L-valine production with pyruvate dehydrogenase complex-deficient Corynebacterium glutamicum

Corynebacterium glutamicum was engineered for the production of L-valine from glucose by deletion of the aceE gene encoding the E1p enzyme of the pyruvate dehydrogenase complex and additional overexpression of the ilvBNCE genes encoding the L-valine biosynthetic enzymes acetohydroxyacid synthase, isomeroreductase, and transaminase B. In the absence of cellular growth, C. glutamicum DeltaaceE showed a relatively high intracellular concentration of pyruvate (25.9 mM) and produced significant amounts of pyruvate, L-alanine, and L-valine from glucose as the sole carbon source. Lactate or acetate was not formed. Plasmid-bound overexpression of ilvBNCE in C. glutamicum DeltaaceE resulted in an approximately 10-fold-lower intracellular pyruvate concentration (2.3 mM) and a shift of the extracellular product pattern from pyruvate and L-alanine towards L-valine. In fed-batch fermentations at high cell densities and an excess of glucose, C. glutamicum DeltaaceE(pJC4ilvBNCE) produced up to 210 mM L-valine with a volumetric productivity of 10.0 mM h(-1) (1.17 g l(-1) h(-1)) and a maximum yield of about 0.6 mol per mol (0.4 g per g) of glucose.     

Enzymatic synthesis of radiolabeled phosphonoacetaldehyde

Phosphonoacetaldehyde (Pald) is formed in a variety of biosynthetic pathways leading to natural phosphonates and is an intermediate in the degradation pathway of the natural product 2-aminoethylphosphonate. To facilitate the investigation of the enzymes catalyzing these pathways, a method for the synthesis of radiolabeled Pald was developed. The enzyme pyruvate phosphate dikinase was used to prepare phosphoenolpyruvate (PEP) from pyruvate, adenosine triphosphate (ATP), and orthophosphate. Then PEP was converted to phosphonopyruvate (Ppyr) with PEP mutase and then to Pald with Ppyr decarboxylase. By using [beta-32P]ATP or [2-14C]pyruvate as precursor, [32P]Pald or [1-14C]Pald was obtained, respectively. The utilization of the synthetic, radiolabeled Pald as a probe of enzyme mechanism was demonstrated with the enzyme phosphonoacetaldehyde hydrolase (trivial name phosphonatase). The single turnover time course for the formation and consumption of radiolabeled covalent enzyme species evidenced a kinetically competent covalent intermediate.     

The regiochemistry and stereochemistry of the biosynthesis of vitamin B6 from triose units

13C and 2H NMR spectroscopy has been employed to probe the biosynthesis of vitamin B6 in Escherichia coli. The 13C NMR spectrum of a sample of pyridoxol derived biosynthetically from D-[1,2,3,4,5,6-13C6]glucose shows that the bonds, C(2)-C(3) and C(4)-C(5), of the pyridine nucleus are the only two carbon-carbon bonds of pyridoxol which are generated de novo in the course of its biosynthesis from glucose. It follows that the pyridoxol skeleton is generated from two intact triose units and a triose-derived two-carbon unit, all of which are supplied by glucose. From the 2H NMR spectra of samples of pyridoxol derived from (R)-[1,1-2H2]glycerol and (S)-[1,1-2H2]glycerol, respectively, it can be deduced that the rehydroxymethyl group of glycerol enters C-2', C-4', and C-5' of the pyridoxol skeleton. It follows that each of the three fragments is derived from glycerol in stereo-specific fashion. These results answer questions concerning the regiochemistry and the stereochemistry of pyridoxol biosynthesis.     

