Threonine Degradation Research Articles

The Fur regulon in anaerobically grown Salmonella enterica sv. Typhimurium: identification of new Fur targets

BackgroundThe Ferric uptake regulator (Fur) is a transcriptional regulator that controls iron homeostasis in bacteria. Although the regulatory role of Fur in Escherichia coli is well characterized, most of the studies were conducted under routine culture conditions, i.e., in ambient oxygen concentration. To reveal potentially novel aspects of the Fur regulon in Salmonella enterica serovar Typhimurium under oxygen conditions similar to that encountered in the host, we compared the transcriptional profiles of the virulent wild-type strain (ATCC 14028s) and its isogenic Δfur strain under anaerobic conditions.ResultsMicroarray analysis of anaerobically grown Δfur S. Typhimurium identified 298 differentially expressed genes. Expression of several genes controlled by Fnr and NsrR appeared to be also dependent on Fur. Furthermore, Fur was required for the activity of the cytoplasmic superoxide disumutases (MnSOD and FeSOD). The regulation of FeSOD gene, sodB, occurred via small RNAs (i.e., the ryhB homologs, rfrA and rfrB) with the aid of the RNA chaperone Hfq. The transcription of sodA was increased in Δfur; however, the enzyme was inactive due to the incorporation of iron instead of manganese in SodA. Additionally, in Δfur, the expression of the gene coding for the ferritin-like protein (ftnB) was down-regulated, while the transcription of the gene coding for the nitric oxide (NO·) detoxifying flavohemoglobin (hmpA) was up-regulated. The promoters of ftnB and hmpA do not contain recognized Fur binding motifs, which indicated their probable indirect regulation by Fur. However, Fur activation of ftnB was independent of Fnr. In addition, the expression of the gene coding for the histone-like protein, H-NS (hns) was increased in Δfur. This may explain the observed down-regulation of the tdc operon, responsible for the anaerobic degradation of threonine, and ftnB in Δfur.ConclusionsThis study determined that Fur is a positive factor in ftnB regulation, while serving to repress the expression of hmpA. Furthermore, Fur is required for the proper expression and activation of the antioxidant enzymes, FeSOD and MnSOD. Finally, this work identified twenty-six new targets of Fur regulation, and demonstrates that H-NS repressed genes are down-regulated in Δfur.

Low Α, Β – Unsaturated Carbonyl Compounds as a Food Risk Factor in Thermal Processing of Grapeseed Oil

Low α,β – unsaturated carbonyl compounds (LUCC) (2-propenal, crotonaldehyde) and other similar structures highly reactive organic chemicals is ubiquitously present in (cooked) foods and in the environment. Chemical reactions responsible for release of acrolein (ACR) include heat-induced dehydration of glycerol, retro-aldol cleavage of dehydrated carbohydrates, lipid peroxidation of polyunsaturated fatty acids, and Strecker degradation of methionine and threonine. Thermal processing (managing or aggressive non-monitored) of grapeseeds oil can become a food-risk factor.

Acrolein: Sources, metabolism, and biomolecular interactions relevant to human health and disease

Acrolein (2-propenal) is ubiquitously present in (cooked) foods and in the environment. It is formed from carbohydrates, vegetable oils and animal fats, amino acids during heating of foods, and by combustion of petroleum fuels and biodiesel. Chemical reactions responsible for release of acrolein include heat-induced dehydration of glycerol, retro-aldol cleavage of dehydrated carbohydrates, lipid peroxidation of polyunsaturated fatty acids, and Strecker degradation of methionine and threonine. Smoking of tobacco products equals or exceeds the total human exposure to acrolein from all other sources. The main endogenous sources of acrolein are myeloperoxidase-mediated degradation of threonine and amine oxidase-mediated degradation of spermine and spermidine, which may constitute a significant source of acrolein in situations of oxidative stress and inflammation. Acrolein is metabolized by conjugation with glutathione and excreted in the urine as mercapturic acid metabolites. Acrolein forms Michael adducts with ascorbic acid in vitro, but the biological relevance of this reaction is not clear. The biological effects of acrolein are a consequence of its reactivity towards biological nucleophiles such as guanine in DNA and cysteine, lysine, histidine, and arginine residues in critical regions of nuclear factors, proteases, and other proteins. Acrolein adduction disrupts the function of these biomacromolecules which may result in mutations, altered gene transcription, and modulation of apoptosis.

