Absence of 5-Hydroxy-4-Ketohexanoate and the α-Ketoglutarate Dehydrogenase Complex in Mutants of Saccharomyces oviformis Incapable of Growing on Ethanol

The roles of the enzyme which forms 5-hydroxy-4-ketohexanoate (HKH) and of related enzymes in the metabolism of ethanol were studied in Saccharomyces oviformis WH92 and its mutants, which grew poorly or not at all on ethanol. The strains, which did not grow on ethanol, did not form HKH from α-ketoglutarate and acetaldehyde enzymatically and were also devoid of the α-ketoglutarate dehydrogenase complex. Acetaldehyde inhibited the activity of α-ketoglutarate dehydrogenase. These mutants did not grow on acetate since they had no acetyl-CoA synthetase activity. The relationship of the formation of HKH with the metabolism of ethanol is discussed.     

Glutamate Biosynthesis in Lactococcus lactis subsp. lactis NCDO 2118

Unlike other lactic acid bacteria, Lactococcus lactis subsp. lactis NCDO 2118 was able to grow in a medium lacking glutamate and the amino acids of the glutamate family. Growth in such a medium proceeded after a lag phase of about 2 days and with a reduced growth rate (0.11 h⁻¹) compared to that in the reference medium containing glutamate (0.16 h⁻¹). The enzymatic studies showed that a phosphoenolpyruvate carboxylase activity was present, while the malic enzyme and the enzymes of the glyoxylic shunt were not detected. As in most anaerobic bacteria, no α-ketoglutarate dehydrogenase activity could be detected, and the citric acid cycle was restricted to a reductive pathway leading to succinate formation and an oxidative branch enabling the synthesis of α-ketoglutarate. The metabolic bottleneck responsible for the limited growth rate was located in this latter pathway. As regards the synthesis of glutamate from α-ketoglutarate, no glutamate dehydrogenase was detected. While the glutamate synthase-glutamine synthetase system was detected at a low level, high transaminase activity was measured. The conversion of α-ketoglutarate to glutamate by the transaminase, the reverse of the normal physiological direction, operated with different amino acids as nitrogen donor. All of the enzymes assayed were shown to be constitutive.     

Procyclic trypanosomes recycle glucose catabolites and TCA cycle intermediates to stimulate growth in the presence of physiological amounts of proline

Trypanosoma brucei, a protist responsible for human African trypanosomiasis (sleeping sickness), is transmitted by the tsetse fly where the procyclic forms of the parasite develop in the proline-rich (1–2 mM) and glucose-depleted digestive tract. Proline is essential for the midgut colonization of the parasite in the insect vector, however other carbon sources could be available and used to feed its central metabolism. Here we show that procyclic trypanosomes can consume and metabolize metabolic intermediates, including those excreted from glucose catabolism (succinate, alanine and pyruvate), with the exception of acetate, which is the ultimate end-product excreted by the parasite. Among the tested metabolites, tricarboxylic acid (TCA) cycle intermediates (succinate, malate and α-ketoglutarate) stimulated growth of the parasite in the presence of 2 mM proline. The pathways used for their metabolism were mapped by proton-NMR metabolic profiling and phenotypic analyses of thirteen RNAi and/or null mutants affecting central carbon metabolism. We showed that (i) malate is converted to succinate by both the reducing and oxidative branches of the TCA cycle, which demonstrates that procyclic trypanosomes can use the full TCA cycle, (ii) the enormous rate of α-ketoglutarate consumption (15-times higher than glucose) is possible thanks to the balanced production and consumption of NADH at the substrate level and (iii) α-ketoglutarate is toxic for trypanosomes if not appropriately metabolized as observed for an α-ketoglutarate dehydrogenase null mutant. In addition, epimastigotes produced from procyclics upon overexpression of RBP6 showed a growth defect in the presence of 2 mM proline, which is rescued by α-ketoglutarate, suggesting that physiological amounts of proline are not sufficient per se for the development of trypanosomes in the fly. In conclusion, these data show that trypanosomes can metabolize multiple metabolites, in addition to proline, which allows them to confront challenging environments in the fly.     

