Use of 13C in biosynthetic studies. The labelling pattern in tenellin enriched from isotope-labelled acetate, methionine, and phenylalanine

The biogenetic origin of the carbon atoms in tenellin has been established by adding 13C-enriched compounds to cultures of Beauveria bassiana, and determining the isotopic distribution in the metabolite by 13C nuclear magnetic resonance spectrometry. Tenellin is formed by condensation of an acetate-derived polyketide chain with a phenylpropanoid unit that may be phenylalanine. Alternate carbon atoms of the polyketide chain were labelled with sodium [1(-13C)]- and [2-(13C]-acetate; sodium [1,2-(13C)]acetate was incorporated as intact two-carbon units, the presence of which in tenellin was apparent from coupling between adjacent 13C-enriched carbons. Substituent methyl groups of the polyketide-derived alkenyl chain were labelled with L-[Me-13C]methionine. The labelling patterns from DL-[carboxy-13C]phenylalanine and DL-[alpha-13C]phenylalanine indicated a rearrangement of the propanoid component at some stage in the synthesis. The mass spectrum of tenellin from cultures administered L-[15N]phenylalanine showed isotopic enrichment similar to that obtained with 13C- or 14C-labelled phenylalanine. During incorporation of L-[carboxy-14C, beta-3H]phenylalanine 96% of the tritium label was lost, discounting the possibility of a 1,2-hydride shift during biosynthesis of the metabolite.     

13C-NMR study of autotrophic CO2 fixation pathways in the sulfur-reducing Archaebacterium Thermoproteus neutrophilus and in the phototrophic Eubacterium Chloroflexus aurantiacus.

The unresolved autotrophic CO2 fixation pathways in the sulfur-reducing Archaebacterium Thermoproteus neutrophilus and in the phototrophic Eubacterium Chloroflexus aurantiacus have been investigated. Autotrophically growing cultures were labelled with [1,4-13C1]succinate, and the 13C pattern in cell constituents was determined by 1H- and 13C-NMR spectroscopy of purified amino acids and other cell constituents. In both organisms succinate contributed to less than 10% of cell carbon, the major part of carbon originated from CO2. All cell constituents became 13C-labelled, but different patterns were observed in the two organisms. This proves that two different cyclic CO2 fixation pathways are operating in autotrophic carbon assimilation in both of which succinate is an intermediate. The 13C-labelling pattern in T. neutrophilus is consistent with the operation of a reductive citric acid cycle and rules out any other known autotrophic CO2 fixation pathway. Surprisingly, the proffered [1,4-13C1]succinate was partially converted to double-labelled [3,4-13C2]glutamate, but not to double-labelled aspartate. These findings suggest that the conversion of citrate to 2-oxoglutarate is readily reversible under the growth conditions used, and a reversible citrate cleavage reaction is proposed. The 13C-labelling pattern in C. aurantiacus disagrees with any of the established CO2 fixation pathways; it therefore demands a novel autotrophic CO2 fixation cycle in which 3-hydroxypropionate and succinate are likely intermediates. The bacterium excreted substantial amounts of 3-hydroxypropionate (5 mM) and succinate (0.5 mM) at the end of autotrophic growth. Autotrophically grown Chloroflexus cells contained acetyl-CoA carboxylase and propionyl-CoA carboxylase activity. These enzymes are proposed to be the main CO2-fixing enzymes resulting in malonyl-CoA and methylmalonyl-CoA formation; from these carboxylation products 3-hydroxypropionate and succinate, respectively, can be formed.     