New Functions for Parts of the Krebs Cycle in Procyclic Trypanosoma brucei, a Cycle Not Operating as a Cycle

We investigated whether substrate availability influences the type of energy metabolism in procyclic Trypanosoma brucei. We show that absence of glycolytic substrates (glucose and glycerol) does not induce a shift from a fermentative metabolism to complete oxidation of substrates. We also show that glucose (and even glycolysis) is not essential for normal functioning and proliferation of pleomorphic procyclic T. brucei cells. Furthermore, absence of glucose did not result in increased degradation of amino acids. Variations in availability of glucose and glycerol did result, however, in adaptations in metabolism in such a way that the glycosome was always in redox balance. We argue that it is likely that, in procyclic cells, phosphoglycerate kinase is located not only in the cytosol, but also inside glycosomes, as otherwise an ATP deficit would occur in this organelle. We demonstrate that procyclic T. brucei uses parts of the Krebs cycle for purposes other than complete degradation of mitochondrial substrates. We suggest that citrate synthase plus pyruvate dehydrogenase and malate dehydrogenase are used to transport acetyl-CoA units from the mitochondrion to the cytosol for the biosynthesis of fatty acids, a process we show to occur in proliferating procyclic cells. The part of the Krebs cycle consisting of alpha-ketoglutarate dehydrogenase and succinyl-CoA synthetase was used for the degradation of proline and glutamate to succinate. We also demonstrate that the subsequent enzymes of the Krebs cycle, succinate dehydrogenase and fumarase, are most likely used for conversion of succinate into malate, which can then be used in gluconeogenesis.

Temporal Response of Hepatic Threonine Dehydrogenase in Chickens to the Initial Consumption of a Threonine-Imbalanced Diet

Amino acid imbalances contribute to higher requirements of amino acids than would occur if the dietary profile of amino acids perfectly matched the requirements. The mechanisms of imbalances have not been fully elucidated. Because threonine dehydrogenase (TDH) activity in liver mitochondria increases in chicks and rats subjected to threonine imbalance, the current study was carried out to determine whether the change in TDH activity occurs rapidly enough after the consumption of an imbalanced diet to be considered a possible primary metabolic response. In a series of experiments, Leghorn chicks were allowed free access to a semipurified basal diet marginally limited in threonine or the same diet containing a mixture of indispensable amino acids (IAA) lacking threonine to cause a threonine imbalance. In the first experiment, dietary supplements of 5.5 and 11.1% IAA were used to determine a level of supplement that would cause a robust response in the specific activity of TDH. Feed intake, body weight gains and efficiency of feed utilization were lower and specific activities of TDH were higher in chicks fed 11.1% IAA than in those fed 5.5% IAA. In subsequent experiments, hepatic TDH activities and plasma amino acid profiles of the control and experimental groups were determined at 1. 5, 3, 6, 12 and 24 h after the first offering of the diet containing 11.1% IAA. The specific activities of TDH in chicks fed the IAA supplement were 40-150% higher (P < 0.05) and plasma threonine concentrations were 42-53% lower (P < 0.05) than in chicks fed the basal diet at all times except 1.5 h. These results indicate that changes in the capacity for threonine degradation via TDH may occur in the liver within a few hours after the consumption of a threonine-imbalanced diet and suggest the possibility that altered TDH activity may contribute to the increased threonine requirement associated with threonine imbalance.

Enrichment of threonine content in Saccharomyces cerevisiae by pathway engineering.

In a previous work, we have investigated the effect of amplifying individually the genes of the threonine biosynthetic pathway on threonine accumulation by yeast. Here, we present the results of the simultaneous amplification of these genes in strains with different genetic backgrounds. These strains carry a mutant HOM3-R2 allele (coding for a feedback-insensitive aspartate kinase), and/or a mutant cha1 allele that makes it defective in threonine degradation by the catabolic L-serine (L-threonine) deaminase. The results show that the amplification of the clustered genes affects threonine and homoserine accumulation only when it includes the HOM3 gene, or when combined with a HOM3-R2 mutation. Similarly, the cha1 mutation is only effective when a certain amount of threonine is reached. Threonine overproduction affects other cellular functions such as the accumulation of other amino acids, the cell growth and metabolite excretion, probably reflecting a redirection of the carbon flux in the central metabolism.

