Threonine metabolism byPseudomonas aeruginosa

83 strains ofPseudomonas aeruginosa were unable to utilizel-threonine as carbon-energy source, although this compound served as sole nitrogen source. Auxotrophs ofP. aeruginosa 9-D2 that requiredl-serine or glycine for growth could grow in the presence ofl-threonine. Extracts ofP. aeruginosa 9-D2 grown in the presence ofl-threonine contained threonine dehydrogenase and alpha-amino beta-ketobutyrate: CoA ligase activities; threonine aldolase was not detectable. Cells grown in the absence ofl-threonine produced no detectable threonine dehydrogenase.l-Leucine neither stimulated nor repressed threonine dehydrogenase levels. Glycine, and to a lesser extentl-serine, repressedl-threonine-mediated threonine dehydrogenase synthesis. A mutant of strain 9-D2 was isolated that could utilizel-threonine as sole carbon-energy source. This strain produced elevated levels of threonine dehydrogenase, but only slightly higher levels of alpha-amino beta-ketobutyrate: CoA ligase activities.     

Metabolism of threonine by Fusaria growth on threonine as the sole carbon and nitrogen source

Three strains ofFusarium supporting aerobic growth onl-threonine as the sole source of energy and carbon and nitrogen, initially metabolised threonine to acetyl-CoA and glycine via induciblel-threonine:NAD⁺ dehydrogenase plus 2-amino-3-oxobutyrate:CoA ligase activities. Comparative enzyme induction patterns after growth of the three strains on a wide range of carbon sources indicated that the glycine produced by the NAD⁺ plus CoASH-dependent cleavage of threonine was subsequently utilised as an energy source and biosynthetic precursor via the glycine-serine pathway, pyruvate carboxylase, and ultimately the TCA cycle. Acetyl-CoA, the second initial C₂ threonine catabolism product, was subsequently assimilated via a combined TCA plus glyoxylate cycle.     

Role of threonine dehydrogenase in Escherichia coli threonine degradation.

Threonine was used as nitrogen source by Escherichia coli K-12 through a pathway beginning with the enzyme threonine dehydrogenase. The 2-amino-3-ketobutyrate formed was converted to glycine, and the glycine was converted to serine, which acted as the actual nitrogen donor. The enzyme formed under anaerobic conditions and known as threonine deaminase (biodegradative) is less widespread than threonine dehydrogenase and may be involved in energy metabolism rather than in threonine degradation per se.     

Multivalent Induction of Biodegradative Threonine Deaminase

To determine the inducer(s) of the biodegradative threonine deaminase in Escherichia coli, the effects of various amino acids on the synthesis of this enzyme were investigated. The complex medium used hitherto for the enzyme induction can be completely replaced by a synthetic medium composed of 18 natural amino acids. In this synthetic medium, the omission of each of the seven amino acids threonine, serine, aspartic acid, methionine, valine, leucine, and arginine resulted in the greatest loss of enzyme formation. These seven amino acids did not significantly influence the uptake of other amino acids into the cells. Furthermore, they did not stimulate the conversion of inactive enzyme into an active form, since they did not affect the enzyme level in cells in which protein synthesis was inhibited by chloramphenicol. Threonine, serine, aspartic acid, and methionine failed to stimulate enzyme production in cells in which messenger ribonucleic acid synthesis was arrested by rifampin, whereas valine, leucine, and arginine stimulated enzyme synthesis under the same conditions. Therefore, the first four amino acids appear to act as inducers of the biodegradative threonine deaminase in E. coli and the last three amino acids appear to be amplifiers of enzyme production. The term "multivalent induction" has been proposed for this type of induction, i.e., enzyme induction only by the simultaneous presence of several amino acids.     

Threonine as a carbon source for Escherichia coli.