Biosynthesis of a neo-epi-verrucosane diterpene in the liverwort Fossombronia alaskana. A retrobiosynthetic NMR study

The biosynthesis of the diterpene 8alpha-acetoxy-13alpha-hydroxy-5-oxo-13-epi- neoverrucosane in the arctic liverwort Fossombronia alaskana was studied by incorporation experiments using [1-(13)C]- and [U-(13)C(6)]glucose as precursors. The (13)C-labeling patterns of acetyl-CoA, pyruvate, and phosphoenolpyruvate in intermediary metabolism were reconstructed from the (13)C NMR data of biosynthetic amino acids (leucine, alanine, phenylalanine) and were used to predict hypothetical labeling patterns for isopentenyl pyrophosphate formed via the mevalonate pathway and the deoxyxylulose pathway. The labeling patterns observed for the neoverrucosane diterpene were consistent with the intermediate formation of geranyllinaloyl pyrophosphate assembled from dimethylallyl pyrophosphate and three molecules of isopentenyl pyrophosphate generated predominantly or entirely via 1-deoxyxylulose 5-phosphate. The experimental data can be integrated into a detailed biosynthetic scheme involving a 1,5-hydride shift. The postulated involvement of the 1,5-hydride shift was confirmed by an incorporation experiment with [6,6-(2)H(2)]glucose.     

EFFECT OF HYPERPHENYLALANINEMIA ON VITAMIN B6, METABOLISM IN DEVELOPING RAT BRAIN

Abstract— The turnover of the different forms of B₆ vitamers in the brains of normal and hyperphenylalaninemic preweanling rats was compared after administration of a load of [¹⁴C]pyridoxol. Metabolic transformations occurred in the following sequence: oxidation of pyridoxol to pyridoxal, which was in turn phosphorylated to the 5'-phosphate ester. No significant amount of pyridoxamine was formed during the 8-h experimental period. Pyridoxamine 5'-phosphate was derived from pyridoxal 5'-phosphate. The specific radioactivity of pyridoxal phosphate in the hyperphenylalaninemic brain was significantly lower and increased at a slower rate than in control brains. This difference could not be accounted for by either a deficient supply or inhibited activity of the enzyme, pyridoxal kinase. The synthesis of pyridoxamine 5'-phosphate in the experimental animals also lagged behind the controls. Decreased activity of enzymes dependent on pyridoxal phosphate as cofactor would explain the slower turnover of this B₆-coenzyme.     

Citrate can partially replace carbon dioxide required for growth of Lactococcus lactis subsp. lactis biovar diacetylactis

Lactococcus lactis subsp. lactis biovar diacetylactis was grown as batch cultures on a chemically defined medium. No growth was observed when the cultures were sparged with pure nitrogen (1.3 l l-1 min-1) whereas the cultures displayed exponential growth in the presence of minute amounts of carbon dioxide (0.035 mol-% of the inlet gas). However, in the former case, the addition of citrate restored growth. This suggested that oxaloacetate required for aspartate biosynthesis can be formed by the carboxylation of pyruvate or by citrate catabolism. When the cultures were heavily sparged with nitrogen (2.6 l l-1 min-1), no growth was observed even in the presence of citrate. This indicated that growth in these conditions was repressed by the absence of carbon dioxide required in some other biosynthetic reaction than in the carboxylation of pyruvate leading to oxaloacetate/aspartate biosynthesis.     

13C-NMR study of acetate assimilation in Thermoproteus neutrophilus

Acetate assimilation into amino acids and the functioning of central biosynthetic pathways in the extremely thermophilic anaerobic archaebacterium Thermoproteus neutrophilus was investigated using 13C NMR as the method for determination of the labelling patterns. Acetate was assimilated via reductive carboxylation of acetyl-CoA to pyruvate and pyruvate conversion to phosphoenolpyruvate which was further carboxylated to oxaloacetate. 2-Oxoglutarate was mainly formed via citrate. However, the labelling patterns of glutamic acid and alanine were in agreement with the concurrent synthesis of about 15% 2-oxoglutarate and 5% pyruvate through the reductive citric acid cycle. A scrambling phenomenon occurring in aspartate and all amino acids derived through oxaloacetate was observed. The labelling patterns of amino acids were in agreement with their standard biosynthetic pathways, with two remarkable exceptions: isoleucine was synthesized via the citramalate pathway and lysine was synthesized via the 2-aminoadipate pathway which has previously been reported only in eukaryotic microorganisms.     