The Campylobacter jejuni NADH:ubiquinone oxidoreductase (complex I) utilizes flavodoxin rather than NADH

Campylobacter jejuni encodes 12 of the 14 subunits that make up the respiratory enzyme NADH:ubiquinone oxidoreductase (also called complex I). The two nuo genes not present in C. jejuni encode the NADH dehydrogenase, and in their place in the operon are the novel genes designated Cj1575c and Cj1574c. A series of mutants was generated in which each of the 12 nuo genes (homologues to known complex I subunits) was disrupted or deleted. Each of the nuo mutants will not grow in amino acid-based medium unless supplemented with an alternative respiratory substrate such as formate. Unlike the nuo genes, Cj1574c is an essential gene and could not be disrupted unless an intact copy of the gene was provided at an unrelated site on the chromosome. A nuo deletion mutant can efficiently respire formate but is deficient in α-ketoglutarate respiratory activity compared to the wild type. In C. jejuni, α-ketoglutarate respiration is mediated by the enzyme 2-oxoglutarate:acceptor oxidoreductase; mutagenesis of this enzyme abolishes α-ketoglutarate-dependent O₂ uptake and fails to reduce the electron transport chain. The electron acceptor for 2-oxoglutarate:acceptor oxidoreductase was determined to be flavodoxin, which was also determined to be an essential protein in C. jejuni. A model is presented in which CJ1574 mediates electron flow into the respiratory transport chain from reduced flavodoxin and through complex I.     

Expression of a Heterologous Glutamate Dehydrogenase Gene in Lactococcus lactis Highly Improves the Conversion of Amino Acids to Aroma Compounds

The first step of amino acid degradation in lactococci is a transamination, which requires an α-keto acid as the amino group acceptor. We have previously shown that the level of available α-keto acid in semihard cheese is the first limiting factor for conversion of amino acids to aroma compounds, since aroma formation is greatly enhanced by adding α-ketoglutarate to cheese curd. In this study we introduced a heterologous catabolic glutamate dehydrogenase (GDH) gene into Lactococcus lactis so that this organism could produce α-ketoglutarate from glutamate, which is present at high levels in cheese. Then we evaluated the impact of GDH activity on amino acid conversion in in vitro tests and in a cheese model by using radiolabeled amino acids as tracers. The GDH-producing lactococcal strain degraded amino acids without added α-ketoglutarate to the same extent that the wild-type strain degraded amino acids with added α-ketoglutarate. Interestingly, the GDH-producing lactococcal strain produced a higher proportion of carboxylic acids, which are major aroma compounds. Our results demonstrated that a GDH-producing lactococcal strain could be used instead of adding α-ketoglutarate to improve aroma development in cheese.     

Oxidoreductases Involved in Cell Carbon Synthesis of Methanobacterium thermoautotrophicum