Threonine dehydrogenase is a minor degradative pathway of threonine catabolism in adult humans.

The threonine dehydrogenase (TDG) pathway is a significant route of threonine degradation, yielding glycine in experimental animals, but has not been accurately quantitated in humans. Therefore, the effect of a large excess of dietary threonine, given either as free amino acid (+Thr) or as a constituent of protein (+P-Thr), on threonine catabolism to CO(2) and to glycine was studied in six healthy adult males using a 4-h constant infusion of L-[1-(13)C]threonine and [(15)N]glycine. Gas chromatography-combustion isotope ratio mass spectrometry was used to determine [(13)C]glycine produced from labeled threonine. Threonine intakes were higher on +Thr and +P-Thr diets compared with control (126, 126, and 50 micromol x kg(-1) x h(-1), SD 8, P < 0.0001). Threonine oxidation to CO(2) increased threefold in subjects on +Thr and +P-Thr vs. control (49, 45, and 15 micromol x kg(-1) x h(-1), SD 6, P < 0.0001). Threonine conversion to glycine tended to be higher on +Thr and +P-Thr vs. control (3.5, 3.4, and 1.6 micromol x kg(-1) x h(-1), SD 1.3, P = 0.06). The TDG pathway accounted for only 7-11% of total threonine catabolism and therefore is a minor pathway in the human adult.

Pyrazine formation from serine and threonine.

The formation of pyrazines from L-serine and L-threonine has been studied. L-Serine and L-threonine, either alone or combined, were heated at 120 degrees C as low temperature for 4 h or at 300 degrees C as high temperature for 7 min. The pyrazines formed from each reaction were identified by GC/MS, and the yields (to the amino acid used, as parts per million) were determined by GC/FID. It was found that pyrazine, methylpyrazine, ethylpyrazine, 2-ethyl-6-methylpyrazine, and 2,6-diethylpyrazine were formed from serine, whereas 2,5-dimethylpyrazine, 2,6-dimethylpyrazine, trimethylpyrazine, 2-ethyl-3,6-dimethylpyrazine, and 2-ethyl-3, 5-dimethylpyrazine were formed from threonine. Mechanistically, it is proposed that the thermal degradation of serine or threonine is composed of various complex reactions. Among these reactions, decarbonylation followed by dehydration is the main pathway to generate the alpha-aminocarbonyl intermediates leading to the formation of the main product, such as pyrazine from serine or 2, 5-dimethylpyrazine from threonine. Also, deamination after decarbonylation generates more reactive intermediates, alpha-hydroxycarbonyls. Furthermore, aldol condensation of these reactive intermediates provides alpha-dicarbonyls. Subsequently, these alpha-dicarbonyls react with the remaining serine or threonine by Strecker degradation to form additional alpha-aminocarbonyl intermediates, which then form additional pyrazines. In addition, decarboxylation and retroaldol reaction may also involve the generation of the intermediates.

Role of Escherichia coli RpoS, LexA and H-NS global regulators in metabolism and survival under aerobic, phosphate-starvation conditions.