Threonine can be used aerobically as the sole source of carbon and energy by mutants of Escherichia coli K-12. The pathway used involves the conversion of threonine via threonine dehydrogenase to aminoketobutyric acid, which is further metabolized by aminoketobutyric acid ligase, forming acetyl coenzyme A and glycine. A strain devoid of serine transhydroxymethylase uses this pathway and excretes glycine as a waste product. Aminoketobutyric acid ligase activity was demonstrated after passage of crude extracts through Sephadex G100.     

Synthesis of biodegradative threonine dehydratase in Escherichia coli: role of amino acids, electron acceptors, and certain intermediary metabolites

The specific activity of inducible biodegradative threonine dehydratase (EC 4.2.1.16) in Escherichia coli K-12 increased significantly when the standard tryptone-yeast extract medium or a synthetic mixture of 18 L-amino acids was supplemented with 10 mM KNO3 or 50 mM fumarate and with 4 mM cyclic AMP. In absolute terms, almost four times as much enzyme was produced in the amino acid medium as in the tryptone-yeast extract medium. Enzyme induction in the amino acid medium was sensitive to catabolite repression by glucose, gluconate, glycerol, and pyruvate. An analysis of amino acid requirements for enzyme induction showed that a combination of only four amino acids, threonine, serine, valine, and isoleucine, produced high levels of threonine dehydratase provided that both fumarate and cyclic AMP were present. Immunochemical data revealed that the enzyme synthesized in the presence of these four amino acids was indistinguishable from that produced in the tryptone-yeast extract or the medium with 18 amino acids. We interpret these results to mean that not the amino acids themselves but some metabolites derived anaerobically in reactions involving an electron acceptor may function as putative regulatory molecule(s) in the anaerobic induction of this enzyme.     

Biosynthesis of amino acids in Clostridium pasteurianum

1. Clostridium pasteurianum was grown on a synthetic medium with the following carbon sources: (a) (14)C-labelled glucose, alone or with unlabelled aspartate or glutamate, or (b) unlabelled glucose plus (14)C-labelled aspartate, glutamate, threonine, serine or glycine. The incorporation of (14)C into the amino acids of the cell protein was examined. 2. In both series of experiments carbon from exogenous glutamate was incorporated into proline and arginine; carbon from aspartate was incorporated into glutamate, proline, arginine, lysine, methionine, threonine, isoleucine, glycine and serine. Incorporations from the other exogenous amino acids indicated the metabolic sequence: aspartate --> threonine --> glycine right harpoon over left harpoon serine. 3. The following activities were demonstrated in cell-free extracts of the organism: (a) the formation of aspartate by carboxylation of phosphoenolpyruvate or pyruvate, followed by transamination; (b) the individual reactions of the tricarboxylic acid route to 2-oxoglutarate from oxaloacetate; glutamate dehydrogenase was not detected; (c) the conversion of aspartate into threonine via homoserine; (d) the conversion of threonine into glycine by a constitutive threonine aldolase; (e) serine transaminase, phosphoserine transaminase, glycerate dehydrogenase and phosphoglycerate dehydrogenase. This last activity was abnormally high. 4. The combined evidence indicates that in C. pasteurianum the biosynthetic role of aspartate and glutamate is generally similar to that in aerobic and facultatively aerobic organisms, but that glycine is synthesized from glucose via aspartate and threonine.     

Study of amino acid utilization by isogenic strains of Escherichia coli in relation to the culture growth and biosynthesis of penicillin amidase

Dynamics of free amino acid utilization by isogenic strains of Escherichia coli differing in intensity of their growth and levels of penicillin acylase biosynthesis in media containing corn steep liquor or peptone was studied. It was shown that in both the media some amino acids such as serine, threonine, glutaminic and asparaginic acids were actively utilized by the strains mainly during the culture intensive growth while others such as glycine, alanine and tyrosine were actively utilized during the enzyme biosynthesis. Intensively utilized arginine and proline were probably used for the growth and biosynthesis. The other amino acids were not utilized completely from the media. The lowest levels of their utilization were observed when the strains were cultivated in the medium with peptone.     