Cell-free extracts of Methanobacterium thermoautotrophicum were found to contain high activities of the following oxidoreductases (at 60°C): pyruvate dehydrogenase (coenzyme A acetylating), 275 nmol/min per mg of protein; α-ketoglutarate dehydrogenase (coenzyme A acylating), 100 nmol/min per mg; fumarate reductase, 360 nmol/min per mg; malate dehydrogenase, 240 nmol/min per mg; and glyceraldehyde-3-phosphate dehydrogenase, 100 nmol/min per mg. The kinetic properties (apparent V_max and K_M values), pH optimum, temperature dependence of the rate, and specificity for electron acceptors/donors of the different oxidoreductases were examined. Pyruvate dehydrogenase and α-ketoglutarate dehydrogenase were shown to be two separate enzymes specific for factor 420 rather than for nicotinamide adenine dinucleotide (NAD), NADP, or ferredoxin as the electron acceptor. Both activities catalyzed the reduction of methyl viologen with the respective α-ketoacid and a coenzyme A-dependent exchange between the carboxyl group of the α-ketoacid and CO₂. The data indicate that the two enzymes are similar to pyruvate synthase and α-ketoglutarate synthase, respectively. Fumarate reductase was found in the soluble cell fraction. This enzyme activity coupled with reduced benzyl viologen as the electron donor, but reduced factor 420, NADH, or NADPH was not effective. The cells did not contain menaquinone, thus excluding this compound as the physiological electron donor for fumarate reduction. NAD was the preferred coenzyme for malate dehydrogenase, whereas NADP was preferred for glyceraldehyde-3-phosphate dehydrogenase. The organism also possessed a factor 420-dependent hydrogenase and a factor 420-linked NADP reductase. The involvement of the described oxidoreductases in cell carbon synthesis is discussed.     

Succinyl-Coenzyme A Synthetase and its Role in delta-Aminolevulinic Acid Biosynthesis in Euglena gracilis

Euglena gracilis cells synthesize the key tetrapyrrole precursor, δ-aminolevulinic acid (ALA), by two routes: plastid ALA is formed from glutamate via the transfer RNA-dependent five-carbon route, and ALA that serves as the precursor to mitochondrial hemes is formed by ALA synthase-catalyzed condensation of succinyl-coenzyme A and glycine. The biosynthetic source of succinyl-coenzyme A in Euglena is of interest because this species has been reported not to contain α-ketoglutarate dehydrogenase and not to use succinyl-coenzyme A as a tricarboxylic acid cycle intermediate. Instead, α-ketoglutarate is decarboxylated to form succinic semialdehyde, which is subsequently oxidized to form succinate. Desalted extract of Euglena cells catalyzed ALA formation in a reaction that required coenzyme A and GTP but did not require exogenous succinyl-coenzyme A synthetase. GTP could be replaced with ATP. Cell extract also catalyzed glycine-and α-ketoglutarate-dependent ALA formation in a reaction that required coenzyme A and GTP, was stimulated by NADP⁺, and was inhibited by NAD⁺. Succinyl-coenzyme A synthetase activity was detected in extracts of dark- and light-grown wild-type and nongreening mutant cells. In vitro succinyl-coenzyme A synthetase activity was at least 10-fold greater than ALA synthase activity. These results indicate that succinyl-coenzyme A synthetase is present in Euglena cells. Even though the enzyme may play no role in the transformation of α-ketoglutarate to succinate in the atypical tricarboxylic acid cycle, it catalyzes succinyl-coenzyme A formation from succinate for use in the biosynthesis of ALA and possibly other products.     

Uptake of α-Ketoglutarate by Citrate Transporter CitP Drives Transamination in Lactococcus lactis

Transamination is the first step in the conversion of amino acids into aroma compounds by lactic acid bacteria (LAB) used in food fermentations. The process is limited by the availability of α-ketoglutarate, which is the best α-keto donor for transaminases in LAB. Here, uptake of α-ketoglutarate by the citrate transporter CitP is reported. Cells of Lactococcus lactis IL1403 expressing CitP showed significant levels of transamination activity in the presence of α-ketoglutarate and one of the amino acids Ile, Leu, Val, Phe, or Met, while the same cells lacking CitP showed transamination activity only after permeabilization of the cell membrane. Moreover, the transamination activity of the cells followed the levels of CitP in a controlled expression system. The involvement of CitP in the uptake of the α-keto donor was further demonstrated by the increased consumption rate in the presence of l-lactate, which drives CitP in the fast exchange mode of transport. Transamination is the only active pathway for the conversion of α-ketoglutarate in IL1403; a stoichiometric conversion to glutamate and the corresponding α-keto acid from the amino acids was observed. The transamination activity by both the cells and the cytoplasmic fraction showed a remarkably flat pH profile over the range from pH 5 to pH 8, especially with the branched-chain amino acids. Further metabolism of the produced α-keto acids into α-hydroxy acids and other flavor compounds required the coupling of transamination to glycolysis. The results suggest a much broader role of the citrate transporter CitP in LAB than citrate uptake in the citrate fermentation pathway alone.     

