Arabidopsis loss-of-function mutant in the lysine pathway points out complex regulation mechanisms

In plants, the amino acids lysine, threonine, methionine and isoleucine have L-aspartate-beta-semialdehyde (ASA) as a common precursor in their biosynthesis pathways. How this ASA precursor is dispersed among the different pathways remains vague knowledge. The proportional balances of free and/or protein-bound lysine, threonine, isoleucine and methionine are a function of protein synthesis, secondary metabolism and plant physiology. Some control points determining the flux through the distinct pathways are known, but an adequate explanation of how the competing pathways share ASA in a fine-tuned amino acid biosynthesis network is yet not available. In this article we discuss the influence of lysine biosynthesis on the adjacent pathways of threonine and methionine. We report the finding of an Arabidopsis thaliana dihydrodipicolinate synthase T-DNA insertion mutant displaying lower lysine synthesis, and, as a result of this, a strongly enhanced synthesis of threonine. Consequences of these cross-pathway regulations are discussed.     

Peptide inhibitors MAP the way towards fighting anthrax pathogenesis

BCAAs (branched-chain amino acids) are indispensable (essential) amino acids that are required for body protein synthesis. Indispensable amino acids cannot be synthesized by the body and must be acquired from the diet. The BCAA leucine provides hormone-like signals to tissues such as skeletal muscle, indicating overall nutrient sufficiency. BCAA metabolism provides an important transport system to move nitrogen throughout the body for the synthesis of dispensable (non-essential) amino acids, including the neurotransmitter glutamate in the central nervous system. BCAA metabolism is tightly regulated to maintain levels high enough to support these important functions, but at the same time excesses are prevented via stimulation of irreversible disposal pathways. It is well known from inborn errors of BCAA metabolism that dysregulation of the BCAA catabolic pathways that leads to excess BCAAs and their alpha-keto acid metabolites results in neural dysfunction. In this issue of Biochemical Journal, Joshi and colleagues have disrupted the murine BDK (branched-chain alpha-keto acid dehydrogenase kinase) gene. This enzyme serves as the brake on BCAA catabolism. The impaired growth and neurological abnormalities observed in this animal show conclusively the importance of tight regulation of indispensable amino acid metabolism.     

Induction of Arabidopsis tryptophan pathway enzymes and camalexin by amino acid starvation, oxidative stress, and an abiotic elicitor.

The tryptophan (Trp) biosynthetic pathway leads to the production of many secondary metabolites with diverse functions, and its regulation is predicted to respond to the needs for both protein synthesis and secondary metabolism. We have tested the response of the Trp pathway enzymes and three other amino acid biosynthetic enzymes to starvation for aromatic amino acids, branched-chain amino acids, or methionine. The Trp pathway enzymes and cytosolic glutamine synthetase were induced under all of the amino acid starvation test conditions, whereas methionine synthase and acetolactate synthase were not. The mRNAs for two stress-inducible enzymes unrelated to amino acid biosynthesis and accumulation of the indolic phytoalexin camalexin were also induced by amino acid starvation. These results suggest that regulation of the Trp pathway enzymes under amino acid deprivation conditions is largely a stress response to allow for increased biosynthesis of secondary metabolites. Consistent with this hypothesis, treatments with the oxidative stress-inducing herbicide acifluorfen and the abiotic elicitor alpha-amino butyric acid induced responses similar to those induced by the amino acid starvation treatments. The role of salicylic acid in herbicide-mediated Trp and camalexin induction was investigated.     

