Regulatory analysis of amino acid synthesis pathway in Escherichia coli: aspartate family

Proteins have various compositions of twenty specific naturally occurring amino acids. In spite of their importance in cellular metabolism, biosynthesis mechanisms, changing control conditions, and affection of effectors are not clearly understood yet. So we have made an effort to elucidate the details of metabolic control mechanisms in amino acid synthesis pathways through examining an extensive database search. In this study, we have newly constructed six amino acid biosynthesis pathways including aspartate, asparagine, methionine, threonine, isoleucine, and lysine, which we call the aspartate family. They contain the major reaction mechanisms, which inhibitory control loops and activating compounds. Moreover, we have tried to collect all of the effectors which might affect the aspartate family biosynthetic networks.     

In Vivo Regulation of Threonine and Isoleucine Biosynthesis in Lemna paucicostata Hegelm. 6746

Little, if any, regulation of threonine synthesis was observed in Lemna paucicostata Hegelm. 6746 supplemented with concentrations of threonine and/or isoleucine that allow for uptake of these amino acids in amounts sufficient for total plant requirements, and that increase tissue concentrations of soluble threonine manyfold. High tissue concentrations of soluble threonine generated endogenously in isoleucine-supplemented plants were no more effective in regulation than a similar concentration of threonine accumulated from the medium. These studies exclude also major regulation of threonine biosynthesis by bivalent repression by threonine plus isoleucine. Isoleucine biosynthesis was severely inhibited by supplementation with isoleucine, but not with threonine or methionine. The fivefold increase in soluble threonine in isoleucine-supplemented plants suggests that threonine dehydratase is a major locus for feedback regulation of isoleucine synthesis. It is concluded that regulation of threonine biosynthesis differs from that of the other amino acids of the aspartate family (isoleucine, methionine, and lysine), each of which strongly feedback regulates its own synthesis. Methionine supplementation had a negligible effect on the tissue concentration of soluble threonine, indicating that threonine is not important in balancing changes of flux into methionine by equivalent changes of flux through the step catalyzed by aspartokinase.     

Photosynthetic formation of the aspartate family of amino acids in isolated chloroplasts

The metabolism of ¹⁴C-labeled aspartic acid, diaminopimelic acid, malic acid and threonine by isolated pea (Pisum sativum L.) chloroplasts was examined. Light enhanced the incorporation of [¹⁴C] aspartic acid into soluble homoserine, isoleucine, lysine, methionine and threonine and protein-bound aspartic acid plus asparagine, isoleucine, lysine, and threonine. Lysine (2 millimolar) inhibited its own formation as well as that of homoserine, isoleucine and threonine. Threonine (2 millimolar) inhibited its own synthesis and that of homoserine but had only a small effect on isoleucine and lysine formation. Lysine and threonine (2 millimolar each) in combination strongly inhibited their own synthesis as well as that of homoserine. Radioactive [1,7-¹⁴C]diaminopimelic acid was readily converted into [¹⁴C]threonine in the light and its labeling was reduced by exogenous isoleucine (2 millimolar) or a combination of leucine and valine (2 millimolar each). The strong light stimulation of amino acid formation illustrates the point that photosynthetic energy is used in situ for amino acid and protein biosynthesis, not solely for CO₂ fixation.     

Shared Metabolic Pathways in a Coevolved Insect-Bacterial Symbiosis

The symbiotic bacterium Buchnera aphidicola lacks key genes in the biosynthesis of five essential amino acids (EAAs), and yet its animal hosts (aphids) depend on the symbiosis for the synthesis of these EAAs (isoleucine, leucine, methionine, phenylalanine, and valine). We tested the hypothesis, derived from genome annotation, that the missing Buchnera reactions are mediated by host enzymes, with the exchange of metabolic intermediates between the partners. The specialized host cells bearing Buchnera were separated into a Buchnera fraction and a Buchnera-free host cell fraction (HF). Addition of HF to isolated Buchnera preparations significantly increased the production of leucine and phenylalanine, and recombinant enzymes mediating the final reactions in branched-chain amino acid and phenylalanine synthesis rescued the production of these EAAs by Buchnera preparations without HF. The likely precursors for the missing proximal reactions in isoleucine and methionine synthesis were identified, and they differed from predictions based on genome annotations: synthesis of 2-oxobutanoate, the aphid-derived precursor of isoleucine synthesis, was stimulated by homoserine and not threonine via threonine dehydratase, and production of the homocysteine precursor of methionine was driven by cystathionine, not cysteine, via reversal of the transsulfuration pathway. The evolution of shared metabolic pathways in this symbiosis can be attributed to host compensation for genomic deterioration in the symbiont, involving changes in host gene expression networks to recruit specific enzymes to the host cell.     