Utilization of l-threonine by a species of Arthrobacter. A novel catabolic role for `aminoacetone synthase''

1. A species of Arthrobacter (designated Arthrobacter 9759) was isolated from soil by its ability to grow aerobically on l-threonine as sole source of carbon atoms, nitrogen atoms and energy; the organism also grew well on other sources of carbon atoms including glycine, but no growth was obtainable on aminoacetone or dl-1-aminopropan-2-ol. 2. During growth on threonine, (14)C from l-[U-(14)C]threonine was rapidly incorporated into glycine and citrate, and thereafter into serine, alanine, aspartate and glutamate. 3. With extracts of threonine-grown cells supplied with l-[U-(14)C]threonine, evidence was obtained of the NAD and CoA-dependent catabolism of l-threonine to produce acetyl-CoA plus glycine. Short-term incorporation studies in which [2-(14)C]acetate and [2-(14)C]glycine were supplied (a) to cultures growing on threonine, and (b) to extracts of threonine-grown cells, showed that the acetyl-CoA was metabolized via the tricarboxylic acid cycle and glyoxylate cycle whereas the glycine was converted into pyruvate via the folate-dependent ;serine pathway'. 4. The threonine-grown organism contained ;biosynthetic' threonine dehydratase and a potent NAD-linked l-threonine dehydrogenase but possessed no l-threonine aldolase activity. 5. Evidence was obtained that the acetyl-CoA and glycine produced from l-threonine had their immediate origin in the alpha-amino-beta-oxobutyrate formed by the threonine dehydrogenase; the CoA-dependent cleavage of this compound was catalysed by an alpha-amino-beta-oxobutyrate CoA-ligase, which was identified with ;aminoacetone synthase'. A continuous spectrophotometric assay of this enzyme was developed, and it was found to be inducibly synthesized only during growth on threonine and not during growth on acetate plus glycine. 6. By using a reconstituted mixture of separately purified l-threonine dehydrogenase and alpha-amino-beta-oxobutyrate CoA-ligase (i.e. ;aminoacetone synthase'), l-[U-(14)C]threonine was broken down to [(14)C]glycine plus [(14)C]acetyl-CoA (trapped as [(14)C]citrate). 7. There was no evidence of aminoacetone metabolism by Arthrobacter 9759 even though a small amount of this amino ketone appeared in the culture medium during growth on threonine.     

Bacterial catabolism of threonine. Threonine degradation initiated by L-threonine-NAD+ oxidoreductase.

1. Isolates representing seven bacterial genera capable of growth on L-threonine medium, and possessing high L-threonine 3-dehydrogenase activity, were examined to elucidate the catabolic route. 2. The results of growth, manometric and enzymic experiments indicated the catabolism of L-threonine by cleavage to acetyl-CoA plus glycine, the glycine being further metabolized via L-serine to pyruvate, in all cases. No evidence was obtained of a role for aminoacetone in threonine catabolism or for the metabolism of glycine by the glycerate pathway. 3. The properties of a number of key enzymes in L-threonine catabolism were investigated. The inducibly formed L-threonine 3-dehydrogenase, purified from Corynebacterium sp. B6 to a specific activity of about 30-35 mumol of product formed/min per mg of protein, exhibited a sigmoid kinetic response to substrate concentration. The half-saturating concentration of substrate, [S]0.5, was 20mM and the Hill constant (h) was 1.50. The Km for NAD+ was 0.8mM. The properties of the enzyme were studied in cell-free extracts of other bacteria. 4. New assays for 2-amino-3-oxobutyrate-CoA ligase were devised. The Km for CoA was determined for the first time and found to be 0.14mM at pH8, for the enzyme from Corynebacterium sp. B6. Evidence was obtained for the efficient linkage of the dehydrogenase and ligase enzymes. Cell-free extracts all possessed high activities of the inducibly formed ligase. 5. L-Serine hydroxymethyltransferase was formed constitutively by all isolates, whereas formation of the 'glycine-cleavage system' was generally induced by growth on L-threonine or glycine. The coenzyme requirements of both enzymes were established, and their linked activity in the production of L-serine from glycine was demonstrated by using extracts of Corynebacterium sp. B6. 6. L-Serine dehydratase, purified from Corynebacterium sp. B6 to a specific activity of about 4mumol of product formed/min per mg of protein, was found to exhibit sigmoid kinetics with an [S]0.5 of about 20mM and h identical to 1.4. Similar results were obtained with enzyme preparations from all isolates. The enzyme required Mg2+ for maximum activity, was different from the L-threonine dehydratase also detectable in extracts, and was induced by growth on L-threonine or glycine.     

