Comparative metabolism of branched-chain amino acids to precursors of juvenile hormone biogenesis in corpora allata of lepidopterous versus nonlepidopterous insects

Comparative studies were performed on the role of branched-chain amino acids (BCAA) in juvenile hormone (JH) biosynthesis using several lepidopterous and nonlepidopterous insects. Corpora cardiaca-corpora allata complexes (CC-CA, the corpora allata being the organ of JH biogenesis) were maintained in culture medium containing a uniformly 14C-labeled BCAA, together with [methyl-3H]methionine as mass marker for JH quantification. BCAA catabolism was quantified by directly analyzing the medium for the presence of 14C-labeled propionate and/or acetate, while JHs were extracted, purified by liquid chromatography, and subjected to double-label liquid scintillation counting. Our results indicate that active BCAA catabolism occurs within the CC-CA of lepidopterans, and this efficiently provides propionyl-CoA (from isoleucine or valine) for the biosynthesis of the ethyl branches of JH I and II. Acetyl-CoA, formed from isoleucine or leucine catabolism, is also utilized by lepidopteran CC-CA for biosynthesizing JH III and the acetate-derived portions of the ethyl-branched JHs. In contrast, CC-CA of nonlepidopterans fail to catabolize BCAA. Consequently, exogenous isoleucine or leucine does not serve as a carbon source for the biosynthesis of JH III by these glands, and no propionyl-CoA is produced for genesis of ethyl-branched JHs. This is the first observation of a tissue-specific metabolic difference which in part explains why these novel homosesquiterpenoids exist in lepidopterans, but not in nonlepidopterans.     

Genetic analysis of pathway regulation for enhancing branched‐chain amino acid biosynthesis in plants

The branched‐chain amino acids (BCAAs) valine, leucine and isoleucine are essential amino acids that play critical roles in animal growth and development. Animals cannot synthesize these amino acids and must obtain them from their diet. Plants are the ultimate source of these essential nutrients, and they synthesize BCAAs through a conserved pathway that is inhibited by its end products. This feedback inhibition has prevented scientists from engineering plants that accumulate high levels of BCAAs by simply over‐expressing the respective biosynthetic genes. To identify components critical for this feedback regulation, we performed a genetic screen for Arabidopsis mutants that exhibit enhanced resistance to BCAAs. Multiple dominant allelic mutations in the VALINE‐TOLERANT 1 (VAT1) gene were identified that conferred plant resistance to valine inhibition. Map‐based cloning revealed that VAT1 encodes a regulatory subunit of acetohydroxy acid synthase (AHAS), the first committed enzyme in the BCAA biosynthesis pathway. The VAT1 gene is highly expressed in young, rapidly growing tissues. When reconstituted with the catalytic subunit in vitro, the vat1 mutant‐containing AHAS holoenzyme exhibits increased resistance to valine. Importantly, transgenic plants expressing the mutated vat1 gene exhibit valine tolerance and accumulate higher levels of BCAAs. Our studies not only uncovered regulatory characteristics of plant AHAS, but also identified a method to enhance BCAA accumulation in crop plants that will significantly enhance the nutritional value of food and feed.     

Dual role of alpha-acetolactate decarboxylase in Lactococcus lactis subsp. lactis.

The alpha-acetolactate decarboxylase gene aldB is clustered with the genes for the branched-chain amino acids (BCAA) in Lactococcus lactis subsp. lactis. It can be transcribed with BCAA genes under isoleucine regulation or independently of BCAA synthesis under the control of its own promoter. The product of aldB is responsible for leucine sensibility under valine starvation. In the presence of more than 10 microM leucine, the alpha-acetolactate produced by the biosynthetic acetohydroxy acid synthase IlvBN is transformed to acetoin by AldB and, consequently, is not available for valine synthesis. AldB is also involved in acetoin formation in the 2,3-butanediol pathway, initiated by the catabolic acetolactate synthase, AlsS. The differences in the genetic organization, the expression, and the kinetics parameters of these enzymes between L. lactis and Klebsiella terrigena, Bacillus subtilis, or Leuconostoc oenos suggest that this pathway plays a different role in the metabolism in these bacteria. Thus, the alpha-acetolactate decarboxylase from L. lactis plays a dual role in the cell: (i) as key regulator of valine and leucine biosynthesis, by controlling the acetolactate flux by a shift to catabolism; and (ii) as an enzyme catalyzing the second step of the 2,3-butanediol pathway.     

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.     

