Biosynthesis of vitamin B-12 in anaerobic bacteria. Experiments with Eubacterium limosum and D-erythrose 14C-labeled in different positions

In anaerobic microorganisms the origin of C atoms 2 and 4-7 of the 5,6-dimethylbenzimidazole moiety of vitamin B-12 is still unknown. In order to tackle this problem we added several 14C-labeled putative precursors to Eubacterium limosum fermentations. The degradation of the isolated vitamin B-12 revealed that only D-erythrose, 14C-labeled in different positions, was efficiently incorporated into the 5,6-dimethylbenzimidazole part. The 5,6-dimethylbenzimidazole obtained from an experiment with D-[U-14C]erythrose was further degraded. It was found that C-2 was unlabeled, whereas half of the label was located in C-5 plus C-6, and the other half in C-4 plus C-7. These results demonstrate that in E. limosum D-erythrose is a precursor of C-atoms 4, 5, 6 and 7 of the 5,6-dimethylbenzimidazole part of vitamin B-12.     

Biosynthesis of riboflavin in Bacillus subtilis: origin of the four-carbon moiety.

We studied the incorporation of [1-13C]ribose and [1,3-13C2]glycerol into the riboflavin precursor 6,7-dimethyl-8-ribityllumazine, using a riboflavin-deficient mutant of Bacillus subtilis. The formation of the pyrazine ring requires the addition of a four-carbon moiety to a pyrimidine precursor. The results show that C-6 alpha, C-6, C-7, and C-7 alpha of 6,7-dimethyl-8-ribityllumazine were biosynthetically equivalent to C-1, C-2, C-3, and C-5 of a pentose phosphate. C-4 of the pentose precursor was lost through an intramolecular skeletal rearrangement. Thus, the last steps in the biosynthesis of 6,7-dimethyl-8-ribityllumazine apparently involve the same mechanism in bacteria as in fungi.     

Microbial synthesis of L-[15N]leucine L-[15N]isoleucine, and L-[3-13C]-and L-[3'-13C]isoleucines studied by nuclear magnetic resonance and gas chromatography-mass spectrometry

The preparation of leucine and isoleucine labeled with 15N and of site-specific 13C-labeled isoleucines is described. This method is based on the induction of the biosynthetic pathways specific for branched chain amino acids in glutamic acid producing bacteria, and controlled provision of stable isotope labeled precursors. Corynebacterium glutamicum (ATCC 13032), a glutamic acid overproducer, was incubated in leucine production medium which consisted of a basal medium supplemented with [15N]ammonium sulfate, glucose, and sodium alpha-ketoisocaproate. production of L-[15N]leucine reached 138 mumol/ml at an isotopic efficiency of 90%. It was purified and checked by proton NMR and GC-MS. The electron impact (EI) spectrum showed 95 atom% enrichment. The cultivation of C. glutamicum in a similar medium containing alpha-ketobutyrate yielded L-[15N]isoleucine at a concentration of 120 mumol/ml. The GC-MS EI and chemical ionization (CI) spectra confirmed enrichment of 96 atom% 15N as that of the labeled precursors. The biosynthesis of L-[13C]isoleucine was carried out by induced cells which were transferred to a similar medium in which [2-13C]- or [3-13C]pyruvic acid replaced glucose. 13C NMR of the product isoleucine revealed single-site enrichment at C-3 or at C-3' respective to the precursor [13C]pyruvate; i.e., C-3 was labeled from [2-13C]pyruvate and C-3' from [3-13C]pyruvate. Mass spectrometric analysis confirmed that all molecules were labeled only in one carbon. This site-specific incorporation of [13C]pyruvate is contrasted with the labeling pattern obtained when producing cells were supplied with [2-13C]acetate, instead of pyruvate, when most label was incorporated into carbons 3 and 3' of the same isoleucine molecule.     