It has been suggested that Escherichia coli can resist aerobic, glucose-starvation conditions by switching rapidly from an aerobic to a fermentative metabolism, thereby preventing the production by the respiratory chain of reactive oxygen species (ROS) that can damage cellular constituents. In contrast, it has been reported that E. coli cannot resist aerobic, phosphate (Pi)-starvation conditions, probably because of the maintenance of an aerobic metabolism and the continuous production of ROS. This paper presents evidence that E. coli cells starved for Pi under aerobic conditions indeed maintain an active aerobic metabolism for about 3 d, which allows the complete degradation of exogenous nutrients such as arginine (metabolized probably to putrescine via the SpeA-initiated pathway) and glucose (metabolized notably to acetate), but cell viability is not significantly affected because of the protection afforded against ROS through the expression of the RpoS and LexA regulons. The involvement of the LexA-controlled RuvAB and RecA proteins with the RecG and RecBCD proteins in metabolism and cell viability implies that DNA double-strand breaks (DSB), and thus hydroxyl radicals that normally generate this type of damage, are produced in Pi-starved cells. It is shown that induction of the LexA regulon, which helps protect Pi-starved cells, is totally prevented by introduction of a recB mutation, which indicates that DSB are actually the main DNA lesion generated in Pi-starved cells. The requirement of RpoS for survival of cells starved for Pi may thus be explained by the role played by various RpoS-controlled gene products such as KatE, KatG and Dps in the protection of DNA against ROS. In the same light, the degradation of arginine and threonine may be accounted for by the synthesis of polyamines (putrescine and spermidine) that protect nucleic acids from ROS. Besides LexA and RpoS, a third global regulator, the nucleoid-associated protein H-NS, is also shown to play a key role in Pi-starved cells. Through a modulation of the metabolism during Pi starvation, H-NS may perform two complementary tasks: it helps maintain a rapid metabolism of glucose and arginine, probably by favouring the activity of aerobic enzymes such as the NAD-dependent pyruvate dehydrogenase complex, and it may enhance the cellular defences against ROS which are then produced by increasing RpoS activity via the synthesis of acetate and presumably homoserine lactone.

Expression of the Escherichia coli catabolic threonine dehydratase in Corynebacterium glutamicum and its effect on isoleucine production.

The catabolic or biodegradative threonine dehydratase (E.C. 4.2.1. 16) of Escherichia coli is an isoleucine feedback-resistant enzyme that catalyzes the degradation of threonine to alpha-ketobutyrate, the first reaction of the isoleucine pathway. We cloned and expressed this enzyme in Corynebacterium glutamicum. We found that while the native threonine dehydratase of C. glutamicum was totally inhibited by 15 mM isoleucine, the heterologous catabolic threonine dehydratase expressed in the same strain was much less sensitive to isoleucine; i.e., it retained 60% of its original activity even in the presence of 200 mM isoleucine. To determine whether expressing the catabolic threonine dehydratase (encoded by the tdcB gene) provided any benefit for isoleucine production compared to the native enzyme (encoded by the ilvA gene), fermentations were performed with the wild-type strain, an ilvA-overexpressing strain, and a tdcB-expressing strain. By expressing the heterologous catabolic threonine dehydratase in C. glutamicum, we were able to increase the production of isoleucine 50-fold, whereas overexpression of the native threonine dehydratase resulted in only a fourfold increase in isoleucine production. Carbon balance data showed that when just one enzyme, the catabolic threonine dehydratase, was overexpressed, 70% of the carbon available for the lysine pathway was redirected into the isoleucine pathway.

Threonine kinetics in preterm infants fed their mothers' milk or formula with various ratios of whey to casein

Plasma threonine concentrations are elevated in infants fed formula containing a whey-to-casein protein ratio of 60:40 compared with concentrations in infants fed formula containing a ratio of 20:80 or human milk (60:40). We studied whether degradation of excess threonine was lower in formula-fed infants than in infants fed their mothers' milk. Threonine kinetics were examined in 17 preterm infants (gestational age: 31+/-2 wk: birth weight: 1720+/-330 g) by using an 18-h oral infusion of [1-13C]threonine at a postnatal age of 21+/-11 d and weight of 1971+/-270 g. Five infants received breast milk. Formula-fed infants (n = 12) were randomly assigned to receive 1 of 3 formulas (5.3 g protein/MJ) that differed only in the whey-to-casein ratio (20:80, 40:60, and 60:40). Threonine intake increased significantly in formula-fed infants with increasing whey content of the formula (48.5, 56.4, and 63.2 micromol.kg(-1).h(-1), respectively; pooled SD: 2.2; P = 0.0001), as did plasma threonine concentrations (228, 344, and 419 micromol/L, respectively; pooled SD: 75; P = 0.03). Despite a generous threonine intake by infants fed breast milk (58.0+/-16.0 micromol.kg(-1).h(-1), plasma threonine concentrations remained low (208+/-41 micromol/L). Fecal threonine excretion and net threonine tissue gain, estimated by nitrogen balance, did not differ significantly among groups. Threonine oxidation did not differ significantly among formula-fed infants but was significantly lower in formula-fed infants fed than in infants fed breast milk (17.1% compared with 24.3% of threonine intake, respectively). Formula-fed infants have a lower capacity to oxidize threonine than do infants fed breast milk.