Biosynthesis of chloramphenicol. Studies on the origin of the dichloroacetyl moiety

Chloramphenicol produced by cultures of Streptomyces species 3022a supplemented with sodium [1,2-13C]acetate was labelled with 13C exclusively in the dichloromethine (2.6 +/- 0.1%) and carbonyl (0.59 +/- 0.05% carbon atoms. Satellite signals from 13C-13C coupling between covalently bonded 13C-enriched carbon atoms were too intense to be attributed to random combination of labelled atoms at the average enrichments measured, but their intensity relative to those of the signals for uncoupled 13C atoms indicated that most of the precursor had been incorporated after 13C-13C bond fission. Since [2,3-13c]succinic acid enriched only the carbonyl carbon atom of chloramphenicol, these results suggest that neither acetate nor a Krebs cycle intermediate is a direct precursor of the dichloroacetyl group. Cultures supplemented with [2-3h]-or [2h2]-dichloroacetic acid incorporated negligible amounts of isotope into the antibiotic; on this evidence, the free acid is not an intermediate in chloramphenicol biosynthesis and the acylation step may precede chlorination.     

Purification and characterization of cytosolic aldolase from carrot storage root.

The time courses of incorporation of 13C from 13C-labelled glucose or acetate into cerebral amino acids (glutamate, glutamine and 4-aminobutyrate) and lactate were monitored by using 13C-n.m.r. spectroscopy. When [1-13C]glucose was used as precursor the C-2 of 4-aminobutyrate was more highly labelled than the analogous C-4 of glutamate, whereas no label was observed in glutamine. A similar pattern was observed with [2-13C]glucose: the C-1 of 4-aminobutyrate was more highly labelled than the analogous C-5 of glutamate. Again, no labelling of glutamine was detected. In contrast, [2-13C]acetate labelled the C-4 of glutamine and the C-2 of 4-aminobutyrate more highly than the C-4 of glutamate; [1-13C]acetate also labelled the C-1 and C-5 positions of glutamine more than the analogous positions of glutamate. These results are consistent with earlier patterns reported from the use of 14C-labelled precursors that led to the concept of compartmentation of neuronal and glial metabolism and now provide the possibility of distinguishing differential effects of metabolic perturbations on the two pools simultaneously. An unexpected observation was that citrate is more highly labelled from acetate than from glucose.     

Impact of the gliotoxin L-serine-O-sulphate on cellular metabolism in cultured rat astrocytes

L-serine-O-sulphate is a member of a group of amino acids collectively called gliotoxins and is a substrate for the high affinity sodium-dependent glutamate transporters. Previous studies have shown that it is toxic to primary cultures of astrocytes but the mode of toxicity is unknown. The current study demonstrates that L-serine-O-sulphate, at a sub-toxic concentration (400 microM), causes significant disruption to glucose and alanine metabolism in cultures of rat cortical astrocytes. More specifically, using (13)C NMR spectroscopy a significant reduction in labelled end products from [1-(13)C]glucose and [3-(13)C]alanine was found in the presence of L-serine-O-sulphate. Additionally, using [2-(13)C]glycine a 27% reduction in de novo glutathione synthesis was observed in the presence of the gliotoxin. Incubation of the cells with L-serine-O-sulphate reduced the activity of alanine and aspartate aminotransferase by 53% and 67%, respectively. Collectively these results show that the gliotoxin, L-serine-O-sulphate, causes major disruptions to metabolic pathways in primary cultures of astrocytes.     

Delayed labelling of brain glutamate after an intra-arterial [13C]glucose bolus: evidence for aerobic metabolism of guinea pig brain glycogen store.

Glycogen in glial cells is the largest store of glucose equivalents in the brain. Here we describe evidence that brain glycogen contributes to aerobic energy metabolism of the guinea pig brain in vivo. Five min after an intra-arterial bolus injection of d-[U-14C]glucose, 28+/-11% of the radioactivity in brain tissue was associated with the glycogen fraction, indicating that a significant proportion of labelled glucose taken up by the brain is converted to glycogen shortly after bolus infusion. Incorporation of 13C-label into lactate generated by brains made ischaemic after d-[1-13C]glucose injection confirms that these glucose equivalents can be mobilised for anaerobic glucose metabolism. Aerobic metabolism was monitored by following the time course of 13C-incorporation into glutamate in guinea pig cortex and cerebellum in vivo. After an intra-arterial bolus injection of d-[1-13C]glucose, glutamate labelling reached a maximum 40-60 min after injection, suggesting that a slowly metabolised pool of labelled glucose equivalents was present. As the concentration of 13C-labelled glucose in blood was shown to decrease below detectable levels within 5 min of bolus injection, this late phase of glutamate labelling must occur with mobilisation of a brain storage pool of labelled glucose equivalents. We interpret this as evidence that glucose equivalents in glycogen may contribute to energy metabolism in the aerobic guinea pig brain.     