Fermentative production of branched chain amino acids: a focus on metabolic engineering

The branched chain amino acids (BCAAs), l-valine, l-leucine, and l-isoleucine, have recently been attracting much attention as their potential to be applied in various fields, including animal feed additive, cosmetics, and pharmaceuticals, increased. Strategies for developing microbial strains efficiently producing BCAAs are now in transition toward systems metabolic engineering from random mutagenesis. The metabolism and regulatory circuits of BCAA biosynthesis need to be thoroughly understood for designing system-wide metabolic engineering strategies. Here we review the current knowledge on BCAAs including their biosynthetic pathways, regulations, and export and transport systems. Recent advances in the development of BCAA production strains are also reviewed with a particular focus on l-valine production strain. At the end, the general strategies for developing BCAA overproducers by systems metabolic engineering are suggested.     

Biochemical Characterization of Branched Chain Amino Acids Uptake in Trypanosoma cruzi

Trypanosoma cruzi is the etiological agent of Chagas disease. During its life cycle, it alternates among vertebrate and invertebrate hosts. Metabolic flexibility is a main biochemical characteristic of this parasite, which is able to obtain energy by oxidizing a variety of nutrients that can be transported from the extracellular medium. Moreover, several of these metabolites, more specifically amino acids, have a variety of functions beyond being sources of energy. Branched chain amino acids (BCAA), beyond their role in ATP production, are involved in sterol biosynthesis; for example, leucine is involved as a negative regulator of the parasite differentiation process occurring in the insect midgut. BCAA are essential metabolites in most nonphotosynthetic eukaryotes, including trypanosomes. In view of this, the metabolism of BCAA in T. cruzi depends mainly on their transport into the cell. In this work, we kinetically characterized the BCAA transport in T. cruzi epimastigotes. Our data point to BCAA as being transported by a single saturable transport system able to recognize leucine, isoleucine and valine. In view of this, we used leucine to further characterize this system. The transport increased linearly with temperature from 10 to 45 °C, allowing the calculation of an activation energy of 51.30 kJ/mol. Leucine uptake was an active process depending on ATP production and a H⁺ gradient, but not on a Na⁺ or K⁺ gradient at the cytoplasmic membrane level.     

Plasma amino acids in four models of experimental liver injury in rats

Summary We studied the plasma amino acid profiles in four models of hepatic injury in rats. In partially hepatectomized rats (65% of liver was removed) we observed significant increase of aromatic amino acids (AAA; i.e. tyrosine and phenylalanine), taurine, aspartate, threonine, serine, asparagine, methionine, ornithine and histidine. Branched-chain amino acids (BCAA; i.e. valine, leucine and isoleucine) concentrations were unchanged. In ischemic and carbon tetrachloride acute liver damage we observed extreme elevation of most of amino acids (BCAA included) and very low concentration of arginine. In carbon tetrachloride induced liver cirrhosis we observed increased levels of AAA, aspartate, asparagine, methionine, ornithine and histidine and decrease of BCAA, threonine and cystine. BCAA/AAA ratio decreased significantly in partially hepatectomized and cirrhotic rats and was unchanged in ischemic and acute carbon tetrachloride liver damage. We conclude that a high increase of most of amino acids is characteristic of fulminant hepatic necrosis; decreased BCAA/AAA ratio is characteristic of liver cirrhosis; and decrease of BCAA/AAA ratio may not be used as an indicator of the severity of hepatic parenchymal damage.Abbreviations BCAA branched-chain amino acids (i.e. valine, leucine and isoleucine) - AAA aromatic amino acids (i.e. tyrosine and phenylalanine)     

Auxotrophy accounts for nodulation defect of most Sinorhizobium meliloti mutants in the branched-chain amino acid biosynthesis pathway

Some Sinorhizobium meliloti mutants in genes involved in isoleucine, valine, and leucine biosynthesis were previously described as being unable to induce nodule formation on host plants. Here, we present a reappraisal of the interconnection between the branched-chain amino acid biosynthesis pathway and the nodulation process in S. meliloti. We characterized the symbiotic phenotype of seven mutants that are auxotrophic for isoleucine, valine, or leucine in two closely related S. meliloti strains, 1021 and 2011. We showed that all mutants were similarly impaired for nodulation and infection of the Medicago sativa host plant. In most cases, the nodulation phenotype was fully restored by the addition of the missing amino acids to the plant growth medium. This strongly suggests that auxotrophy is the cause of the nodulation defect of these mutants. However, we confirmed previous findings that ilvC and ilvD2 mutants in the S. meliloti 1021 genetic background could not be restored to nodulation by supplementation with exogenous amino acids even though their Nod factor production appeared to be normal.     

Desilicification from the High Silica Containing Kraft Black Liquor by the Improved Carbon Dioxide Method

Addition of NADH to crude but not to pure branched-chain α-keto acid decarboxylase decreased the CO₂ production from α-keto-β-methylvalerate (KMV) suggesting the presence of an NADH dependent inhibitor in the crude enzyme from Bacillus subtilis. This NADH-dependent decarboxylase inhibitor was purified to homogeneity by a fast protein liquid chromatography system.