The anaerobic degradation of L-serine and L-threonine in enterobacteria: networks of pathways and regulatory signals.

The mechanisms controlling the biosynthesis and degradation of l-serine and l-threonine are remarkably complex. Their metabolism forms a network of pathways linking several amino acids, central primary metabolites such as pyruvate, oxaloacetate and 3-phosphoglycerate, and C1 metabolism. Studies on the degradation of these amino acids in Escherichia coli have revealed the involvement of fascinating enzymes that utilise quite diverse catalytic mechanisms. Moreover, it is emerging that both environmental and metabolic signals have a major impact in controlling enzyme synthesis This is exemplified by the anaerobically regulated tdc operon, which encodes a metabolic pathway for the degradation of serine and threonine. Studies on this pathway are beginning to provide insights into how an organism adapts its genetic makeup to meet the physiological demands of the cell.

Hydrolysis of tannery wastes to protein meal for animal feedstuffs: A process and product evaluation

AbstractHydrolysis of protein solid wastes from tanneries to yield protein meal for animal nutrition has been performed in a pilot assembly designed to process 400 kg h−1 of disposal material under optimum energy consumption and product yield conditions. Evaluation of the significance of amino acid degradation caused by the process, in relation to the product cost and quality in animal nutrition, was the purpose. A nutrition bioassay was carried out to study the incidence of dietary substitution of the above protein meal for soybean meal on growth performance by early weaned piglets. The results show that degradation of serine, proline, arginine and threonine occurs during collagen hydrolysis, and that only the threonine concentration in the product falls below the dietary requirement. However, integration of protein meal diets with the necessary amino acid content may be accomplished cheaply. Compared with other vegetable and animal commercial protein meals, the cost of the product obtained from tanned hides appears competitive. Processing of other tannery disposal material is not recommended, due to the low protein content of the starting material.

In Vitro Testing of a Glyoxalase I Inhibitor

Glyoxalase I (S-D-lactoylglutathione methylglyoxal lyase (isomerising), EC 4.4.1.5) and glyoxalase I1 (S-2hydroxyacylglutathione hydrolase, EC 3.1.2.6) catalyze the conversion of toxic a-ketoaldehydes to nontoxic ahydroxycarboxylic acids. Endogenously formed methylglyoxal is converted to D-lactic acid by the tandem action of these two ubiquitous enzymes and catalytic quantities of reduced glutathione (1). The primary biological role of the glyoxalase system is not known but the enzyme system may be involved in regulation of cell division (2), threonine degradation (11, regulation of heme biosynthesis (3) and methylglyoxal detoxification (1). Recent evidence indicates that methylglyoxal is formed as a side product (0.4 mM/cell/day) during the conversion of dihydroxyacetone phosphate to glyceraldehyde 3phosphate by triosephosphate isomerase (43). Methylglyoxal is also formed enzymatically from triose phosphates (6) and from acetone by hepatic acetoneinducible cytochrome P,,, (7). Hence, the glyoxalase system serves as one of the major pathways for aketoaldehyde metabolism. Inhibitors of the glyoxalase system may prove to be useful in controlling or eliminating certain pathologies (cancer and inflammatory processes). Such inhibitors may also be useful as probes of the glyoxalase system. For example, it was recently shown that the malarial parasite Plasmodium falciparum consumes large amounts of glucose during the intraerythrocyte stage of its lifecycle and that 6-7% of the glucose consumed is converted to D-lactate via the glyoxalase system (8). Several glutathione analogues were tested against yeast glyoxalase I. Under our assay conditions (9), the compound S-(N-phenethylthiocarbamoy1)-L-glutathione (Figure 1) was determined to have an IC,, of approximately 156 pM (10). As this compound was

High Levels of Acetaldehyde in Nonalcoholic Liver Injury After Threonine or Ethanol Administration