Carbon flux through citric acid cycle pathways in perfused heart by 13C NMR spectroscopy

Mathematical models of the TCA cycle derived previously for 14C tracer studies have been extended to 13C NMR to measure the 13C fractional enrichment of [2-13C]acetyl-CoA entering the cycle and the relative activities of the oxidative versus anaplerotic pathways. The analysis is based upon the steady-state enrichment of 13C into the glutamate carbons. Hearts perfused with [2-13C]acetate show low but significant activity of the anaplerotic pathways. Activation of two different anaplerotic pathways is demonstrated by addition of unlabeled propionate or pyruvate to hearts perfused with [2-13C]acetate. In each case, the amount of [2-13C]acetate being oxidized and the relative carbon flux through anaplerotic versus oxidative pathways are evaluated.     

Biosynthetic studies on the tropane ring system of the tropane alkaloids from Datura stramonium

Isotopic labelling experiments have been carried out in Datura stramonium root cultures with the following isotopically labelled precursors; [2H3]- [2-13C, 2H3]-, [1-13C, 18O2]-acetates, 2H2O, [2H3-methyl]-methionine, [2-13C]-phenyllactate, [3-2H]-tropine and [2'-13C, 3-2H]-littorine. The study explored the incorporation of isotope into the tropane ring system of littorine 1 and hyoscyamine 2 and revealed that deuterium from acetate is incorporated only into C-6 and C-7, and not into C-2 and C-4 as previously reported. Oxygen-18 was not retained at a detectable level into the C(3)-O bond from [1-13C, 18O2]-acetate. The intramolecular nature of the rearrangement of littorine 1 to hyoscyamine 2 is revealed again by a labelling study using [2'-13C, 3-2H]-littorine, [2-13C]-phenyllactate and [3-2H]-tropine.     

Resonance Raman study on the flavin in the purple intermediates of D-amino acid oxidase

Resonance Raman (RR) spectra were measured for the purple intermediates of D-amino acid oxidase reconstituted with isotopically labelled FAD's, i.e., [4a-13C]-, [4,10a-13C2]-, [2-13C]-, [5-15N]-, and [1,3-15N2]flavin adenine dinucleotides, and compared with those with the native enzyme. The RR lines around 1605 cm-1 with D-alanine or D-proline as a substrate and at 1548 cm-1 with D-alanine undergo isotopic shifts upon [4a-13C]- and [4,10a-13C2]-labelling. These lines are assigned to the vibrational modes associated with C(10a) = C(4a) - C(4) = O moiety of reduced flavin, providing the first assignment of RR lines of reduced flavin and conclusive evidence that reduced flavin is involved in this intermediate.     

A 13C-NMR study on the influxes into the tricarboxylic acid cycle of a renal epithelial cell line, LLC-PK1/Cl4: the metabolism of [2-13C]glycine, L-[3-13C]alanine and L-[3-13C]aspartic acid in renal epithelial cells