Pentose cycle and reducing equivalents in rat mammary-gland slices

1. Slices of mammary gland of lactating rats were incubated with glucose labelled uniformly with (14)C and in positions 1, 2, 3 and 6, and with (3)H in all six positions. Glucose carbon atoms are incorporated into CO(2), fatty acids, lipid glycerol, the glucose and galactose moieties of lactose, lactate, soluble amino acids and proteins. C-3 of glucose appears in fatty acids. The incorporation of (3)H into fatty acids is greatest from [3-(3)H]glucose. (3)H from [5-(3)H]glucose appears, apart from in lactose, nearly all in water. 2. The specific radioactivity of the galactose moiety of lactose from [1-(14)C]- and [6-(14)C]-glucose was less, and that from [2-(14)C]- and [3-(14)C]-glucose more, than that of the glucose moiety. There was no randomization of carbon atoms in the glucose moiety, but it was extensive in galactose. 3. The pentose cycle was calculated from (14)C yields in CO(2) and fatty acids, and from the degradation of galactose from [2-(14)C]glucose. A method for the quantitative determination of the contribution of the pentose cycle, from incorporation into fatty acids from [3-(14)C]glucose, is derived. The rate of the reaction catalysed by hexose 6-phosphate isomerase was calculated from the randomization pattern in galactose. 4. Of the utilized glucose, 10-20% is converted into lactose, 20-30% is metabolized via the pentose cycle and the rest is metabolized via the Embden-Meyerhof pathway. About 10-15% of the triose phosphates and pyruvate is derived via the pentose cycle. 5. The pentose cycle is sufficient to provide 80-100% of the NADPH requirement for fatty acid synthesis. 6. The formation of reducing equivalents in the cytoplasm exceeds that required for reductive biosynthesis. About half of the cytoplasmic reducing equivalents are probably transferred into mitochondria. 7. In the Appendix a concise derivation of the randomization of C-1, C-2 and C-3 as a function of the pentose cycle is described.     

13C NMR study of the biosynthesis of toxins by Fusarium graminearum

13C NMR spectroscopic investigations on the biosynthesis of mycotoxins produced by Fusarium graminearum (M69) were carried out through the incorporation of [1-13C]- and [2-13C]acetate precursors. The major secondary metabolites produced by this species in still culture were deoxynivalenol (3,7,15-trihydroxy-12,13-epoxytrichothec-9-en-one), 15-acetyldeoxynivalenol, zearalenone, and butenolide. [1-13C]- and [2-13C]acetate were incorporated in alternate carbon atoms in zearalenone, consistent with the head to tail condensation of nine acetate units. The trichothecenes were enriched in a manner consistent with the condensation of three mevalonate units. 13C/13C couplings, observed between C-5 and C-12, as well as between C-6 and C-15 of 15-acetyldeoxynivalenol, confirms the current hypothesis of formation of the trichothecene ring system by cyclization of farnesyl pyrophosphate. The incorporation pattern in ergosterol is also consistent with a mevalonate origin, while the adjacent incorporation of acetate methyl groups in butenolide suggests a glutamate precursor. The degree of enrichment in the secondary metabolites, which ranged from 3 to 10% at each carbon site, was observed in the 13C NMR spectra of the crude fungal extracts to be dependent on the timing of acetate addition to the culture. The specific toxins produced together with the quantity of each, were also found to be dependent on the timing of acetate addition. Competition between the three biosynthetic pathways of secondary metabolism, i.e. polyketide, mevalonate, and amino acid for the labeled acetate in this organism is a complex function of culture conditions.     

Biosynthesis of vitamin B(6) in rhizobium

The biosynthetic pathway of pyridoxol (vitamin B(6)) in Rhizobium was clarified by studies on the incorporation of (13)C- or (15)N-labeled precursors into pyridoxol or its biosynthetic intermediates. Pyridoxol was formed by ring closure of two compounds, 1-deoxy-D-xylulose and 4-hydroxy-L-threonine. The former was formed from D-glyceraldehyde and pyruvate through decarboxylation of pyruvate, and the latter from glycine and glycolaldehyde.     