Molecular identification of a further branched-chain aminotransferase 7 (BCAT7) in tomato plants

Although the branched-chain amino acids (BCAAs) are essential components of the mammalian diet, our current understanding of their metabolism in plants is still limited. It is however well known that the branched-chain amino acid transaminases (BCATs) play a crucial role in both the synthesis and degradation of the BCAAs leucine, isoleucine and valine. We previously characterized the BCAT gene family in tomato, revealing it to be highly diverse in subcellular localization, substrate preference, and expression. Here we performed further characterization of this family and provide evidence for the presence of another member, BCAT7. On mapping the chromosomal location of this enzyme, it was possible to define the exact chromosome map position of the gene. Although in Arabidopsis thaliana the AtBCAT7 has been considered a pseudo-gene, quantitative evaluation of the expression levels of this gene revealed that the expression profile of the BCAT7 in different tissues of tomato (Solanum lycopersicum cv. M82) plants is highly variable with the highest expression found in developed flowers. By using a C-terminal E-GFP gene fusion we demonstrate that the BCAT7 is extraplastidial and in combination with the kinetic characterization of BCAT7 our results suggest that it most likely operates in BCAA degradation in vivo and support our hypothesis of another functional member of BCAT family. The combined data presented are discussed within the context of BCAA metabolism and its functions in higher plants.     

Effects of branched-chain amino acids on plasma amino acids in amyotrophic lateral sclerosis

Summary Although the cause of amyotrophic lateral sclerosis (ALS) remains unknown, biological findings suggest that the excitatory amino acid glutamate contributes to the pathogenesis of ALS. In previous studies of ALS, the therapeutic effect of the branched-chain amino acids (BCAAs) leucine, valine and isoleucine has been evaluated. The present study aimed at investigating the acute effect of BCAAs on plasma glutamate levels in ALS patients. Following two oral doses of BCAAs, significantly increased plasma levels were seen for valine (500%), isoleucine (1,377%) and leucine (927%), however the plasma level of glutamate was not affected. The plasma level of several other amino acids (tryptophan, tyrosine, phenylalanine and methionine) were found decreased after oral BCAAs, which may indicate a diminution in the rate of degradation of muscle protein and/or an increase in tissue disposal of amino acids.     

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.     

Changes in free amino acids and polyamine levels in Satsuma leaves in response to Asian citrus psyllid infestation and water stress

The effects of biotic and abiotic stresses on changes in amino acids and polyamine levels in Satsuma orange （Citrus unshiu; cultivar Owari） leaves were inves- tigated. Asian citrus psyllids Diaphorina citri （Kuwayama） （ACP） infestation was used to induce biotic stress while a water deficit was imposed to induce abiotic stress. Potted trees were infested by placing 50 psyllids on 3 citrus leaves enclosed in nylon mesh bags for 5 d. A parallel set of plants were kept water stressed by maintaining the soil at 20% water holding capacity for 5 d. Levels of total free amino acids were higher in water stressed and ACP infested leaves. Polyamine putrescine increased in infested leaves but not in water stressed leaves. Proline was the most abundant amino acid and its levels significantly increased by both biotic and abiotic stresses. Proline levels in infested leaves were significantly higher than the water stressed leaves. Histidine, methionine, asparagine, arginine, serine, and leucine levels also increased significantly in infested leaves, but in water stressed leaves only leucine, methionine, and threonine increased. Levels of amino acids, such as tyrosine, isoleucine, phenylalanine, glutamic acid, and alanine, declined in infested leaves. Under water stress asparagine, phenylalanine, serine, and histidine also declined compared to controls. This indicates that while proteolysis occurred under both stresses, metabolic conversion of amino acids was different under the two stresses. In ACP infested leaves some amino acids may be used as feeding material and/or converted into secondary metabolites for defense.     

Regulation of branched-chain amino acid transport in Escherichia coli.

The repression and derepression of leucine, isoleucine, and valine transport in Escherichia coli K-12 was examined by using strains auxotrophic for leucine, isoleucine, valine, and methionine. In experiments designed to limit each of these amino acids separately, we demonstrate that leucine limitation alone derepressed the leucine-binding protein, the high-affinity branched-chain amino acid transport system (LIV-I), and the membrane-bound, low-affinity system (LIV-II). This regulation did not seem to involve inactivation of transport components, but represented an increase in the differential rate of synthesis of transport components relative to total cellular proteins. The apparent regulation of transport by isoleucine, valine, and methionine reported elsewhere was shown to require an intact leucine, biosynthetic operon and to result from changes in the level of leucine biosynthetic enzymes. A functional leucyl-transfer ribonucleic acid synthetase was also required for repression of transport. Transport regulation was shown to be essentially independent of ilvA or its gene product, threonine deaminase. The central role of leucine or its derivatives in cellular metabolism in general is discussed.     