Ammonium ions enhance the flow through the pyruvate dehydrogenase in Ehrlich ascites tumor cells

The flow through pyruvate dehydrogenase was assayed in glycolysing cells by the evolution of 14CO2 from [1-14C] pyruvate. Parallel incubations were carried out in high bicarbonate buffer (25 mM) and in bicarbonate-free buffer. The activation of the complex by NH+4 was only observed in high bicarbonate buffer, because the dilution of labelled CO2 in the presence of an excess of bicarbonate enables the quantitative determination of labelled CO2 evolved from pyruvate in the decarboxylase step. In the bicarbonate-free buffer the activation of the complex was not observed, because the 14CO2 evolved from pyruvate was consumed by biosynthetic processes inside the cell. On the contrary in isolated hepatocytes the NH+4 activation of the pyruvate dehydrogenase was observed in both buffers. In Ehrlich ascites cells, in common with other mammalian tissues, pyruvate dehydrogenase activity was found to be inversely correlated to the intramitochondrial ATP/ADP ratio.     

The enzymic degradation of alkyl-substituted gentisates, maleates and malates

1. Cell-free extracts, prepared from a non-fluorescent Pseudomonas grown on m-cresol, oxidized gentisate and certain alkyl-substituted gentisates with the consumption of 1 mol of oxygen and the formation of 1 mol of pyruvate from 1 mol of substrate. 2. In addition to pyruvate, malate was formed from gentisate; citramalate was formed from 3-methylgentisate and 4-methylgentisate; 2,3-dimethylmalate was formed from 3,4-dimethylgentisate. 3. One enantiomer, d-(-)-citramalate, was formed enzymically from 3-methylgentisate, 4-methylgentisate and citraconate. l-(+)-Citramalate was formed from mesaconate by the same extracts. When examined as its dimethyl ester by gas-liquid chromatography, enzymically formed 2,3-dimethylmalate showed the same behaviour as one of the two racemates prepared from the synthetic compound. 4. Maleate, citraconate and 2,3-dimethylmaleate were rapidly hydrated by cell extracts, but ethylfumarate and 2,3-dimethylfumarate were not attacked. 5. Cell extracts oxidized 1,4-dihydroxy-2-naphthoate to give pyruvate and phthalate. 6. Alkylgentisates were oxidized by a gentisate oxygenase (EC 1.13.1.4) present in Pseudomonas 2,5. The ring-fission products were attacked by maleylpyruvase, but not by fumarylpyruvase, and their u.v.-absorption spectra were those expected for alkyl-substituted maleylpyruvates. 7. When supplemented with ATP, CoA, succinate and Mg(2+) ions, an enzyme system from cells grown with 2,5-xylenol formed pyruvate from d- but not from l-citramalate. Extracts from cells grown with dl-citramalate or with itaconate attacked both d- and l-citramalate; other alkylmalates were cleaved in similar fashion to give pyruvate or 2-oxobutyrate. 8. These results accord with a general sequence of reactions in which the benzene nucleus of an alkylgentisate is cleaved to give an alkyl-substituted maleylpyruvate. The ring-fission products are hydrolysed to give pyruvate, plus alkylmalic acids which then undergo aldol fissions, probably as their CoA esters. In Pseudomonas 2,5 several homologous sequences of this general type appear to be catalysed by a single battery of enzymes with broad substrate specificities, whereas the metabolic capabilities of the fluorescent Pseudomonas 3,5 are more restricted. 9. Intact cells of both organisms metabolize d-malic acid by reactions that have not been elucidated, but are different from those which degrade alkylmalates.     