6-Ethyl-5-hydroxy-2,7-dimethoxy-1,4-naphthoquinone from Hendersonula toruloidea: A biosynthetic study using C labels detected by nuclear magnetic resonance and C tracers

Production of 6-ethyl-5-hydroxy-2,7-dimethoxy-1,4-naphthoquinone was obtained by growth of Hendersonula toruloidea on Czapek-Dox broth supplemented with malt extract. Stationary cultures were grown at 28°C for 21–22 days yielding about 6 mg of metabolite per 700 ml of culture fluid. The best incorporations of isotopic tracers were obtained by addition at the 20th day of growth, followed by harvest 24–48 hr later. With [2-¹⁴C]acetate, incorporation values were in the range of 0.1–0.3% with dilution values from 2000 to 5900. With [1-¹⁴C]propionate, incorporations were much lower (0.04%) and dilutions much higher (120,000). Activity from [¹⁴CH₃]methionine was incorporated only into the OCH₃ groups (incorporation values, 0.5–0.7%). Nuclear magnetic resonance studies confirmed that propionate was not a precursor. Using [1,2-¹³C]acetate, substantial enrichments were obtained at all carbon atoms except those of the OCH₃ groups. The following pairs of carbon atoms were shown to be derived from acetate units: C-1 + 2, C-3 + 4, C-5 + 10, C-6 + 7, C-8 + 9, C-11 + 12. The biosynthetic pathway is clearly that of acetate plus polymalonate. Experiments with [2-¹³C²H₃]acetate suggested that the “starter” acetate unit was located at positions C-12 + 11.     

6-Ethyl-5-hydroxy-2,7-dimethoxy-1,4-naphthoquinone from Hendersonula toruloidea: A biosynthetic study using 13C labels detected by nuclear magnetic resonance and 14C tracers

Production of 6-ethyl-5-hydroxy-2,7-dimethoxy-1,4-naphthoquinone was obtained by growth of Hendersonula toruloidea on Czapek-Dox broth supplemented with malt extract. Stationary cultures were grown at 28°C for 21–22 days yielding about 6 mg of metabolite per 700 ml of culture fluid. The best incorporations of isotopic tracers were obtained by addition at the 20th day of growth, followed by harvest 24–48 hr later. With [2-¹⁴C]acetate, incorporation values were in the range of 0.1–0.3% with dilution values from 2000 to 5900. With [1-¹⁴C]propionate, incorporations were much lower (0.04%) and dilutions much higher (120,000). Activity from [${}^{14}\text{CH}{}_3$]methionine was incorporated only into the OCH₃ groups (incorporation values, 0.5–0.7%). Nuclear magnetic resonance studies confirmed that propionate was not a precursor. Using [1,2-¹³C]acetate, substantial enrichments were obtained at all carbon atoms except those of the OCH₃ groups. The following pairs of carbon atoms were shown to be derived from acetate units: C-1 + 2, C-3 + 4, C-5 + 10, C-6 + 7, C-8 + 9, C-11 + 12. The biosynthetic pathway is clearly that of acetate plus polymalonate. Experiments with [2-¹³C²H₃]acetate suggested that the “starter” acetate unit was located at positions C-12 + 11.     

The activity of the pentose phosphate pathway in isolated liver cells

Isolated liver cells have been used to assess the relative contribution of the pentose phosphate pathway to glucose metabolism. The incorporation of carbon from specifically labelled glucose into ¹⁴CO₂ by isolated cells gave values (μg.atoms/g.cells/hr) of: C-1, 7.9; C-6, 1.3; C-U, 3.4. The corresponding figures for liver slices were: C-1, 2.3; C-6, 1.6; C-U, 3.0. The most striking difference was the 3.5-fold increase in the oxidation of C-1 of glucose. Isolated cells retain more than 50% of ATP and have a content of intermediates of the glycolytic pathway closely similar to freeze-clamped liver. The relative importance of the pentose phosphate pathway in isolated liver cells, approximately 16% of glucose catabolised, is consistent with the enzyme profile of liver and the reductive synthetic reactions of the tissue.     

Glial Formation of Pyruvate and Lactate from TCA Cycle Intermediates: Implications for the Inactivation of Transmitter Amino Acids?