Derivation of glycine from threonine in Escherichia coli K-12 mutants.

Escherichia coli AT2046 has been shown previously to lack the enzyme serine transhydroxymethylase and to require exogenous glycine for growth as a consequence. Strains JEV73 and JEV73R, mutants derived from strain AT2046, are shown here to be serine transhydroxymethylase deficient, but able to derive their glycine from endogenously synthesized threonine. Leucine is shown to be closely involved in the regulation of biosynthesis of glycine, to spare glycine in strain AT2046T, to replace glycine in strain JEV73, and to increase threonine conversion to glycine in a representative prototroph of E. coli. An interpretation of strains JEV73 and JEV73R as regulatory mutants of strain AT2046 is given. A hypothesis as to the role of leucine as a signal for nitrogen scavenging is suggested.     

4-Hydroxybenzoate-coenzyme A ligase from Rhodopseudomonas palustris: purification, gene sequence, and role in anaerobic degradation.

Anaerobic metabolism of most aromatic acids is initiated by coenzyme A thioester formation. Rhodopseudomonas palustris grows well under anaerobic, phototrophic conditions with many aromatic acids, including benzoate and 4-hydroxybenzoate, as a carbon source. A coenzyme A ligase that reacts with 4-hydroxybenzoate was purified from 4-hydroxybenzoate-grown cells of R. palustris. This enzyme required MgATP, reduced coenzyme A, and 4-hydroxybenzoate, benzoate, or cyclohex-1,4-dienecarboxylate for optimal activity but also used phosphopantetheine, cyclohex-2,5-dienecarboxylate, and 4-fluorobenzoate at lower rates. The 4-hydroxybenzoate-coenzyme A ligase differed in molecular characteristics from a previously described benzoate-coenzyme A ligase from R. palustris, and the two ligases did not cross-react immunologically. The gene encoding the 4-hydroxybenzoate enzyme was cloned and sequenced. The deduced gene product showed about 20% amino acid identity with bacterial coenzyme A ligases involved in aerobic degradation of aromatic acids. An R. palustris mutant carrying a disrupted 4-hydroxybenzoate-coenzyme A ligase gene was unable to grow with 4-hydroxybenzoate under anaerobic conditions, indicating that the enzyme is essential for anaerobic degradation of this compound.     

Overproduction of threonine aldolase circumvents the biosynthetic role of pyruvate decarboxylase in glucose-limited chemostat cultures of Saccharomyces cerevisiae