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.     

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.     

Prevention of DNA damage by l-carnitine induced by metabolites accumulated in maple syrup urine disease in human peripheral leukocytes in vitro

Maple syrup urine disease (MSUD) is an inherited aminoacidopathy caused by a deficiency in branched-chain α-keto acid dehydrogenase complex activity that leads to the accumulation of the branched-chain amino acids (BCAAs) leucine (Leu), isoleucine, and valine and their respective α-keto-acids, α-ketoisocaproic acid (KIC), α keto-β-methylvaleric acid, and α-ketoisovaleric acid. The major clinical features presented by MSUD patients include ketoacidosis, failure to thrive, poor feeding, apnea, ataxia, seizures, coma, psychomotor delay, and mental retardation; however, the pathophysiology of this disease is poorly understood. MSUD treatment consists of a low protein diet supplemented with a mixture containing micronutrients and essential amino acids but excluding BCAAs. Studies have shown that oxidative stress may be involved in the neuropathology of MSUD, with the existence of lipid and protein oxidative damage in affected patients. In recent years, studies have demonstrated the antioxidant role of l-carnitine (l-Car), which plays a central function in cellular energy metabolism and for which MSUD patients have a deficiency. In this work, we investigated the in vitro effect of Leu and KIC in the presence or absence of l-Car on DNA damage in peripheral whole blood leukocytes using the alkaline comet assay with silver staining and visual scoring. Leu and KIC resulted in a DNA damage index that was significantly higher than that of the control group, and l-Car was able to significantly prevent this damage, mainly that due to KIC.     

Effects of amino acids on Thiobacillus acidophilus. I. Growth studies with special reference to valine.

The heterotrophic growth of Thiobacillus acidophilus was inhibited by branched-chain amino acids; valine, isoleucine, and leucine. The inhibition by valine and leucine were partially reversed by isoleucine, and the inhibition by isoleucine was partially reversed by valine. Inhibitions by methionine or threonine were partially reversed when both amino acids were present in the growth medium. Inhibition by tyrosine was increased by phenylalanine or tryptophan. Cystine completely inhibited growth. Other amino acids tested produced little or no inhibition. Acetohydroxy acid synthetase (AHAS) activity was demonstrated in crude extracts of T. acidophilus. In crude extracts the optimum pH was 8.5 with a shift to 9.0 in the presence of valine. Valine was the only branched-chain amino acid which inhibited the AHAS activity. The presence of only one peak of AHAS activity upon centrifugation in linear glycerol density gradients demonstrated that the AHAS activity sediments as one component.     

Comparative studies of Acyl-CoA dehydrogenases for monomethyl branched chain substrates in amino acid metabolism

Short/branched chain acyl-CoA dehydrogenase (SBCAD), isovaleryl-CoA dehydrogenase (IVD), and isobutyryl-CoA dehydrogenase (IBD) are involved in metabolism of isoleucine, leucine, and valine, respectively. These three enzymes all belong to acyl-CoA dehydrogenase (ACD) family, and catalyze the dehydrogenation of monomethyl branched-chain fatty acid (mmBCFA) thioester derivatives. In the present work, the catalytic properties of rat SBCAD, IVD, and IBD, including their substrate specificity, isomerase activity, and enzyme inhibition, were comparatively studied. Our results indicated that SBCAD has its catalytic properties relatively similar to those of straight-chain acyl-CoA dehydrogenases in terms of their isomerase activity and enzyme inhibition, while IVD and IBD are different. IVD has relatively broader substrate specificity than those of the other two enzymes in accommodating various substrate analogs. The present study increased our understanding for the metabolism of monomethyl branched-chain fatty acids (mmBCFAs) and branched-chain amino acids (BCAAs), which should also be useful for selective control of a particular reaction through the design of specific inhibitors.     