Methionine-lysine-threonine-isoleucine interrelationships in the amino acid nutrition of rice callus tissue

Growth of rice callus tissue is discouraged when methionineis excluded from CMAA medium. While determining the methionineelimination effect, the amino acid interrelationships amongmethionine, lysine, threonine and isoleucine in the nutritionof the callus tissue were found. Poor growth, found in cultureson methionine deficient media was seen only when the media containedboth threonine and lysine, simultaneously. The substitutionof homoserine for methionine was also observed. Determination of free amino acid composition in tissues revealedthat free methionine was barely detectable in tissues grownwith sufficient amounts of threonine and lysine. When the concentrationof either threonine or lysine was reduced, the free methioninecontent of the tissue increased. When the methionine deficientmedium was supplemented with homoserine, the free methioninein the tissue increased, although the tissue retained a considerableamount of free threonine and lysine. Cultivation of tissue onan isoleucine deficient medium resulted in a significant decreasein free threonine content. These experimental results suggest that the biosynthetic pathwayto methionine is cooperatively inhibited by threonine and lysine,and that threonine decomposition is inhibited by its end productisoleucine. (Received February 19, 1970; )     

The aspartic acid metabolic pathway, an exciting and essential pathway in plants

Summary. Aspartate is the common precursor of the essential amino acids lysine, threonine, methionine and isoleucine in higher plants. In addition, aspartate may also be converted to asparagine, in a potentially competing reaction. The latest information on the properties of the enzymes involved in the pathways and the genes that encode them is described. An understanding of the overall regulatory control of the flux through the pathways is undisputedly of great interest, since the nutritive value of all cereal and legume crops is reduced due to low concentrations of at least one of the aspartate-derived amino acids. We have reviewed the recent literature and discussed in this paper possible methods by which the concentrations of the limiting amino acids may be increased in the seeds.     

Expression and purification of aspartate beta-semialdehyde dehydrogenase from infectious microorganisms

l-Aspartate-beta-semialdehyde dehydrogenase (ASA DH) lies at the first branch point in the aspartate metabolic pathway that leads to the formation of the amino acids lysine, isoleucine, methionine, and threonine in most plants, bacteria, and fungi. Since the aspartate pathway is not found in humans, but is necessary for bacterial cell wall biosynthesis, the enzymes in this pathway are potential targets for the development of new antibiotics. The asd gene that encodes for ASA DH has been obtained from several infectious organisms and ligated into a pET expression vector. ASA DHs from Haemophilus influenza, Pseudomonas aeruginosa, and Vibrio cholerae were expressed as soluble proteins in Escherichia coli, while ASA DH from Helicobacter pylori was obtained primarily as inclusion bodies. The V. cholerae genome contains two asd genes. Both enzymes have been expressed and purified, and each displays significant ASA DH activity. The purification of highly active ASA DH from each of these organisms has been achieved for the first time, in greater than 95% purity and high overall yield. Kinetic parameters have been determined for each purified enzyme, and the values have been compared to those of E. coli ASA DH.     

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.     

Amino Acid Metabolism of Lemna minor L. : III. Responses to Aminooxyacetate

Aminooxyacetate, a known inhibitor of transaminase reactions and glycine decarboxylase, promotes rapid depletion of the free pools of serine and aspartate in nitrate grown Lemna minor L. This compound markedly inhibits the methionine sulfoximine-induced accumulation of free ammonium ions and greatly restricts the methionine sulfoximine-induced depletion of amino acids such as glutamate, alanine, and asparagine. These results suggest that glutamate, alanine, and asparagine are normally catabolized to ammonia by transaminase-dependent pathways rather than via dehydrogenase or amidohydrolase reactions. Aminooxyacetate does not inhibit the methionine sulfoximine-induced irreversible deactivation of glutamine synthetase in vivo, indicating that these effects cannot be simply ascribed to inhibition of methionine sulfoximine uptake by amino-oxyacetate. This transaminase inhibitor promotes extensive accumulation of several amino acids including valine, leucine, isoleucine, alanine, glycine, threonine, proline, phenylalanine, lysine, and tyrosine. Since the aminooxyacetate induced accumulations of valine, leucine, and isoleucine are not inhibited by the branched-chain amino acid biosynthesis inhibitor, chlorsulfuron, these amino acid accumulations most probably involve protein turnover. Depletions of soluble protein bound amino acids are shown to be approximately stoichiometric with the free amino acid pool accumulations induced by aminooxyacetate. Aminooxyacetate is demonstrated to inhibit the chlorsulfuron-induced accumulation of α-amino-n-butyrate in L. minor, supporting the notion that this amino acid is derived from transamination of 2-oxobutyrate.     