Transamination Is Required for α-Ketoisocaproate but Not Leucine to Stimulate Insulin Secretion

It remains unclear how α-ketoisocaproate (KIC) and leucine are metabolized to stimulate insulin secretion. Mitochondrial BCATm (branched-chain aminotransferase) catalyzes reversible transamination of leucine and α-ketoglutarate to KIC and glutamate, the first step of leucine catabolism. We investigated the biochemical mechanisms of KIC and leucine-stimulated insulin secretion (KICSIS and LSIS, respectively) using BCATm^−/− mice. In static incubation, BCATm disruption abolished insulin secretion by KIC, d,l-α-keto-β-methylvalerate, and α-ketocaproate without altering stimulation by glucose, leucine, or α-ketoglutarate. Similarly, during pancreas perfusions in BCATm^−/− mice, glucose and arginine stimulated insulin release, whereas KICSIS was largely abolished. During islet perifusions, KIC and 2 mm glutamine caused robust dose-dependent insulin secretion in BCATm^+/+ not BCATm^−/− islets, whereas LSIS was unaffected. Consistently, in contrast to BCATm^+/+ islets, the increases of the ATP concentration and NADPH/NADP⁺ ratio in response to KIC were largely blunted in BCATm^−/− islets. Compared with nontreated islets, the combination of KIC/glutamine (10/2 mm) did not influence α-ketoglutarate concentrations but caused 120 and 33% increases in malate in BCATm^+/+ and BCATm^−/− islets, respectively. Although leucine oxidation and KIC transamination were blocked in BCATm^−/− islets, KIC oxidation was unaltered. These data indicate that KICSIS requires transamination of KIC and glutamate to leucine and α-ketoglutarate, respectively. LSIS does not require leucine catabolism and may be through leucine activation of glutamate dehydrogenase. Thus, KICSIS and LSIS occur by enhancing the metabolism of glutamine/glutamate to α-ketoglutarate, which, in turn, is metabolized to produce the intracellular signals such as ATP and NADPH for insulin secretion.     

Characterization of the transport of oxaloacetate by pea leaf mitochondria

Mitochondria isolated from pea (Pisum sativum L.) leaves are able to transport the keto acid, oxaloacetate, from the reaction medium into he mitochondrial matrix at high rates. The rate of uptake by the mitochondria was measured as the rate of disappearance of oxaloacetate from the reaction medium as it was reduced by matrix malate dehydrogenase using NADH provided by glycine oxidation. The oxaloacetate transporter was identifed as being distinct from the dicarboxylate and the α-ketoglutarate transporters because of its inhibitor sensitivities and its inability to interact with other potential substrates. Phthalonate and phthalate were competitive inhibitors of oxaloacetate transport with K_i values of 60 micromolar and 2 millimolar, respectively. Butylmalonate, an inhibitor of the dicarboxylate and α-ketoglutarate transporters, did not alter the rate of oxaloacetate transport. In addition, a 1000-fold excess of malate, malonate, succinate, α-ketoglutarate, or phosphate had little effect on the rate of oxaloacetate transport. The K_m for the oxaloacetate transporter was about 15 micromolar with a maximum velocity of over 500 nanomoles per milligram mitochondrial protein/min at 25°C. No requirement for a counter ion to move against oxaloacetate was detected and the highest rates of uptake occurred at alkaline pH values. An equivalent transporter has not been reported in animal mitochondria.     