Regulation of branched-chain, and sulfur-containing amino acid metabolism by glutathione during ultradian metabolic oscillation of Saccharomyces cerevisiae

Autonomous ultradian metabolic oscillation (T approximately or =50 min) was detected in an aerobic chemostat culture of Saccharomyces cerevisiae. A pulse injection of GSH (a reduced form of glutathione) into the culture induced a perturbation in metabolic oscillation, with respiratory inhibition caused by H2S burst production. As the production of H2S in the culture was controlled by different amino acids, we attempted to characterize the effects of GSH on amino acid metabolism, particularly with regard to branched chain and sulfur-containing amino acids. During stable metabolic oscillation, concentrations of intracellular glutamate, aspartate, threonine, valine, leucine, isoleucine, and cysteine were observed to oscillate with the same periods of dissolved O2 oscillation, although the oscillation amplitudes and maximal phases were shown to differ. The methionine concentration was stably maintained at 0.05 mM. When GSH (100 microM) was injected into the culture, cellular levels of branched chain amino acids increased dramatically with continuous H2S production, whereas the cysteine and methionine concentrations were noticeably reduced. These results indicate that GSH-dependent perturbation occurs as the result of the promotion of branched chain amino acid synthesis and an attenuation of cysteine and methionine synthesis, both of which activate the generation of H2S. In a low sulfate medium containing 2.5 mM sulfate, the GSH injections did not result in perturbations of dissolved O2, NAD(P)H redox oscillations without burst H2 production. This suggests that GSH-dependent perturbation is intimately linked with the metabolism of branched-chain amino acids and H2 generation, rather than with direct GSH-GSSG redox control.     

Branched-chain amino acid metabolism in higher plants

Valine, leucine and isoleucine contain short branched carbohydrate residues responsible for their classification as branched-chain amino acids (BCAA). Among the proteinogenic amino acids, BCAA show the highest hydrophobicity and are accordingly the major constituents of transmembrane regions of membrane proteins. BCAA cannot be synthesized by humans and thus belong to the essential amino acids. In contrast, plants are able to synthesize these amino acids de novo and are an important source for these compounds in the human diet. However, BCAA cannot only be synthesized in plants, leucine and probably also valine and isoleucine can also be degraded. Many enzymes operating in turnover are found in mitochondria, while some catabolizing activities are located in peroxisomes. The breakdown of BCAA is physically separated from their biosynthesis in chloroplasts. Additionally, in the order of the Capparales, enzymes of the leucine metabolism seem to be evolutionary related to or may even participate in the methionine chain elongation pathway, the early part of the biosynthesis of aliphatic glucosinolates. In summary, in higher plants a complex network of pathways interferes with the homeostasis of Val, Leu and Ile.     

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.     

Relationships between amino acid catabolism and protein anabolism in the ruminant

The observed relation found in sheep between the flux rate of an amino acid and the proportion found in whole-body protein suggests that the major immediate fate of an amino acid is its incorporation into tissue protein. This may be true even for dispensable amino acids. In ruminants, there is substantial utilization of several amino acids (serine, glycine, threonine, histidine, and methionine) for the synthesis of methyl groups; the use of these amino acids for gluconeogenesis is limited. There is little evidence that demands of gluconeogenesis limit the availability of amino acids for protein synthesis. Most amino acids are catabolized in the liver but there may be significant catabolism of alanine, aspartate, and glutamate in peripheral tissues, especially muscle. Normally, peripheral catabolism of branched-chain amino acids is significantly less in ruminants than other species. Nevertheless, there is some oxidation of leucine by muscle and this may be substantially increased in the diabetic state. Catabolism of leucine (and perhaps isoleucine and valine) might be inversely related to use for protein synthesis, but there is no evidence of such a relation for other amino acids.