Abstract: Cerebral formation of lactate via the tricarboxylic acid (TCA) cycle was investigated through the labeling of lactate from [2-¹³C]acetate and [1-¹³C]glucose as shown by ¹³C NMR spectroscopy. In fasted mice that had received [2-¹³C]acetate intravenously, brain lactate C-2 and C-3 were labeled at 5, 15, and 30 min, reflecting formation of pyruvate and hence lactate from TCA cycle intermediates. In contrast, [1-¹³C]glucose strongly labeled lactate C-3, reflecting glycolysis, whereas lactate C-2 was weakly labeled only at 15 min. These data show that formation of pyruvate, and hence lactate, from TCA cycle intermediates took place predominantly in the acetate-metabolizing compartment, i.e., glia. The enrichment of total brain lactate from [2-¹³C]acetate reached ∼1% in both the C-2 and the C-3 position in fasted mice. It was calculated that this could account for 20% of the lactate formed in the glial compartment. In fasted mice, there was no significant difference between the labeling of lactate C-2 and C-3 from [2-¹³C]acetate, whereas in fed mice, lactate C-3 was more highly labeled than the C-2, reflecting adaptive metabolic changes in glia in response to the nutritional state of the animal. It is hypothesized that conversion of TCA cycle intermediates into pyruvate and lactate may be operative in the glial metabolism of extracellular glutamate and GABA in vivo. Given the vasodilating effect of lactate on cerebral vessels, which are ensheathed by astrocytic processes, conversion of glutamate and GABA into lactate could be one mechanism mediating increases in cerebral blood flow during nervous activity.     

Ribose biosynthesis in methanogenic bacteria

NMR spectroscopy was used to determine the labeling patterns of the ribose moieties of ribonucleosides purified from Methanospirillum hungatei, Methanococcus voltae, Methanobrevibacter smithii, Methanosphaera stadtmanae, Methanosarcina barkeri and Methanobacterium bryantii labeled with ¹³C-precursors. In most methanogens tested ribose was labeled in a manner consistent with the operation of the oxidative branch of the pentose phosphate pathway. In contrast, transaldolase and transketolase reactions typical of a partial nonoxidative pentose phosphate pathway are hypothesized to explain the different labeling patterns and enrichments of carbon atoms observed in the ribose moiety of Methanococcus voltae. The source of erythrose 4-phosphate needed for the transaldolase reaction proposed in Methanococcus voltae, and for biosynthesis of aromatic amino acids in methanogenic bacteria in general, was assessed. Phenylalanine carbon atom C-7 was labeled by [1-¹³C]pyruvate in Methanospirillum hungatei, Methanococcus voltae, and Methanococcus jannaschii, the only methanogens which incorporated sufficient label from pyruvate for testing. Reductive carboxylation of a triose precursor (derived from pyruvate) to synthesize erythrose 4-phosphate is consistent with the labeling patterns observed in phenylalanine and ribose.Abbreviation TCA Tricarboxylic acid Issued as NRCC Publication No. 37382     

Pyruvate carboxylation as an anaplerotic mechanism in the isolated perfused rat heart.

1. The role of pyruvate carboxylation in the net synthesis of tricarboxylic acid-cycle intermediates during acetate metabolism was studied in isolated rat hearts perfused with [1-14C]pyruvate. 2. The incorporation of the 14C label from [1-14C]pyruvate into the tricarboxylic acid-cycle intermediates points to a carbon input from pyruvate via enzymes in addition to pyruvate dehydrogenase and citrate synthase. 3. On addition of acetate, the specific radioactivity of citrate showed an initial maximum at 2 min, with a subsequent decline in labelling. The C-6 of citrate (which is removed in the isocitrate dehydrogenase reaction) and the remainder of the molecule showed differential labelling kinetics, the specific radioactivity of C-6 declining more rapidly. Since this carbon is lost in the isocitrate dehydrogenase reaction, the results are consistent with a rapid inactivation of pyruvate dehydrogenase after the addition of acetate, which was confirmed by measuring the 14CO2 production from [1-14C]pyruvate. 4. The results can be interpreted to show that carboxylation of pyruvate to the C4 compounds of the tricarboxylic acid cycle occurs under conditions necessitating anaplerosis in rat myocardium, although the results do not identify the enzyme involved. 5. The specific radioactivity of tissue lactate was too low to allow it to be used as an indicator of the specific radioactivity of the intracellular pyruvate pool. The specific radioactivity of alanine was three times that of lactate. When the hearts were perfused with [1-14C]lactate, the specific radioactivity of alanine was 70% of that of pyruvate. The results suggest that a subcompartmentation of lactate and pyruvate occurs in the cytosol.     