Diacetyl and α-Acetolactate Overproduction by Lactococcus lactis subsp. lactis Biovar Diacetylactis Mutants That Are Deficient in α-Acetolactate Decarboxylase and Have a Low Lactate Dehydrogenase Activity

Lactococcus lactis subsp. lactis biovar diacetylactis strains are utilized in several industrial processes for producing the flavoring compound diacetyl or its precursor α-acetolactate. Using random mutagenesis with nitrosoguanidine, we selected mutants that were deficient in α-acetolactate decarboxylase and had low lactate dehydrogenase activity. The mutants produced large amounts of α-acetolactate in anaerobic milk cultures but not in aerobic cultures, except when the medium was supplemented with catalase, yeast extract, or hemoglobin.     

Haloxyfop Inhibition of the Pyruvate and the alpha-Ketoglutarate Dehydrogenase Complexes of Corn (Zea mays L.) and Soybean (Glycine max [L.] Merr.)

The grass-specific herbicide haloxyfop, ((±)-2-[4-((3-chloro-5-(trifluoromethyl)-2-pyridinyl)oxy)-phenoxy] propionic acid) has been shown to inhibit lipid synthesis and respiration, to cause the accumulation of amino acids, and not to affect cellular sugar or ATP levels. Thus studies were carried out with enzyme activities from corn (Zea mays L.) (haloxyfop sensitive) and soybean (Glycine max [L.] Merr.) (haloxyfop tolerant) to locate the possible inhibition sites among the glycolytic and tricarboxylic acid (TCA) cycle enzymes. Following along the oxidative metabolism pathway of sugars, the pyruvate dehydrogenase complex (PDC) was the first enzyme among the glycolytic enzymes that demonstrated noticeable inhibition by 1 millimolar haloxyfop. Kinetic studies with corn and soybean PDC from both purified etioplasts and mitochondria gave K_i values of from 1 to 10 millimolar. Haloxyfop also inhibited the activity of the TCA cycle enzyme, the α-ketoglutarate dehydrogenase complex (α-KGDC) which carries out the same reaction as PDC except for the substitution of α-ketoglutarate for pyruvate as one of the substrates. The K_i values were somewhat lower in this case (near 1 millimolar). The relatively high K_i values for both enzyme complexes would indicate that these may not be the herbicidal sites of inhibition, but it is possible that the herbicide could be concentrated in compartments and/or the substrate concentrations may be well below optimal. Likewise little difference was seen in the haloxyfop inhibition of the enzyme activities from the sensitive species, corn, and from the tolerant species, soybean, so the selectivity of the herbicide is not evident from these results. The inhibition of the PDC and α-KGDC as the mode of action of haloxyfop is, however, consistent with the observed physiological effects of the herbicide, and these are the only enzymic activities so far found to be sensitive to haloxyfop.     

The Characterization of α-Glycerophosphate Dehydrogenase Mutants in DROSOPHILA MELANOGASTER

Thirty mutants of α-glycerophosphate dehydrogenase (αGPDH, EC 1.1.1.8) from Drosophila melanogaster were produced with the chemical mutagen ethyl methanesulfonate (EMS). These mutants and nine others previously obtained have been characterized with respect to level of enzymatic activity, viability, flight ability, and presence of cross-reacting material (CRM). The presence of αGPDH mRNA in several of the mutants has been tested by in vitro translation. There are strong correlations between the level of enzyme activity, viability and flight ability. Thirteen of the mutants are CRM^- by solution immunoprecipitation experiments, but of these, only three are CRM^- by a more sensitive ¹²⁵ I-protein A-based radioimmune gel assay. The viability of the three CRM^- mutants suggests that the absence of αGPDH protein is not a lethal condition. The immunoprecipitated protein of the low activity mutant, αGpdh^nGL3, has a smaller apparent molecular weight on polyacrylamide-SDS gels than does the protein from wild type. Criteria for the identification of nonsense mutations in Drosophila are discussed.     