Pyruvate decarboxylase-negative (Pdc(-)) mutants of Saccharomyces cerevisiae require small amounts of ethanol or acetate to sustain aerobic, glucose-limited growth. This nutritional requirement has been proposed to originate from (i) a need for cytosolic acetyl coenzyme A (acetyl-CoA) for lipid and lysine biosynthesis and (ii) an inability to export mitochondrial acetyl-CoA to the cytosol. To test this hypothesis and to eliminate the C(2) requirement of Pdc(-) S. cerevisiae, we attempted to introduce an alternative pathway for the synthesis of cytosolic acetyl-CoA. The addition of L-carnitine to growth media did not restore growth of a Pdc(-) strain on glucose, indicating that the C(2) requirement was not solely due to the inability of S. cerevisiae to synthesize this compound. The S. cerevisiae GLY1 gene encodes threonine aldolase (EC 4.1.2.5), which catalyzes the cleavage of threonine to glycine and acetaldehyde. Overexpression of GLY1 enabled a Pdc(-) strain to grow under conditions of carbon limitation in chemostat cultures on glucose as the sole carbon source, indicating that acetaldehyde formed by threonine aldolase served as a precursor for the synthesis of cytosolic acetyl-CoA. Fractionation studies revealed a cytosolic localization of threonine aldolase. The absence of glycine in these cultures indicates that all glycine produced by threonine aldolase was either dissimilated or assimilated. These results confirm the involvement of pyruvate decarboxylase in cytosolic acetyl-CoA synthesis. The Pdc(-) GLY1 overexpressing strain was still glucose sensitive with respect to growth in batch cultivations. Like any other Pdc(-) strain, it failed to grow on excess glucose in batch cultures and excreted pyruvate when transferred from glucose limitation to glucose excess.     

Growth Inhibition of Streptomyces Species by l-Serine and Its Effect on Tetracycline Biosynthesis

The addition of serine to minimal medium inhibited the growth of Streptomyces aureofaciens and Streptomyces rimosus. Both the outgrowth of spores and the growth of vegetative cells were inhibited by l-serine. This effect was independent of the carbon source used. In rich nutrient medium, however, the serine effect was not observed. The presence of glycine and methionine in minimal medium reversed the growth inhibition imposed by serine, suggesting that a metabolic block related to the synthesis of these two amino acids was involved. A serine-tolerant mutant of S. aureofaciens isolated after ultraviolet irradiation showed a level of serine deaminase comparable to that of the wild-type strain, which indicated that tolerance to serine was not due to the presence of a more active deaminating enzyme in the mutant. Serine markedly reduced tetracycline and oxytetracycline biosynthesis with the parental strains of Streptomyces spp. The serine-tolerant mutant, however, produced almost the same amount of tetracycline in the presence or absence of serine. The final cell population in fermentation broth was not significantly reduced by l-serine, and the addition of glycine and methionine did not increase the tetracycline yields, which suggested that l-serine inhibition of antibiotic biosynthesis was by a mechanism different from that related to growth inhibition.     

Benzoate-coenzyme A ligase, encoded by badA, is one of three ligases able to catalyze benzoyl-coenzyme A formation during anaerobic growth of Rhodopseudomonas palustris on benzoate.

The first step of anaerobic benzoate degradation is the formation of benzoyl-coenzyme A by benzoate-coenzyme A ligase. This enzyme, purified from Rhodopseudomonas palustris, is maximally active with 5 microM benzoate. To study the molecular basis for this reaction, the benzoate-coenzyme A ligase gene (badA) was cloned and sequenced. The deduced amino acid sequence of badA showed substantial similarity to other coenzyme A ligases, with the highest degree of similarity being that to 4-hydroxybenzoate-coenzyme A ligase (50% amino acid identity) from R. palustris. A badA mutant that was constructed had barely detectable levels of ligase activity when cell extracts were assayed at 10 microM benzoate. Despite this, the mutant grew at wild-type rates on benzoate under laboratory culture conditions (3 mM benzoate), and mutant cell extracts had high levels of ligase activity when assayed at a high concentration of benzoate (1 mM). This suggested that R. palustris expresses, in addition to BadA, a benzoate-activating enzyme(s) with a relatively low affinity for benzoate. A possible role of 4-hydroxybenzoate-coenzyme A ligase (encoded by hbaA) in this capacity was investigated by constructing a badA hbaA double mutant. Although the double mutant grew more slowly on benzoate than badA cells, growth rates were still significant, suggesting the involvement of a third enzyme in benzoate activation. Competition experiments involving the addition of a small amount of cyclohexanecarboxylate to ligase assay mixtures implicated cyclohexanecarboxylate-coenzyme A ligase as being this third enzyme. These results show that wild-type R. palustris cells synthesize at least three enzymes that can catalyze the initial step in anaerobic benzoate degradation during growth on benzoate. This observation supports previous suggestions that benzoyl-coenzyme A formation plays a central role in anaerobic aromatic compound biodegradation.     