The purified inhibitor was identical with leucine dehydrogenase as to N-terminal amino acid squence (35 residues) and molecular weight, and catalyzed the oxidative deamination of three branched chain amino acids (BCAAs), valine, leucine, and isoleucine. The decarboxylase inhibitor was therefore identified as leucine dehydrogenase. A decreased substrate availability caused by leucine dehydrogenase thus reasonably accounted for the NADH dependent inhibition of the decarboxylation. In turn, the observation that leucine dehydrogenase competes with the decarboxylase for branched-chain α-keto acid (BCKA) suggested an involvement of this enzyme in the branched chain fatty acid (BCFA) biosynthesis. This view was supported by the observation that addition of NAD to crude fatty acid synthetase increased the incorporation of isoleucine into BCFAs. Pyridoxal-5′-phosphate and α-ketoglutarate, cofactors for BCAA transaminase, modulated BCFA biosynthesis from isoleucine in vitro, suggesting also the involvement of transaminase reaction in BCFA biosynthesis.     

Multivalent Repression of Isoleucine-Valine Biosynthesis in Saccharomyces cerevisiae

Regulation of the biosynthesis of four of the five enzymes of the isoleucine-valine pathway was studied in Saccharomyces cerevisiae. A method is described for limiting the growth of a leucine auxotroph by using valine as a competitor for the permease. Limitation for isoleucine and valine was accomplished by the use of peptides containing these amino acids conjugated with glycine as nutritional supplements for auxotrophs. The enzymes were repressed on synthetic medium containing isoleucine, valine, and leucine, as well as on broth supplemented with these amino acids. Limitation for any of the three branched-chain amino acids led to derepression of the isoleucine-valine biosynthetic pathway. Maximal derepression ranged from 3-fold for threonine deaminase to approximately 10-fold for acetohydroxyacid synthase. (Two of the enzymes, acetohydroxyacid synthase and dihydroxyacid dehydrase, may be controlled by a mechanism different from that regulating threonine deaminase.) Possible molecular mechanisms for multivalent repression are discussed.     

Changes in plasma phenylalanine, isoleucine, leucine, and valine are associated with significant changes in intracranial pressure and jugular venous oxygen saturation in patients with severe traumatic brain injury

Changes in plasma aromatic amino acids (AAA?=?phenylalanine, tryptophan, tyrosine) and branched chain amino acids (BCAA?=?isoleucine, leucine, valine) levels possibly influencing intracranial pressure (ICP) and cerebral oxygen consumption (SjvO(2)) were investigated in 19 sedated patients up to 14?days following severe traumatic brain injury (TBI). Compared to 44 healthy volunteers, jugular venous plasma BCAA were significantly decreased by 35% (p?相似文献   

Metabolism of branched-chain amino acids in starved rats: the role of hepatic tissue

Parameters of branched-chain amino acids (BCAA; leucine, isoleucine and valine) and protein metabolism were evaluated using L-[1-(14)C]leucine and alpha-keto[1-(14)C]isocaproate (KIC) in the whole body and in isolated perfused liver (IPL) of rats fed ad libitum or starved for 3 days. Starvation caused a significant increase in plasma BCAA levels and a decrease in leucine appearance from proteolysis, leucine incorporation into body proteins, leucine oxidation, leucine-oxidized fraction, and leucine clearance. Protein synthesis decreased significantly in skeletal muscle and the liver. There were no significant differences in leucine and KIC oxidation by IPL. In starved animals, a significant increase in net release of BCAA and tyrosine by IPL was observed, while the effect on other amino acids was non-significant. We conclude that the protein-sparing phase of uncomplicated starvation is associated with decreased whole-body proteolysis, protein synthesis, branched-chain amino acid (BCAA) oxidation, and BCAA clearance. The increase in plasma BCAA levels in starved animals results in part from decreased BCAA catabolism, particularly in heart and skeletal muscles, and from a net release of BCAA by the hepatic tissue.     

Alternative pathways for biosynthesis of leucine and other amino acids in Bacteroides ruminicola and Bacteroides fragilis