Perchloric acid extracts of LLC-PK1/Cl4 cells, a renal epithelial cell line, incubated with either [2-13C]glycine L-[3-13C]alanine, or D,L-[3-13C]aspartic acid were investigated by 13C-NMR spectroscopy. All amino acids, except labelled glycine, gave rise to glycolytic products and tricarboxylic acid cycle (TCA) intermediates. For the first time we also observed activity of gamma-glutamyltransferase activity and glutathione synthetase activity in LLC-PK1 cells, as is evident from enrichment of reduced glutathione. Time courses showed that only 6% of the labelled glycine was utilized in 30 min, whereas 31% of L-alanine and 60% of L-aspartic acid was utilized during the same period. 13C-NMR was also shown to be a useful tool for the determination of amino acid uptake in LLC-PK1 cells. These uptake experiments indicated that glycine, alanine and aspartic acid are transported into Cl4 cells via a sodium-dependent process. From the relative enrichment of the glutamate carbons, we calculated the activity of pyruvate dehydrogenase to be about 61% when labelled L-alanine was the only carbon source for LLC-PK1/Cl4 cells. Experiments with labelled D,L-aspartic, however, showed that about 40% of C-3-enriched oxaloacetate (arising from a de-amination of aspartic acid) reached the pyruvate pool.     

13C NMR evidence for bacteriochlorophyll c formation by the C5 pathway in green sulfur bacterium, Prosthecochloris

The 13C NMR spectra of the pheophorbide of bacteriochlorophyll c, formed in the presence of L-[1-13C]glutamate and [2-13C]glycine and [13C]bicarbonate in Prosthecochloris aestaurii, were analysed. The isotope in the glutamate was specifically incorporated into the eight carbon atoms in the tetrapyrrole macrocycle derived from the C-5 of 5-aminolevulinic acid, while no specific enrichment of these eight carbon atoms was observed in the spectrum of the pigment formed in the presence of [2-13C]glycine. These labelling patterns provide evidence for the operation of the C5 pathway of 5-aminolevulinic acid synthesis for bacteriochlorophyll c formation in the bacterium. The labelling of bacteriochlorophyll c by [13C]bicarbonate is consistent with its formation from 5-[1,4,5-13C]aminolevulinic acid formed by the C5 pathway from [1,2,5-13C]glutamic acid. It is proposed that this glutamate is the transamination product of 2-[1,2,5-13C]oxoglutaric acid, arising by carboxylation of [1,4-13C]succinyl-CoA with 13CO2 catalysed by 2-oxoglutaric acid synthase activity, and that the labelled succinyl-CoA is, in turn, derived by the fixation of 13CO2 by the reductive tricarboxylic acid cycle. The 13C chemical shifts of two sp2 quaternary carbons of bacteriopheophorbide c methyl ester (C-2 and C-4) were reassigned.     

Brain metabolism of exogenous pyruvate

Pyruvate given in large doses may be neuroprotective in stroke, but it is not known to what degree the brain metabolizes pyruvate. Intravenous injection of [3-13C]pyruvate led to dose-dependent labelling of cerebral metabolites so that at 5 min after injection of 18 mmoles [3-13C]pyruvate/kg (2 g sodium pyruvate/kg), approximately 20% of brain glutamate and GABA were labelled, as could be detected by 13C nuclear magnetic resonance spectrometry ex vivo. Pyruvate, 9 mmoles/kg, was equivalent to glucose, 9 mmoles/kg, as a substrate for cerebral tricarboxylic acid (TCA) cycle activity. Inhibition of the glial TCA cycle with fluoroacetate did not affect formation of [4-13C]glutamate or [2-13C]GABA from [3-13C]pyruvate, but reduced formation of [4-13C]glutamine by 50%, indicating predominantly neuronal metabolism of exogenous pyruvate. Extensive formation of [3-13C]lactate from [2-13C]pyruvate demonstrated reversible carboxylation of pyruvate to malate and equilibration with fumarate, presumably in neurones, but anaplerotic formation of TCA cycle intermediates from exogenous pyruvate could not be detected. Too rapid injection of large amounts of pyruvate led to seizure activity, respiratory arrest and death. We conclude that exogenous pyruvate is an excellent energy substrate for neurones in vivo, but that care must be taken to avoid the seizure-inducing effect of pyruvate given in large doses.     

