inorganic pyrophosphatase (no H+ gradient present) synthesizes pyrophosphate in amounts similar to those synthesized by the chromatophores in totally aqueous medium during illumination, (H+ gradient present). The pH, MgCl2 and Pi dependence for the synthesis of pyrophosphate by the chromatophores at steady-state is similar to that observed at equilibrium with the soluble enzyme in the presence of organic solvents. The possibility is raised that a decrease in water activity may play a role in the mechanism by which the energy derived from the electrochemical H+ gradient is used for the synthesis of pyrophosphate in chromatophores of R. rubrum.     

Peroxisomal metabolism of propionic acid and isobutyric acid in plants

The subcellular sites of branched-chain amino acid metabolism in plants have been controversial, particularly with respect to valine catabolism. Potential enzymes for some steps in the valine catabolic pathway are clearly present in both mitochondria and peroxisomes, but the metabolic functions of these isoforms are not clear. The present study examined the possible function of these enzymes in metabolism of isobutyryl-CoA and propionyl-CoA, intermediates in the metabolism of valine and of odd-chain and branched-chain fatty acids. Using (13)C NMR, accumulation of beta-hydroxypropionate from [2-(13)C]propionate was observed in seedlings of Arabidopsis thaliana and a range of other plants, including both monocots and dicots. Examination of coding sequences and subcellular targeting elements indicated that the completed genome of A. thaliana likely codes for all the enzymes necessary to convert valine to propionyl-CoA in mitochondria. However, Arabidopsis mitochondria may lack some of the key enzymes for metabolism of propionyl-CoA. Known peroxisomal enzymes may convert propionyl-CoA to beta-hydroxypropionate by a modified beta-oxidation pathway. The chy1-3 mutation, creating a defect in a peroxisomal hydroxyacyl-CoA hydrolase, abolished the accumulation of beta-hydroxyisobutyrate from exogenous isobutyrate, but not the accumulation of beta-hydroxypropionate from exogenous propionate. The chy1-3 mutant also displayed a dramatically increased sensitivity to the toxic effects of excess propionate and isobutyrate but not of valine. (13)C NMR analysis of Arabidopsis seedlings exposed to [U-(13)C]valine did not show an accumulation of beta-hydroxypropionate. No evidence was observed for a modified beta-oxidation of valine. (13)C NMR analysis showed that valine was converted to leucine through the production of alpha-ketoisovalerate and isopropylmalate. These data suggest that peroxisomal enzymes for a modified beta-oxidation of isobutyryl-CoA and propionyl-CoA could function for metabolism of substrates other than valine.