Why Threonine Is an Essential Amino Acid in Mammals and Birds: Studies at the Enzyme Level

The only pathway for the synthesis of essential amino acids in vertebrates is reversible transamination of their keto analogs with glutamic acid. At the same time, it is commonly accepted that such essential amino acids as lysine and threonine are not involved in transamination and, therefore, cannot be synthesized from their keto analogs. However, using radio-labeled isotopes, synthesis of threonine was demonstrated in rat liver and in a reaction mixture containing chicken liver threonine dehydrogenase. In the review, we discuss why threonine is an essential amino acid in mammals and birds based on the pathways of threonine biosynthesis in these two classes of vertebrates.     

Amino Acid Biosynthesis in the Halophilic Archaeon Haloarcula hispanica

Biosynthesis of proteinogenic amino acids in the extremely halophilic archaeon Haloarcula hispanica was explored by using biosynthetically directed fractional 13C labeling with a mixture of 90% unlabeled and 10% uniformly 13C-labeled glycerol. The resulting 13C-labeling patterns in the amino acids were analyzed by two-dimensional 13C,1H correlation spectroscopy. The experimental data provided evidence for a split pathway for isoleucine biosynthesis, with 56% of the total Ile originating from threonine and pyruvate via the threonine pathway and 44% originating from pyruvate and acetyl coenzyme A via the pyruvate pathway. In addition, the diaminopimelate pathway involving diaminopimelate dehydrogenase was shown to lead to lysine biosynthesis and an analysis of the 13C-labeling pattern in tyrosine indicated novel biosynthetic pathways that have so far not been further characterized. For the 17 other proteinogenic amino acids, the data were consistent with data for commonly found biosynthetic pathways. A comparison of our data with the amino acid metabolisms of eucarya and bacteria supports the theory that pathways for synthesis of proteinogenic amino acids were established before ancient cells diverged into archaea, bacteria, and eucarya.     

Nutritional factors affecting nisin production by Lactococcus lactis subsp. actis NIZO 22186 in a synthetic medium

L. DE VUYST. 1995 A minimal synthetic medium (SM8) for nisin-producing Lactococcus lactis subsp. lactis strains has been designed; it consists of eight growth-stimulating amino acids (glutamic acid, methionine, valine, leucine, threonine, arginine, isoleucine and histidine), five vitamins (biotin, calcium pantothenate, nicotinic acid, pyridoxine and riboflavin) and the mineral salts dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, magnesium sulphate and trisodium citrate. Nisin biosynthesis is strongly dependent on the presence of a sulphur source, either an inorganic salt (magnesium sulphate or sodium thiosulphate) or the amino acids methionine, cysteine or cystathionine. The amino acids serine, threonine and cysteine highly stimulate nisin production without affecting the final cell yield, indicating their precursor role during nisin biosynthesis.     

Regulatory mechanisms after short‐ and long‐term perturbed lysine biosynthesis in the aspartate pathway: the need for isogenes in Arabidopsis thaliana

The aspartate‐derived amino acid pathway in plants is an intensively studied metabolic pathway, because of the biosynthesis of the four essential amino acids lysine, threonine, isoleucine and methionine. The pathway is mainly controlled by the key regulatory enzymes aspartate kinase (AK; EC 2.7.2.4), homoserine dehydrogenase (HSDH; EC 1.1.1.3) and 4‐hydroxy‐tetrahydrodipicolinate synthase (EC 4.3.3.7), formerly referred to as dihydrodipicolinate synthase (DHDPS). They are encoded by isoenzyme families and it is not known why such families are evolutionarily maintained. To gain more insight into the specific roles and regulation of the isoenzymes, we inhibited DHDPS in Arabidopsis thaliana with the chemical compound (N,N‐dimethylglycinatoboranyloxycarbonylmethyl)‐dimethylamine‐borane (DDAB) and compared the short‐term effects on the biochemical and biomolecular level to the long‐term adaptations in dhdps knockout mutants. We found that DHDPS2 plays a crucial role in controlling lysine biosynthesis, thereby stabilizing flux through the whole aspartate pathway. Moreover, DHDPS2 was also shown to influence the threonine level to a large extent. In addition, the lysine‐sensitive AKs, AKLYS1 and AKLYS3 control the short‐ and long‐term responses to perturbed lysine biosynthesis in Arabidopsis thaliana.     