Acetaldehyde, a product of ethanol oxidation which forms adducts with proteins, has been incriminated in the pathogenesis of alcoholic liver injury. High serum antibody titers against acetaldehyde-protein adducts have been found not only in alcoholics but also in patients with nonalcoholic liver disease, suggesting a contribution of acetaldehyde derived from sources other than exogenous ethanol. To investigate the effect of liver injury on the removal and the production of acetaldehyde, we produced fibrosis and cirrhosis (by chronic administration of carbon tetrachloride) and fatty liver (with very small doses of dimethylnitrosamine) in rats. Endogenous blood acetaldehyde levels increased by 38% in rats with severe liver injury (p less than 0.005), but not significantly in rats with fatty liver. However, an i.v. load of threonine (a physiological source of acetaldehyde), in amounts equivalent to the daily intake of this amino acid, increased blood and hepatic acetaldehyde levels in the rats with both types of liver injury more than in controls. Threonine dehydrogenase and dehydratase activities, involved in the major pathways for threonine degradation in mitochondria and cytosol, respectively, were markedly decreased in rats with liver injury with a resulting increase in hepatic threonine concentration. Moreover, the threonine aldolase activity, which splits threonine into glycine and acetaldehyde, remained unaffected or even slightly increased. Liver injury was also associated with impaired mitochondrial functions, including a 10 to 23% decrease in acetaldehyde oxidation (depending upon the severity of the lesions). As a consequence, administration of ethanol (an exogenous source of acetaldehyde) resulted in striking elevations in the levels of acetaldehyde in carbon tetrachloride-treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)

THE SIGNIFICANCE OF SILAGE PROTEIN DEGRADATION AND PLASMA AMINO ACID RATIOS IN THE CONTROL OF FOOD INTAKE BY LAMBS FED ENSILED AND FRESH ALFALFA

Alfalfa (30% dry matter) was harvested with a particle length of 0.6 cm, conserved fresh (frozen) or as silage and fed to eight lambs during two 27-d periods. The ensiling process resulted in substantial degradation of lysine, threonine, histidine, arginine, tryptophan and tyrosine in the original herbage. Isoleucine, leucine and valine resisted degradation during ensilage. As a consequence of these changes, the ratios of tyrosine: large neutral amino acids and tyrosine: phenylalanine (TYR:LNAA and TYR:PHE) were markedly lower in ensiled compared to fresh alfalfa (0.035 vs. 0.051 and 0.235 vs. 0.313, respectively) but these ratios in plasma were higher (P &lt; 0.05) with silage than with fresh forage (0.114 vs. 0.095 and 1.020 vs. 0.997). Ensiling of alfalfa resulted in a 19% reduction in silage intake but the response did not correlate with changes in plasma ratios of TYR:LNAA and TYR:PHE. Nitrogen retention was 27% lower with ensiled than with fresh alfalfa. Plasma levels of branched chain and other essential amino acids tended to be higher with fresh than ensiled forage but the difference was not significant. It is concluded that the reduction in food intake with alfalfa silage was unrelated to changes in specific ratios of amino acids in plasma but could be related to the supply of essential amino acids to the intestine of lambs. Key words: Silage intake, lambs, nitrogen balance, plasma amino acids, alfalfa

Amino acid catabolism by perfused rat hindquarters: Degradation of threonine and isoleucine

Rat hindquarters were perfused without added substrate other than trace amounts of [U- 14C]threonine or [U- 14C]isoleucine. Comparison of incorporation of radiolabel into some nonessential amino acids, citrate cycle intermediates, and lactate is presented. Activities of three enzymes for the initial reactions in threonine degradation are reported. It is concluded that skeletal muscle catabolizes threonine, and that the latter is a potential source of carbon for glucogenic precursors for the liver. In contrast, label from isoleucine was incorporated into glutamate, glutamine, and alanine much more than was that from threonine. Large amounts of organic acids accumulated, and more than 60% of total radioactivity was lost as CO 2 during a 2-h perfusion period.