Labelling of chlorophylls and precursors by [2-14C]glycine and 2-[1-14C]oxoglutarate in Rhodopseudomonas spheroides and Zea mays. Resolution of the C5 and Shemin pathways of 5-aminolaevulinate biosynthesis by thin-layer radiochromatography

The C-5 of 5-aminolaevulinate, a tetrapyrrole precursor which accumulates when inhibitory laevulinate is present, is derived from either the C-2 of glycine by the 5-aminolaevulinate-synthase-mediated Shemin pathway or the C-1 of 2-oxoglutarate by the C5 pathway. Thin-layer-radiochromatographic procedures are described for determining whether [2-14C]glycine or 2-[1-14C]oxoglutarate labelled the macrocycle of bacteriochlorophyll a, in addition to or rather than the methyl ester or phytyl ester moieties of the side-chains. The method was also used for detecting whether the same substrates label the formaldehyde (C-5) or the succinate (C-1 to C-4) fragments, obtained by periodate cleavage of 5-aminolaevulinate. These methods therefore can readily distinguish between the Shemin and C5 pathways as was demonstrated by using Rhodopseudomonas spheroides and Zea mays (maize), respectively, as examples of each pathway. Both [2-14C]glycine and, to a lesser extent 2-[1-14C]oxoglutarate labelled the macrocycle of bacteriochlorophyll a formed during adaptation of respiring R. spheroides cells to photosynthetic (anaerobic, illuminated) conditions. This and earlier evidence suggested augmentation of the Shemin pathway by a minor C5 pathway contribution. The present studies revealed only Shemin pathway activity: with laevulinate present, [2-14C]glycine formed 5-[5-14C]aminolaevulinate as proved by H14CHO production during periodate cleavage. These methods were sufficiently sensitive also to detect the incorporation of 14CO2, from degradation of either substrate, into 5-aminolaevulinate via the Shemin pathway thus labelling the succinate fragment produced with periodate: this explains bacteriochlorophyll a labelling by 2-[1-14C]oxoglutarate and proves double labelling of 5-aminolaevulinate by [2-14C]glycine. The same techniques were applied to etiolated maize leaves exposed to aerobic illuminated conditions with laevulinate and either 2-[1-14C]oxoglutarate or [2-14C]glycine as substrates. Only the C5 pathway was detected: 2-[1-14C]oxoglutarate was converted to 5-[5-14C]aminolaevulinate, which yielded H14CHO on periodate cleavage. This is not inconsistent with our earlier 13C-NMR studies [Porra, R.J., Klein, O. and Wright, P. E. (1983) Eur. J. Biochem. 130, 509-516] showing that the C5 pathway formed all the 5-aminolaevulinate for chlorophyll biosynthesis.(ABSTRACT TRUNCATED AT 400 WORDS)     

Breath [13CO2] recovery from an oral glucose load during exercise: comparison between [U-13C] and [1,2-13C]glucose.

The purpose of the present experiment was to compare 13CO2 recovery at the mouth, and the corresponding exogenous glucose oxidation computed, during a 100-min exercise at 63 +/- 3% maximal O2 uptake with ingestion of glucose (1.75 g/kg) in six active male subjects, by use of [U-13C] and [1,2-13C]glucose. We hypothesized that 13C recovery and exogenous glucose oxidation could be lower with [1,2-13C] than [U-13C]glucose because both tracers provide [13C]acetate, with possible loss of 13C in the tricarboxylic acid (TCA) cycle, but decarboxylation of pyruvate from [U-13C]glucose also provides 13CO2, which is entirely recovered at the mouth during exercise. The recovery of 13C (25.8 +/- 2.3 and 27.4 +/- 1.2% over the exercise period) and the amounts of exogenous glucose oxidized computed were not significantly different with [1,2-13C] and [U-13C]glucose (28.9 +/- 2.6 and 30.7 +/- 1.3 g, between minutes 40 and 100), suggesting that no significant loss of 13C occurred in the TCA cycle. This stems from the fact that, during exercise, the rate of exogenous glucose oxidation is probably much larger than the flux of the metabolic pathways fueled from TCA cycle intermediates. It is thus unlikely that a significant portion of the 13C entering the TCA cycle could be diverted to these pathways. From a methodological standpoint, this result indicates that when a large amount of [13C]glucose is ingested and oxidized during exercise, 13CO2 production at the mouth accurately reflects the rate of glucose entry in the TCA cycle and that no correction factor is needed to compute the oxidative flux of exogenous glucose.     