Regulation of gluconeogenesis and lipogenesis. The regulation of mitochondrial pyruvate metabolism in guinea-pig liver synthesizing precursors for gluconeogenesis

1. The carboxylation of pyruvate to oxaloacetate by pyruvate carboxylase in guinea-pig liver mitochondria was determined by measuring the amount of (14)C from H(14)CO(3) (-) fixed into organic acids in the presence of pyruvate, ATP, Mg(2+) and P(i). The main products of pyruvate carboxylation were malate, fumarate and citrate. Pyruvate utilization, metabolite formation and incorporation of (14)C from H(14)CO(3) (-) into these metabolites in the presence and the absence of ATP were examined. The synthesis of phosphoenolpyruvate from pyruvate and bicarbonate is minimal during continued oxidation of pyruvate. Larger amounts of phosphoenolpyruvate are formed from alpha-oxoglutarate than from pyruvate. Addition of glutamate, alpha-oxoglutarate or fumarate did not appreciably increase formation of phosphoenolpyruvate when pyruvate was used as substrate. With alpha-oxoglutarate as substrate addition of fumarate resulted in increased formation of phosphoenolpyruvate, whereas addition of succinate inhibited phosphoenolpyruvate formation. In the presence of added oxaloacetate guinea-pig liver mitochondria synthesized phosphoenolpyruvate in amount sufficiently high to play an appreciable role in gluconeogenesis. 2. Addition of fatty acids of increasing carbon chain length caused a strong inhibition of pyruvate oxidation and phosphoenolpyruvate formation, and greatly promoted carbon dioxide fixation and malate, citrate and acetoacetate accumulation. The incorporation of (14)C from H(14)CO(3) (-), [1-(14)C]pyruvate and [2-(14)C]pyruvate into organic acids formed was examined. 3. It is concluded that guinea-pig liver pyruvate carboxylase contributes significantly to gluconeogenesis and that fatty acids and metabolites play an important role in its regulation.     

Metabolic flux analysis of the mixotrophic metabolisms in the green sulfur bacterium Chlorobaculum tepidum

The photosynthetic green sulfur bacterium Chlorobaculum tepidum assimilates CO(2) and organic carbon sources (acetate or pyruvate) during mixotrophic growth conditions through a unique carbon and energy metabolism. Using a (13)C-labeling approach, this study examined biosynthetic pathways and flux distributions in the central metabolism of C. tepidum. The isotopomer patterns of proteinogenic amino acids revealed an alternate pathway for isoleucine synthesis (via citramalate synthase, CimA, CT0612). A (13)C-assisted flux analysis indicated that carbons in biomass were mostly derived from CO(2) fixation via three key routes: the reductive tricarboxylic acid (RTCA) cycle, the pyruvate synthesis pathway via pyruvate:ferredoxin oxidoreductase, and the CO(2)-anaplerotic pathway via phosphoenolpyruvate carboxylase. During mixotrophic growth with acetate or pyruvate as carbon sources, acetyl-CoA was mainly produced from acetate (via acetyl-CoA synthetase) or citrate (via ATP citrate lyase). Pyruvate:ferredoxin oxidoreductase converted acetyl-CoA and CO(2) to pyruvate, and this growth-rate control reaction is driven by reduced ferredoxin generated during phototrophic growth. Most reactions in the RTCA cycle were reversible. The relative fluxes through the RTCA cycle were 80～100 units for mixotrophic cultures grown on acetate and 200～230 units for cultures grown on pyruvate. Under the same light conditions, the flux results suggested a trade-off between energy-demanding CO(2) fixation and biomass growth rate; C. tepidum fixed more CO(2) and had a higher biomass yield (Y(X/S), mole carbon in biomass/mole substrate) in pyruvate culture (Y(X/S) = 9.2) than in acetate culture (Y(X/S) = 6.4), but the biomass growth rate was slower in pyruvate culture than in acetate culture.     

Diacetyl Biosynthesis in Streptococcus diacetilactis and Leuconostoc citrovorum

Pyruvate was shown to be the precursor of diacetyl and acetoin in Streptococcus diacetilactis, but dialyzed cell-free extracts of S. diacetilactis and Leuconostoc citrovorum that had been treated with anion-exchange resin to remove coenzyme A (CoA) formed only acetoin from pyruvate in the presence of thiamine pyrophosphate (TPP) and Mg(++) or Mn(++) ions. The ability to produce diacetyl was restored by the addition of acetyl-CoA. Acetyl-phosphate did not replace the acetyl-CoA. Neither diacetyl nor acetoin was formed when the otherwise complete reaction system was modified by using boiled extract or by omitting the extract, pyruvate, TPP, or the metal ions. Free acetaldehyde was not involved in the biosynthesis of diacetyl or acetoin from pyruvate, dialyzed cell-free extracts of the bacteria produced only acetoin (besides CO(2)) from alpha-acetolactate, and acetoin was not involved in the biosynthesis of diacetyl. Only one of the optical isomers present in racemic alpha-acetolactate was attacked by the extracts, and there was no appreciable spontaneous decarboxylation of the alpha-acetolactate at the pH (4.5) used in experiments.     