Biosynthesis of riboflavin. Enzymatic formation of the xylene moiety from [14C]ribulose 5-phosphate

We have studied the enzymatic formation of the xylene ring of riboflavin using cell extracts from the flavinogenic yeast Candida guilliermondii. 5-Amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione or its 5′-phosphate could serve as substrates. In addition, a pentose phosphate or pentulose phosphate was required. Experiments with [¹⁴C]ribulose 5-phosphate gave evidence for the incorporation of the ribulose carbon atoms except C-4 into the xylene ring of the vitamin.     

The precursors of the xylene ring in riboflavine

1. The nature of the precursors of the xylene ring in riboflavine was reinvestigated with growing as well as resting cells of Eremothecium ashbyii. 2. The incorporation of acetoin into riboflavine was very low; further, [2-(14)C]pyruvate and [1-(14)C]acetate were equally effective as precursors of lumichrome, and pyruvate was much more active as a precursor of acetoin. These results exclude acetoin as a direct precursor of riboflavine. 3. Addition of unlabelled glucose decreased the incorporation of [(14)C]acetate into riboflavine more than it decreased the conversion of acetate into carbon dioxide, indicating that acetate is not a direct riboflavine precursor. 4. The incorporation of various sugars and dilution experiments suggest that a derivative of the intermediates of the pentose phosphate cycle is the precursor of the xylene ring in riboflavine.     

Alternative Pathways of Glucose Utilization in Brain; Changes in the Pattern of Glucose Utilization in Brain Resulting from Treatment of Rats with 6-Aminonicotinamide

The effect of 6-aminonicotinamide (6AN) treatment on the activities of alternative pathways of glucose metabolism in 20-day-old rat brain was evaluated by measurements of yields of 14CO2 from glucose labeled with 14C on carbons 1, 2, 3 + 4, or 6 and uniformly labeled glucose, and from the incorporation of 14C from specifically labeled glucose into lipids by brain slices from cerebral hemispheres and cerebellum. At the highest dose of 6AN used (35 mg/kg body weight) there was a significant decrease in the 14CO2 yields via the pentose phosphate pathway, the glycolytic route, tricarboxylic acid (TCA) cycle, and via the glutamate-gamma-aminobutyric acid pathway. Giving a graded series of doses (20-35 mg 6AN/kg body weight) revealed a hierarchy of responses in which the pentose phosphate pathway, lactate, glyceride-glycerol, and fatty acid formation were most sensitive, followed, in sequence, by the pyruvate dehydrogenase reaction, the glutamate-gamma-aminobutyrate route and, finally, the TCA cycle. The nature of the blocks in the various pathways was examined by the use of metabolite profiles.     

Carbon-13 Nuclear Magnetic Resonance Study of Metabolism of Propionate by Escherichia coli

We have evaluated the use of [1,2-13C2]propionate for the analysis of propionic acid metabolism, based on the ability to distinguish between the methylcitrate and methylmalonate pathways. Studies using propionate-adapted Escherichia coli MG1655 cells were performed. Preservation of the 13C-13C-12C carbon skeleton in labeled alanine and alanine-containing peptides involved in cell wall recycling is indicative of the direct formation of pyruvate from propionate via the methylcitrate cycle, the enzymes of which have recently been demonstrated in E. coli. Additionally, formation of 13C-labeled formate from pyruvate by the action of pyruvate-formate lyase is also consistent with the labeling of pyruvate C-1. Carboxylation of the labeled pyruvate leads to formation of [1,2-13C2]oxaloacetate and to multiply labeled glutamate and succinate isotopomers, also consistent with the flux through the methylcitrate pathway, followed by the tricarboxylic acid (TCA) cycle. Additional labeling of TCA intermediates arises due to the formation of [1-13C]acetyl coenzyme A from the labeled pyruvate, formed via pyruvate-formate lyase. Labeling patterns in trehalose and glycine are also interpreted in terms of the above pathways. The information derived from the [1, 2-13C2]propionate label is contrasted with information which can be derived from singly or triply labeled propionate and shown to be more useful for distinguishing the different propionate utilization pathways via nuclear magnetic resonance analysis.     