Glutamate racemization and catabolism in Fusobacterium varium

The pathways of glutamate catabolism in the anaerobic bacterium Fusobacterium varium, grown on complex, undefined medium and chemically defined, minimal medium, were investigated using specifically labelled (13)C-glutamate. The metabolic end-products acetate and butyrate were isolated from culture fluids and derivatized for analysis by nuclear magnetic resonance and mass spectrometry. On complex medium, labels from L-[1-(13)C]glutamate and L-[4-(13)C]glutamate were incorporated into C1 of acetate and equally into C1/C3 of butyrate, while label derived from L-[5-(13)C]glutamate was not incorporated. The isotopic incorporation results and the detection of glutamate mutase and 3-methylaspartate ammonia lyase in cell extracts are most consistent with the methylaspartate pathway, the best known route of glutamate catabolism in Clostridium species. When F. varium was grown on defined medium, label from L-[4-(13)C]glutamate was incorporated mainly into C4 of butyrate, demonstrating a major role for the hydroxyglutarate pathway. Upon addition of coenzyme B(12) or cobalt ion to the defined medium in replicate experiments, isotope was located equally at C1/C3 of butyrate in accord with the methylaspartate pathway. Racemization of D-glutamate and subsequent degradation of L-glutamate via the methylaspartate pathway are supported by incorporation of label into C2 of acetate and equally into C2/C4 of butyrate from D-[3-(13)C]glutamate and the detection of a cofactor-independent glutamate racemase in cell extracts. Together the results demonstrate a major role for the methylaspartate pathway of glutamate catabolism in F. varium and substantial participation of the hydroxyglutarate pathway when coenzyme B(12) is not available.     

13C n.m.r. studies of gluconeogenesis in rat liver suspensions and perfused mouse livers

Our early 31P n.m.r. studies of compartmentation in suspensions of rat liver cells have been extended by following fructose-1-phosphate peaks, known to be in the cytosol, which gave the same pH as the Pi peak previously assigned to the cytosol. Gluconeogenesis have been followed from [13C]glycerol labelled at C1,3 or at C2 and from labelled [3-13C]alanine. With the glycerol substrate it was possible to follow the label into alpha-glycerophosphate and to determine its distribution in the glucose formed. To a first approximation (i.e. 90%) the glucose level could be followed from its original glycerol position, e.g. [1,3-13C]glycerol to strongly labelled positions 1, 3, 4 and 6 of glucose. Slightly more than 10% of the label was scrambled (i.e. 10% movement of C2 to C1 and ca. 10% of C1 was lost, the remainder being unchanged). These are consistent with a flux through the pentose shunt, dominated by the transketolase pathway. With [3-13C]alanine, about 14 resonances are assigned to different carbons of the intermediates beta-hydroxybutyrate, acetoacetate, lactate, pyruvate, glutamate, glutamine, asparate, as well as C2-alanine, while another 7 resonances are observed from the different anomeric carbons of glucose. The effects of thyroid hormone treatment of the rats upon numerous in vivo rates are clearly observed and will be illustrated.     

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.     

Enrichment for auxotrophic and heat-sensitive mutants of Neurospora crassa by tritium suicide.