Characterising the Role of GABA and Its Metabolism in the Wheat Pathogen Stagonospora nodorum

A reverse genetics approach was used to investigate the role of γ-aminobutyric acid metabolism in the wheat pathogenic fungus Stagonospora nodorum. The creation of mutants lacking Sdh1, the gene encoding succinic semialdehyde dehydrogenase, resulted in strains that grew poorly on γ-aminobutyric acid as a nitrogen source. The sdh1 mutants were more susceptible to reactive oxygen stress but were less affected by increased growth temperatures. Pathogenicity assays revealed that the metabolism of γ-aminobutyric acid is required for complete pathogenicity. Growth assays of the wild-type and mutant strains showed that the inclusion of γ-aminobutyric acid as a supplement in minimal media (i.e., not as a nitrogen or carbon source) resulted in restricted growth but increased sporulation. The addition of glutamate, the precursor to GABA, had no effect on either growth or sporulation. The γ-aminobutyric acid effect on sporulation was found to be dose dependent and not restricted to Stagonospora nodorum with a similar effect observed in the dothideomycete Botryosphaeria sp. The positive effect on sporulation was assayed using isomers of γ-aminobutyric acid and other metabolites known to influence asexual development in Stagonospora nodorum but no effect was observed. These data demonstrate that γ-aminobutyric acid plays an important role in Stagonospora nodorum in responding to environmental stresses while also having a positive effect on asexual development.     

Partial Purification and Reconstitution of the alpha-Ketoglutarate Carrier from Corn (Zea mays L.) Mitochondria

The α-ketoglutarate carrier from corn shoot mitochondria (Zea mays L., B 73) was solubilized in Triton X-114 and partially purified by chromatography on hydroxyapatite and celite in the presence of cardiolipin. On SDS-gel electrophoresis, the hydroxyapatite/celite eluate showed various protein bands between 12 and 70 kilodaltons. When reconstituted into liposomes, the α-ketoglutarate transport protein catalyzed a phthalonate-sensitive α-ketoglutarate/α-ketoglutarate exchange. The protein was purified 60-fold with a recovery of 88% with respect to the mitochondrial extract. The protein yield was 0.6%. The properties of the reconstituted carrier, i.e. requirement for a counter-anion, substrate specificity, and inhibitor sensitivity, were similar to those of the α-ketoglutarate transport system as characterized in plant and animal mitochondria.     

Replication of the Shrimp Virus WSSV Depends on Glutamate-Driven Anaplerosis

Infection with the white spot syndrome virus (WSSV) induces a metabolic shift in shrimp that resembles the “Warburg effect” in mammalian cells. This effect is triggered via activation of the PI3K-Akt-mTOR pathway, and it is usually accompanied by the activation of other metabolic pathways that provide energy and direct the flow of carbon and nitrogen. Here we show that unlike the glutamine metabolism (glutaminolysis) seen in most cancer cells to double deaminate glutamine to produce glutamate and the TCA cycle intermediate α-ketoglutarate (α-KG), at the WSSV genome replication stage (12 hpi), although glutaminase (GLS) expression was upregulated, only glutamate was taken up by the hemocytes of WSSV-infected shrimp. At the same time, we observed an increase in the activity of the two enzymes that convert glutamate to α-KG, glutamate dehydrogenase (GDH) and aspartate aminotransferase (ASAT). α-ketoglutarate concentration was also increased. A series of inhibition experiments suggested that the up-regulation of GDH is regulated by mTORC2, and that the PI3K-mTORC1 pathway is not involved. Suppression of GDH and ASAT by dsRNA silencing showed that both of these enzymes are important for WSSV replication. In GDH-silenced shrimp, direct replenishment of α-KG rescued both ATP production and WSSV replication. From these results, we propose a model of glutamate-driven anaplerosis that fuels the TCA cycle via α-KG and ultimately supports WSSV replication.     