Insights into the autotrophic CO2 fixation pathway of the archaeon Ignicoccus hospitalis: comprehensive analysis of the central carbon metabolism

Ignicoccus hospitalis is an autotrophic hyperthermophilic archaeon that serves as a host for another parasitic/symbiotic archaeon, Nanoarchaeum equitans. In this study, the biosynthetic pathways of I. hospitalis were investigated by in vitro enzymatic analyses, in vivo (13)C-labeling experiments, and genomic analyses. Our results suggest the operation of a so far unknown pathway of autotrophic CO(2) fixation that starts from acetyl-coenzyme A (CoA). The cyclic regeneration of acetyl-CoA, the primary CO(2) acceptor molecule, has not been clarified yet. In essence, acetyl-CoA is converted into pyruvate via reductive carboxylation by pyruvate-ferredoxin oxidoreductase. Pyruvate-water dikinase converts pyruvate into phosphoenolpyruvate (PEP), which is carboxylated to oxaloacetate by PEP carboxylase. An incomplete citric acid cycle is operating: citrate is synthesized from oxaloacetate and acetyl-CoA by a (re)-specific citrate synthase, whereas a 2-oxoglutarate-oxidizing enzyme is lacking. Further investigations revealed that several special biosynthetic pathways that have recently been described for various archaea are operating. Isoleucine is synthesized via the uncommon citramalate pathway and lysine via the alpha-aminoadipate pathway. Gluconeogenesis is achieved via a reverse Embden-Meyerhof pathway using a novel type of fructose 1,6-bisphosphate aldolase. Pentosephosphates are formed from hexosephosphates via the suggested ribulose-monophosphate pathway, whereby formaldehyde is released from C-1 of hexose. The organism may not contain any sugar-metabolizing pathway. This comprehensive analysis of the central carbon metabolism of I. hospitalis revealed further evidence for the unexpected and unexplored diversity of metabolic pathways within the (hyperthermophilic) archaea.     

Synthesis of Pyridoxine by a Pyridoxal Auxotroph of Escherichia coli

Dempsey, Walter B. (University of Florida, Gainesville). Synthesis of pyridoxine by a pyridoxal auxotroph of Escherichia coli. J. Bacteriol. 92:333-337. 1966.-A pyridoxal auxotroph of Escherichia coli B produced pyridoxol and pyridoxol 5'-phosphate during starvation for pyridoxal. The identification of these compounds was made both by bioassay and by ion-exchange chromatography. Pyridoxol 5'-phosphate oxidase activity was absent in extracts of the auxotroph. The rate of synthesis of total pyridoxine by a pyridoxal-starved culture of this auxotroph was 6.0 x 10(-6) moles per mg per hr. Cellular content of pyridoxine was constant at 4.0 x 10(-10) moles/mg.     

Genome-enabled determination of amino acid biosynthesis in Xanthomonas campestris pv. campestris and identification of biosynthetic pathways for alanine, glycine, and isoleucine by 13C-isotopologue profiling