Tritium suicide is shown to be an effective technique for mutant enrichment in Neurospora crassa. When mutagenized conidia were labelled to a high specific radioactivity either with a tritiated amino acid mixture or with [5-3H]uridine at a non-permissive temperature and stored at 4 degrees C to accumulate decays, there was a 13-15 fold enrichment for temperature-sensitive mutants relative to the original mutagenized cultures. For a wild type culture of Neurospora crassa labelled with [5-3H]uridine at 35 degrees C the probability of cell killing per tritium decay was calculated to be 3.64 X 10(-5).     

Metabolic pathways leading from amino acids to vitamin B12 in Propionibacterium shermanii, and the sources of the seven methyl carbons

The metabolic pathways leading from l-[2-13C]aspartic acid, [2-13C]glycine and l-[methyl-13C]methionine to vitamin B12 were investigated, focusing on the biosynthetic pathways leading to the aminopropanol moiety of vitamin B12 and on the role of the Shemin pathway leading to delta-aminolevulinic acid (a biosynthetic intermediate of tetrapyrrole), by means of feeding experiments with Propionibacterium shermanii in combination with 13C-NMR spectroscopy. The 13C-methylene carbons of l-[2-(13)C]aspartic acid, which is transformed to [2-13C]glycine via l-[2-13C]threonine, and [2-13C]glycine added to the culture medium served mainly to enrich the seven methyl carbons of the corrin ring through C-methylation by S-adenosyl-l-[methyl-13C]methionine derived from catabolically generated l-[methyl-13C]methionine in the presence of tetrahydrofolic acid. The results indicate that the catabolism of these amino acids predominates over pathways leading to (2R)-1-amino-2-propanol or delta-aminolevulinic acid in P. shermanii. Feeding of l-[methyl-13C]methionine efficiently enriched all seven methyl carbons. In the cases of [2-13C]glycine and l-[methyl-13C]methionine, the 13C-enrichment ratio of the methyl carbon at C-25 (the site of the first C-methylation) was less than those of the other six methyl carbons, probably due to the influence of endogenous d-glucose in P. shermanii. The almost identical 13C-enrichment ratios of the other six methyl carbons indicated that these C-methylations during vitamin B12 biosynthesis were completed before the amino acids were completely consumed. However, in the case of l-[2-13C]aspartic acid, the 13C-enrichment ratios of five methyl carbons were low and similar, whereas the last two sites of C-methylation (C-53 and C-35) were not labeled, presumably because of complete consumption of the smaller amount of added label. The ratios of 13C-incorporation into the seven methyl carbons are influenced by the conditions of amino acid feeding experiments in a manner that is dependent upon the order of C-methylation in the corrin ring of vitamin B12.     

A multinuclear magnetic resonance study of a cls11 mutant showing the Pet- phenotype of Saccharomyces cerevisiae

Energetic and intermediary metabolism was studied in a Pet- mutant of Saccharomyces cerevisiae with a calcium-sensitive phenotype that shows an inability to grow when cultured in a medium containing non-fermentable substrates. The perchloric acid extracts were prepared from suspensions of cls11 mutant and wild-type cells incubated with [1,3-13C]glycerol or [2-13]acetate, and analyzed by 31P, 13C and 1H NMR. 31P- and 1H-NMR spectra showed significant differences between cls11 and wild-type cells at the level of amino acids, the storage carbohydrate trehalose (higher in mutant cells), and sugar phosphates (higher in wild-type cells). 13C-NMR spectra revealed major differences in the steady-state labelling of glutamate carbons. For incubations with [1,3-13C]glycerol, we estimated from the relative 13C enrichment of glutamate carbons that acetyl-CoA C2 is 43% C13 labelled in wild-type and 10% 13C labelled in mutant cells, respectively. For incubations with [2-13C]acetate, we calculated that the ratio of the relative flux through the glyoxylate shunt versus oxidative reactions is 58% in wild-type cells and 44% in the cls11 mutant cells. Again, a dilution of the relative enrichment of C2 of acetyl-CoA was observed in the mutant cells (89%) compared to the wild-type cells (97%). These results are discussed in terms of pleiotropic defects in non-fermentable carbon metabolism in mutant cells.