Biosynthesis of domoic acid by the diatom Pseudo-nitzschia multiseries

The biosynthesis of the neurotoxin domoic acid (DA) in the diatom Pseudo-nitzschia multiseries was investigated using 13C- and 14C-labelled precursors. The labelling pattern determined by NMR spectroscopy following incorporation of [1,2-13C2]-acetate showed enrichment of every carbon of DA. The enrichment levels were consistent with a biosynthetic pathway involving two different intermediate precursor units. Addition of labelled acetate either early or late during exponential growth gave similar patterns and levels of incorporation. Analysis of the labelling pattern indicated that DA is biosynthesised by condensation of an isoprenoid intermediate with another intermediate derived from the tricarboxylic acid (TCA) cycle. The absence of deuterium at C2 in DA following incorporation of [2-13C, 2H3]-acetate is consistent with alpha-ketoglutarate or a derivative as the TCA cycle-derived intermediate. The different incorporation efficiencies of acetate into the putative precursor intermediates suggest that either each unit is biosynthesized in a different part of the diatom cell, or that the isoprene chain is not assembled by the usual acetate-mevalonate pathway. The latter proposal is supported by the complete absence of deuterium retention in the isoprenoid-derived portion following incorporation of [2-13C, 2H3]-acetate.     

Probing the in vivo biosynthesis of scytonemin, a cyanobacterial ultraviolet radiation sunscreen, through small scale stable isotope incubation studies and MALDI-TOF mass spectrometry

Scytonemin is a dimeric indole phenolic pigment found in the sheaths of many cyanobacteria. This pigment absorbs UV radiation protecting subtending cyanobacterial cells from harmful effects. Based on scytonemin's unique chemical structure, the pathway to its biosynthesis is uncertain, thus motivating the current investigation. Herein, we report the incorporation of both tyrosine and tryptophan into scytonemin, and provide in vivo data supporting the tryptophan origin of the ketone carbon involved in the condensation of the two biosynthetic precursors. This study also reports on the new use of a small-scale, MALDI-TOF mass spectrometry technique to monitor the incorporation of isotopically labeled tyrosine during scytonemin biosynthesis.     

Restoration of glycogenesis in hepatocytes from starved rats.

Hepatocytes isolated from the liver of rats starved for two days synthesized glycogen only when incubated in the presence of both glucose and glucogenic precursors (combinations of alanine, glycerol, pyruvate, lactate or fructose). Unlabeled glucogenic precursors facilitated the incorporation of [U-14C]glucose into glycogen. Unlabeled glucose likewise greatly enhanced glycogen synthesis from isotopically labeled lactate and other glucogenic precursors.In those systems which contained no added endocrines glucose dampened glycogen phosphorylase activity in a cAMP-independent fashion. Fructose is unable to mimic the effects of glucose on glycogen deposition and on glycogen phosphorylase activity.     

Interrelationship and control of glucose metabolism and lipogenesis in isolated fat-cells. Effect of the amount of glucose uptake on the rates of the pentose phosphate cycle and of fatty acid synthesis

In order to study the quantitative relationship between fatty acid synthesis and pentose phosphate-cycle activity under different hormonal and dietary conditions affecting the extent of glucose uptake, cells isolated from rat epididymal adipose tissue were incubated in bicarbonate buffer containing [U-(14)C]-, [1-(14)C]- or [6-(14)C]-glucose. From the amount of glucose taken up, the production of lactate and pyruvate, and the incorporation of (14)C from differently labelled [(14)C]glucose into CO(2), fatty acids and glyceride glycerol, the rates of glucose metabolism via different pathways and the extent of lipogenesis under various experimental conditions were determined. The contribution of the pentose phosphate-cycle to glucose metabolism under normal conditions was calculated to be 8%. Starvation and re-feeding, and the presence of insulin, caused an enhancement of glucose uptake, pentose phosphate-cycle activity and fatty acid synthesis. Plots of both pentose phosphate-cycle activity and fatty acid synthesis versus glucose uptake revealed that the extent of glucose uptake, over a wide range, determines the rates of fatty acid synthesis and glucose metabolism via the pentose phosphate cycle. A balance of formation and production of nicotinamide nucleotides in the cytoplasm was established. The total amount of cytoplasmic NADH and NADPH formed was only in slight excess over the hydrogen equivalents required for the synthesis of fatty acids, glyceride glycerol and lactate. Except in cells from starved animals, the pentose phosphate cycle was found to provide only about 60% of the NADPH required for fatty acid synthesis. The results are discussed with respect to an overall control of the different metabolic and biosynthetic reactions in the fat-cells by the amount of glucose transported into the cell.