To elucidate the biosynthetic pathways for all proteinogenic amino acids in Xanthomonas campestris pv. campestris, this study combines results obtained by in silico genome analysis and by (13)C-NMR-based isotopologue profiling to provide a panoramic view on a substantial section of bacterial metabolism. Initially, biosynthesis pathways were reconstructed from an improved annotation of the complete genome of X. campestris pv. campestris B100. This metabolic reconstruction resulted in the unequivocal identification of biosynthesis routes for 17 amino acids in total: arginine, asparagine, aspartate, cysteine, glutamate, glutamine, histidine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Ambiguous pathways were reconstructed from the genome data for alanine, glycine, and isoleucine biosynthesis. (13)C-NMR analyses supported the identification of the metabolically active pathways. The biosynthetic routes for these amino acids were derived from the precursor molecules pyruvate, serine, and pyruvate, respectively. By combining genome analysis and isotopologue profiling, a comprehensive set of biosynthetic pathways covering all proteinogenic amino acids was unraveled for this plant pathogenic bacterium, which plays an important role in biotechnology as a producer of the exopolysaccharide xanthan. The data obtained lay ground for subsequent functional analyses in post-genomics and biotechnology, while the innovative combination of in silico and wet lab technology described here is promising as a general approach to elucidate metabolic pathways.     

Transpulmonary pyruvate kinetics

Shuttling of intermediary metabolites, such as pyruvate, contributes to the dynamic energy and biosynthetic needs of tissues. Tracer kinetic studies offer a powerful tool to measure the metabolism of substrates like pyruvate that are simultaneously taken up from and released into the circulation by organs. However, we understood that during each circulatory passage, the entire cardiac output transits the pulmonary circulation. Therefore, we examined the transpulmonary pyruvate kinetics in an anesthetized rat model during an unstimulated (Con), lactate clamp (LC), and epinephrine infusion (Epi) conditions using a primed-continuous infusion of [U-13C]pyruvate. Compared with Con and Epi stimulation, LC significantly increased mixed central venous ([v]) and arterial ([a]) pyruvate concentrations (P < 0.05). We hypothesized that the lungs, specifically the pulmonary capillary beds are sites of simultaneous production and removal of pyruvate and contributes significantly to whole body carbohydrate intermediary metabolism. Transpulmonary net pyruvate balances were positive during all three conditions, indicating net pyruvate uptake. Net balance was significantly greater during epinephrine stimulation compared with the unstimulated control (P < 0.05). Tracer-measured pyruvate fractional extraction averaged 42.8 ± 5.8% for all three conditions and was significantly higher during epinephrine stimulation (P < 0.05) than during either Con or LC conditions, that did not differ from each other. Pyruvate total release (tracer measured uptake - net balance) was significantly higher during epinephrine stimulation (400 ± 100 μg/min) vs. Con (30 ± 20 μg/min) (P < 0.05). These data are interpreted to mean that significant pyruvate extraction occurs during circulatory transport across lung parenchyma. The extent of pulmonary parenchymal pyruvate extraction predicts high expression of monocarboxylate (lactate/pyruvate) transporters (MCTs) in the tissue. Western blot analysis of whole lung homogenates detected three isoforms, MCT1, MCT2, and MCT4. We conclude that a major site of circulating pyruvate extraction resides with the lungs and that during times of elevated circulating lactate, pyruvate, or epinephrine stimulation, pyruvate extraction is increased.     

Menaquinone biosynthesis in Escherichia coli: identification of 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate as a novel intermediate and re-evaluation of MenD activity

Menaquinone is an electron carrier in the respiratory chain of Escherichia coli during anaerobic growth. Its biosynthesis involves (1R,6R)-2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylic acid (SHCHC) as an intermediate, which is believed to be derived from isochorismate and 2-ketoglutarate by one of the biosynthetic enzymes-MenD. However, we found that the genuine MenD product is 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylic acid (SEPHCHC), rather than SHCHC. This is supported by the following findings: (i) isochorismate consumption and SHCHC formation are not synchronized in the enzymic reaction, (ii) the rate of SHCHC formation is independent of the enzyme concentration, (iii) SHCHC is not formed in weakly acidic or neutral solutions in which the isochorismate substrate is readily consumed by MenD, and (iv) the MenD turnover product, formed under conditions disabling SHCHC formation, possesses spectroscopic characteristics consistent with the structure of SEPHCHC and spontaneously undergoes 2,5-elimination to form SHCHC and pyruvate in weakly basic solutions. Two properties of the intermediate, ultraviolet transparency and chemical instability, provide a rationale for the fact that SHCHC has been consistently mistaken as the MenD product. In accordance with these findings, MenD was rediscovered to be a highly efficient enzyme with a high second-order rate constant and should be renamed SEPHCHC synthase. Intriguingly, the enzymatic activity responsible for conversion of SEPHCHC into SHCHC appears not to associate with any of the known enzymes in menaquinone biosynthesis but is present in the crude extract of E. coli K12, suggesting that a genuine SHCHC synthase remains to be identified to fully elucidate the ubiquitous biosynthetic pathway.     