The lpd gene product functions as the L protein in the Escherichia coli glycine cleavage enzyme system.

The lpd-encoded lipoamide dehydrogenase, common to the pyruvate and 2-oxoglutarate dehydrogenase multienzyme complexes, also functions as the lipoamide dehydrogenase (L protein) in the Escherichia coli glycine cleavage (GCV) enzyme complex. Inducible GCV enzyme activity was not detected in an lpd deletion mutant; lpd+ transductants had normal levels of inducible GCV enzyme activity. A serA lpd double mutant was unable to utilize glycine as a serine source and lacked detectable GCV enzyme activity, the phenotype of a serA gcv mutant. Transformation of the double mutant with a plasmid encoding a functional lpd gene restored the ability of the mutant to use glycine as a serine source and restored inducible GCV enzyme activity to normal levels. The presence of acetate and succinate in the growth medium of a strain wild type for lpd and gcv resulted in a 50% reduction in inducible GCV enzyme activity. Enzyme levels were restored to normal under these growth conditions when the strain was transformed with a plasmid encoding a functional lpd gene.     

Cobalamin deficiency results in severe metabolic disorder of serine and threonine in rats

Dietary cobalamin (vitamin B12; Cbl) deficiency caused significant increases in plasma serine, threonine, glycine, alanine, tyrosine, lysine and histidine levels in rats. In particular, the serine and threonine levels were over five and eight times, respectively, higher in the Cbl-deficient rats than those in the sufficient controls. In addition, some amino acids, including serine and threonine, were excreted into urine at significantly higher levels in the deficient rats. When Cbl was supplemented into the deficient rats for 2 weeks, in coincidence with the disappearance of the urinary excretion of methylmalonic acid (an index of Cbl deficiency), the plasma serine and threonine levels were normalized. These results indicate that Cbl deficiency results in metabolic disorder of certain amino acids, including serine and threonine. The expression level of hepatic serine dehydratase (SDH), which catalyzes the conversion of serine and threonine to pyruvate and 2-oxobutyrate, respectively, was significantly lowered by Cbl deficiency, even though Cbl does not participate directly in the enzyme reaction. The SDH activity in the deficient rats was less than 20% of that in the sufficient controls, and was normalized 2 weeks after the Cbl supplementation. It is thus suggested that the decrease of the SDH expression relates closely with the abnormalities in the plasma and urinary levels of serine and threonine in the Cbl-deficient rats.     

Branched-Chain Amino Acid Biosynthesis in Salmonella typhimurium: a Quantitative Analysis

We report here the first quantitative study of the branched-chain amino acid biosynthetic pathway in Salmonella typhimurium LT2. The intracellular levels of the enzymes of the pathway and of the 2-keto acid intermediates were determined under various physiological conditions and used for estimation of several of the fluxes in the cells. The results led to a revision of previous ideas concerning the way in which multiple acetohydroxy acid synthase (AHAS) isozymes contribute to the fitness of enterobacteria. In wild-type LT2, AHAS isozyme I provides most of the flux to valine, leucine, and pantothenate, while isozyme II provides most of the flux to isoleucine. With acetate as a carbon source, a strain expressing AHAS II only is limited in growth because of the low enzyme activity in the presence of elevated levels of the inhibitor glyoxylate. A strain with AHAS I only is limited during growth on glucose by the low tendency of this enzyme to utilize 2-ketobutyrate as a substrate; isoleucine limitation then leads to elevated threonine deaminase activity and an increased 2-ketobutyrate/2-ketoisovalerate ratio, which in turn interferes with the synthesis of coenzyme A and methionine. The regulation of threonine deaminase is also crucial in this regard. It is conceivable that, because of fundamental limitations on the specificity of enzymes, no single AHAS could possibly be adequate for the varied conditions that enterobacteria successfully encounter.