The synthesis of amino acids by Methanobacterium omelianskii

1. Methanobacterium omelianskii was grown on (14)CO(2) and unlabelled ethanol, or on [1-(14)C]- or [2-(14)C]-ethanol and unlabelled carbon dioxide. The cell protein was hydrolysed and certain of the amino acids were isolated and degraded. 2. Carbon from both carbon dioxide and ethanol is used for biosynthesis of amino acids, and in most cases ethanol is incorporated as a C(2) unit. Ethanol carbon atoms and carbon dioxide carbon atoms apparently enter the same range of compounds. Ethanol and carbon dioxide are equally important as sources of cell carbon. 3. The origins of carbon atoms of aspartate, alanine, glycine, serine and threonine are consistent with the synthesis of these amino acids, by pathways known to exist in aerobic organisms, from pyruvate arising by a C(2)+C(1) condensation. The proportion of total radioactivity found in C-1 of lysine, proline, methionine and valine is consistent with synthesis of these amino acids by pathways similar to those found in Escherichia coli. Isoleucine is probably formed by carboxylation of a C(5) precursor formed entirely from ethanol. Glutamate is formed by an unknown pathway.     

Methionine inhibits developmental aggregation of Myxococcus xanthus by blocking the biosynthesis of S-adenosyl methionine.

Previous studies showed that high concentrations of methionine (> 1 mM) inhibited aggregation and fruiting body formation in Myxococcus xanthus (E. Rosenberg, D. Filer, D. Zafriti, and S. H. Kindler, J. Bacteriol. 115: 29-34, 1973, and J. M. Campos and D. R. Zusman, Proc. Natl. Acad. Sci. USA 72:518-522, 1975). However, the mechanism for the inhibition was unclear. In this study, we found that high levels of methionine inhibited the biosynthesis of S-adenosylmethionine (SAM) and that reduced intracellular levels of SAM are correlated with defective chemotactic movements and reduced developmental gene expression. In addition, we found that methionine analogs and high concentrations of amino acids which are known to affect SAM synthesis in other bacteria, such as threonine, lysine, and isoleucine, also caused reduced cellular levels of SAM and blocked fruiting body formation in M. xanthus. These results indicate that SAM is required for development of M. xanthus and the inhibitory effect of methionine on development results, at least in part, from its blocking of the biosynthesis of SAM.     

Differential amino acid requirements for sporulation in Bacillus subtilis

The amino acid requirements for sporulation were studied by use of auxotrophic mutants of Bacillus subtilis 168. Cells were grown to T(0) in medium containing the test amino acid and were then transferred to a minimal medium lacking that amino acid. Omission of leucine caused no reduction in sporulation. Omission of methionine, lysine, and phenylalanine appeared to cause reduced levels of sporulation, and sporulation was completely inhibited when isoleucine, tryptophan, and threonine were omitted. The amino acids in this third class showed a sequence of requirements, with tryptophan required earlier than isoleucine, which in turn was required earlier in the sporulation process than threonine. Isoleucine omission did not affect the early sporulation functions of extracellular protease formation or septum formation, but prevented the increased levels of protein synthesis and oxygen consumption that normally accompany early sporulation stages. Isoleucine did not appear to be metabolized to other compounds in significant amounts during sporulation. The role of isoleucine in the sporulation process remains unclear.     

Aspartate kinase and the synthesis of aspartate-derived amino acids in wheat

Aspartate kinase (EC 2.7.2.4.) has been purified from 7 day etiolated wheat (Triticum aestivum L. var. Maris Freeman) seedlings and from embryos imbibed for 8 h. The enzyme was 50% inhibited by 0.25 mM lysine. In this study wheat aspartate kinase was not inhibited by threonine alone or cooperatively with lysine; these results contrast with those published previously. In vivo regulation of the synthesis of aspartate-derived amino acids was examined by feeding [¹⁴C]acetate and [³⁵S]sulphate to 2–3 day germinating wheat embryos in culture in the presence of exogenous amino acids. Lysine (1 mM) inhibited lysine synthesis by 86%. Threonine (1 mM) inhibited threonine synthesis by 79%. Lysine (1 mM) plus threonine (1 mM) inhibited threonine synthesis by 97%. Methionine synthesis was relatively unaffected by these amino acids, suggesting that there are important regulatory sites other than aspartate kinase and homoserine dehydrogenase. [³⁵S]sulphate incorporation into methionine was inhibited 50% by lysine (2 mM) plus threonine (2 mM) correlating with the reported 50% inhibition of growth by these amino acids in this system. The synergistic inhibition of growth, methionine synthesis and threonine synthesis by lysine plus threonine is discussed in terms of lysine inhibition of aspartate kinase and threonine inhibition of homoserine dehydrogenase.Abbreviations AEC S-(2-aminoethyl) cysteine