Formation of 2-methyl-2,4-thiazolidinedicarboxylic acid from L-cysteine in rat tissues

The adduct formed non-enzymatically from L-cysteine and pyruvate: 2-methyl-2,4-thiazolidinecarboxylic acid (CP) was isolated, and identified by the electron impact mass spectroscopy. It was found that CP is formed (by cysteine transformation) and is metabolized in rat tissues. Formation of CP from cysteine or cystine was catalysed by partially purified rat liver gamma-cystathionase.     

Proliferating bloodstream-form Trypanosoma brucei use a negligible part of consumed glucose for anabolic processes

Our quantitative knowledge of carbon fluxes in the long slender bloodstream form (BSF) Trypanosoma brucei is mainly based on non-proliferating parasites, isolated from laboratory animals and kept in buffers. In this paper we present a carbon balance for exponentially growing bloodstream form trypanosomes. The cells grew with a doubling time of 5.3h, contained 46 μ mol of carbon (10(8) cells)(-1) and had a glucose consumption flux of 160 nmol min(-1) (10(8) cells)(-1). The molar ratio of pyruvate excreted versus glucose consumed was 2.1. Furthermore, analysis of the (13)C label distribution in pyruvate in (13)C-glucose incubations of exponentially growing trypanosomes showed that glucose was the sole substrate for pyruvate production. We conclude that the glucose metabolised in glycolysis was hardly, if at all, used for biosynthetic processes. Carbon flux through glycolysis in exponentially growing trypanosomes was 10 times higher than the incorporation of carbon into biomass. This biosynthetic carbon is derived from other precursors present in the nutrient rich growth medium. Furthermore, we found that the glycolytic flux was unaltered when the culture went into stationary phase, suggesting that most of the ATP produced in glycolysis is used for processes other than growth.     

Pyruvate-Derived Amino Acids in Spinach Chloroplasts : Synthesis and Regulation during Photosynthetic Carbon Metabolism

A probable carbon flow from the Calvin cycle to branched chain amino acids and lipids via phosphoenolpyruvate (PEP) and pyruvate was examined in spinach (Spinacia oleracea) chloroplasts. The interpendence of metabolic pathways in and outside chloroplasts as well as product and feedback inhibition were studied. It was shown that alanine, aromatic, and small amounts of branched chain amino acids were formed from bicarbonate in purified intact chloroplasts. Addition of PEP only favored formation of aromatic amino acids. Mechanisms of regulation remained unclear. Concentrations of PEP and pyruvate within the chloroplast impermeable space during photosynthetic carbon fixation were 15 times higher than in the reaction medium. A direct carbon flow to pyruvate was identified (0.1 micromoles per milligram chlorophyll per hour). Pyruvate was taken up by intact chloroplasts slowly, leading to the formation of lysine, alanine, valine, and leucine plus isoleucine (approximate ratios, 100-500:60-100:40-100:2-10). The K_m for the formation of valine and leucine plus isoleucine was estimated to be 0.1 millimolar. Ten micromolar glutamate optimized the transamination reaction regardless of whether bicarbonate or pyruvate was being applied. Alanine and valine formation was enhanced by the addition of acetate to the reaction mixture. The enhancement probably resulted from an inhibition of pyruvate dehydrogenase by acetyl-S-coenzyme A formed from acetate, and resulting accumulation of hydroxyethylthiamine diphosphate and pyruvate. High concentrations of valine and isoleucine inhibited their own and each others synthesis and enhanced alanine formation. When pyruvate was applied, only amino acids were formed; when complemented with bicarbonate, fatty acids were formed as well. This is probably the result of a requirement of acetyl-S-coenzyme A-carboxylase for bicarbonate.