Localization of malate dehydrogenase, adenylate kinase and glycolytic enzymes in glycosomes and the threonine pathway in the mitochondrion of cultured procyclic trypomastigotes of Trypanosoma brucei

Procyclic culture forms of Trypanosoma brucei stock 427 have been screened for the presence of enzymes involved in glycolysis, mitochondrial energy metabolism and threonine degradation. The enzyme activities in the procyclics were compared with those of the bloodstream forms. The specific activities of glycolytic enzymes represented 30–70% of the respective levels in the bloodstream form, except for hexokinase which was 25-fold reduced. Cell fractionation showed that the enzymes involved in the early sequence of the glycolytic pathway, i.e. from hexokinase to phosphoglycerate kinase, and the enzymes NAD +-linked glycerol-3-phosphate dehydrogenase and glycerol kinase were all present in glycosomes equilibrating at a density of 1.23 g/cm 3 in sucrose gradients. Malate dehydrogenase was 8-fold more active in procyclics than in bloodstream forms. This increase in activity was the result of the appearance of malate dehydrogenase in the glycosomes of the procyclics, in addition to mitochondrial and cell-sap activities which were present in both stages of the life cycle. Glycosomes contained part of the adenylate kinase activity, which was also associated with the mitochondrion. Succinate dehydrogenase and sn-glycerol-3-phosphate dehydrogenase, together with oligomycin-sensitive ATPase, were located in the mitochondrion which had a density in sucrose ranging from 1.16 to 1.18 g/cm 3. This organelle also contained L-threonine 3-dehydrogenase and carnitine acetyltransferase, two enzymes involved in threonine catabolism. The latter two enzymes had activities which were, respectively, 15-and 13-fold higher in the procyclics than in the bloodstream form. Mitochondrial sn-glycerol-3-phosphate dehydrogenase was decreased 4-fold.

Hydroxy amino acid metabolism in Pseudomonas cepacia: role of L-serine deaminase in dissimilation of serine, glycine, and threonine.

Growth of Pseudomonas cepacia (P. multivorans) on serine depended upon induction of a previously undescribed L-serine deaminase distinct from threonine deaminase. Formation of the enzyme was induced during growth on serine, glycine, or threonine. The induction pattern reflected a role of the enzyme in catabolism of these three amino acids. Both threonine and glycine supported growth of serine auxotrophs and were presumably converted to serine and pyruvate in the course of their degradation. Mutant strains deficient in serine deaminase, or unable to use pyruvate as a carbon source, failed to utilize serine or glycine and grew poorly with threonine, whereas strains deficient in threonine dehydrogenase or alpha-amino beta-ketobutyrate:coenzyme A ligase (which together convert threonine to glycine and acetyl coenzyme A) failed to utilize threonine or derepress serine deaminase in the presence of this amino acid. The results confirm for the first time the role of alpha-amin beta-ketobutyrate:coenzyme A ligase in threonine degradation and indicate that threonine does not mimic serine as an inducer of serine deaminase.

Control of biodegradative threonine dehydratase inducibility by cyclic AMP in energy-restricted Escherichia coli

To explain the requirement for anaerobic conditions in the induction of biodegradative L-threonine dehydratase in Escherichia coli, Crookes strain, measurements of cyclic AMP (cAMP) were made during aerobic and anaerobic growth and upon an aerobic-to-anaerobic transition. Internal cAMP levels were similar (5 to 10 muM) throughout exponential growth, whether aerobic or anaerobic, but only during anaerobiosis was threonine dehydratase synthesized. When an exponentially growing aerobic culture was made anaerobic, a sharp increase in internal cAMP was noted, reaching 300 muM within 10 min and declining thereafter to normal anaerobic levels. Threonine dehydratase synthesis was detected immediately after the attainment of peak cAMP levels and continued for several generations. A similar pattern but with less accumulation of cAMP and less threonine dehydratase production was also noted upon treatment of an aerobically growing culture with KCN. Pyruvate addition at the time of anaerobic shock severely affected both cAMP accumulation and threonine dehydratase synthesis; however, externally added cAMP could partially counter the pyruvate effect on enzyme synthesis. The conclusion was reached that conditions which resulted in a temporary energy deficit brought about the major accumulation of cAMP, and this elevated level served as a signal for initiation of threonine dehydratase synthesis to supply energy by the nonoxidative degradation of threonine.