Quantification of NADH:ubiquinone oxidoreductase (complex I) content in biological samples

Impairments in mitochondrial energy metabolism have been implicated in human genetic diseases associated with mitochondrial and nuclear DNA mutations, neurodegenerative and cardiovascular disorders, diabetes, and aging. Alteration in mitochondrial complex I structure and activity has been shown to play a key role in Parkinson''s disease and ischemia/reperfusion tissue injury, but significant difficulty remains in assessing the content of this enzyme complex in a given sample. The present study introduces a new method utilizing native polyacrylamide gel electrophoresis in combination with flavin fluorescence scanning to measure the absolute content of complex I, as well as α-ketoglutarate dehydrogenase complex, in any preparation. We show that complex I content is 19 ± 1 pmol/mg of protein in the brain mitochondria, whereas varies up to 10-fold in different mouse tissues. Together with the measurements of NADH-dependent specific activity, our method also allows accurate determination of complex I catalytic turnover, which was calculated as 10⁴ min⁻¹ for NADH:ubiquinone reductase in mouse brain mitochondrial preparations. α-ketoglutarate dehydrogenase complex content was determined to be 65 ± 5 and 123 ± 9 pmol/mg protein for mouse brain and bovine heart mitochondria, respectively. Our approach can also be extended to cultured cells, and we demonstrated that about 90 × 10³ complex I molecules are present in a single human embryonic kidney 293 cell. The ability to determine complex I content should provide a valuable tool to investigate the enzyme status in samples after in vivo treatment in mutant organisms, cells in culture, or human biopsies.     

Role of β-Oxidation Enzymes in γ-Decalactone Production by the Yeast Yarrowia lipolytica

Some microorganisms can transform methyl ricinoleate into γ-decalactone, a valuable aroma compound, but yields of the bioconversion are low due to (i) incomplete conversion of ricinoleate (C₁₈) to the C₁₀ precursor of γ-decalactone, (ii) accumulation of other lactones (3-hydroxy-γ-decalactone and 2- and 3-decen-4-olide), and (iii) γ-decalactone reconsumption. We evaluated acyl coenzyme A (acyl-CoA) oxidase activity (encoded by the POX1 through POX5 genes) in Yarrowia lipolytica in lactone accumulation and γ-decalactone reconsumption in POX mutants. Mutants with no acyl-CoA oxidase activity could not reconsume γ-decalactone, and mutants with a disruption of pox3, which encodes the short-chain acyl-CoA oxidase, reconsumed it more slowly. 3-Hydroxy-γ-decalactone accumulation during transformation of methyl ricinoleate suggests that, in wild-type strains, β-oxidation is controlled by 3-hydroxyacyl-CoA dehydrogenase. In mutants with low acyl-CoA oxidase activity, however, the acyl-CoA oxidase controls the β-oxidation flux. We also identified mutant strains that produced 26 times more γ-decalactone than the wild-type parents.     

Organelle-specific Isozymes of Aspartate-alpha-Ketoglutarate Transaminase in Spinach Leaves

Four distinct isozymes of aspartate-α-ketoglutarate transaminase in a spinach (Spinacia oleracea L.) leaf extract were separated by starch gel electrophoresis. Of the total aspartate-α-ketoglutarate transaminase activity, approximately 45% was represented by the chloroplast isozyme, 26% by the cytosol isozyme, 19% by the mitochondrial isozyme, and 3 to 10% by the peroxisomal isozyme. The aspartate-α-ketoglutarate transamination activity in the four subcellular compartments behaved similarly. It was freely reversible and α-ketoglutarate was preferred to pyruvate or glyoxylate as the amino group acceptor. With glutamate as the amino group donor, oxaloacetate was superior to pyruvate or glyoxylate as the acceptor in chloroplasts, mitochondria, and cytosol, while pyruvate or glyoxylate was preferred to oxaloacetate as the acceptor in peroxisomes.