Bacteroides ruminicola is one of several species of anaerobes that are able to reductively carboxylate isovalerate (or isovaleryl-coenzyme A) to synthesize alpha-ketoisocaproate and thus leucine. When isovalerate was not supplied to growing B. ruminicola cultures, carbon from [U-14C]glucose was used for the synthesis of leucine and other cellular amino acids. When unlabeled isovalerate was available, however, utilization of [U-14C]glucose or [2-14C]acetate for leucine synthesis was markedly and specifically reduced. Enzyme assays indicated that the key enzyme of the common isopropylmalate (IPM) pathway for leucine biosynthesis, IPM synthase, was present in B. ruminicola cell extracts. The specific activity of IPM synthase was reduced when leucine was added to the growth medium but was increased by the addition of isoleucine plus valine, whereas the addition of isovalerate had little or no effect. The activity of B. ruminicola IPM synthase was strongly inhibited by leucine, the end product of the pathway. It seems unlikely that the moderate inhibition of the enzyme by isovalerate adequately explains the regulation of carbon flow by isovalerate in growing cultures. Bacteroides fragilis apparently also uses either the isovalerate carboxylation or the IPM pathway for leucine biosynthesis. Furthermore, both of these organisms synthesize isoleucine and phenylalanine, using carbon from 2-methylbutyrate and phenylacetate, respectively, in preference to synthesis of these amino acids de novo from glucose. Thus, it appears that these organisms have the ability to regulate alternative pathways for the biosynthesis of certain amino acids and that pathways involving reductive carboxylations are likely to be favored in their natural habitats.     

Alternative pathways for biosynthesis of leucine and other amino acids in Bacteroides ruminicola and Bacteroides fragilis.

Bacteroides ruminicola is one of several species of anaerobes that are able to reductively carboxylate isovalerate (or isovaleryl-coenzyme A) to synthesize alpha-ketoisocaproate and thus leucine. When isovalerate was not supplied to growing B. ruminicola cultures, carbon from [U-14C]glucose was used for the synthesis of leucine and other cellular amino acids. When unlabeled isovalerate was available, however, utilization of [U-14C]glucose or [2-14C]acetate for leucine synthesis was markedly and specifically reduced. Enzyme assays indicated that the key enzyme of the common isopropylmalate (IPM) pathway for leucine biosynthesis, IPM synthase, was present in B. ruminicola cell extracts. The specific activity of IPM synthase was reduced when leucine was added to the growth medium but was increased by the addition of isoleucine plus valine, whereas the addition of isovalerate had little or no effect. The activity of B. ruminicola IPM synthase was strongly inhibited by leucine, the end product of the pathway. It seems unlikely that the moderate inhibition of the enzyme by isovalerate adequately explains the regulation of carbon flow by isovalerate in growing cultures. Bacteroides fragilis apparently also uses either the isovalerate carboxylation or the IPM pathway for leucine biosynthesis. Furthermore, both of these organisms synthesize isoleucine and phenylalanine, using carbon from 2-methylbutyrate and phenylacetate, respectively, in preference to synthesis of these amino acids de novo from glucose. Thus, it appears that these organisms have the ability to regulate alternative pathways for the biosynthesis of certain amino acids and that pathways involving reductive carboxylations are likely to be favored in their natural habitats.     

Evolution of the biosynthesis of the branched-chain amino acids

The origin of the biosynthetic pathways for the branched-chain amino acids cannot be understood in terms of the backwards development of the present acetolactate pathway because it contains unstable intermediates. We propose that the first biosynthesis of the branched-chain amino acids was by the reductive carboxylation of short branched chain fatty acids giving keto acids which were then transaminated. Similar reaction sequences mediated by nonspecific enzymes would produce serine and threonine from the abundant prebiotic compounds glycolic and lactic acids. The aromatic amino acids may also have first been synthesized in this way, e.g. tryptophan from indole acetic acid. The next step would have been the biosynthesis of leucine from -ketoisovaleric acid. The acetolactate pathway developed subsequently. The first version of the Krebs cycle, which was used for amino acid biosynthesis, would have been assembled by making use of the reductive carboxylation and leucine biosynthesis enzymes, and completed with the development of a single new enzyme, succinate dehydrogenase. This evolutionary scheme suggests that there may be limitations to inferring the origins of metabolism by a simple back extrapolation of current pathways.     

Branched-chain amino acids improve glucose metabolism in rats with liver cirrhosis

It is well established that impaired glucose metabolism is a frequent complication in patients with hepatic cirrhosis. We previously showed that leucine, one of the branched-chain amino acids (BCAA), promotes glucose uptake under insulin-free conditions in isolated skeletal muscle from normal rats. The aim of the present study was to evaluate the effects of BCAA on glucose metabolism in a rat model of CCl(4)-induced cirrhosis (CCl(4) rats). Oral glucose tolerance tests were performed on BCAA-treated CCl(4) rats. In the CCl(4) rats, treatment with leucine or isoleucine, but not valine, improved glucose tolerance significantly, with the effect of isoleucine being greater than the effect of leucine. Glucose uptake experiments using isolated soleus muscle from the CCl(4) rats revealed that leucine and isoleucine, but not valine, promoted glucose uptake under insulin-free conditions. To clarify the mechanism of the blood glucose-lowering effects of BCAA, we collected soleus muscles from BCAA-treated CCl(4) rats with or without a glucose load. These samples were used to determine the subcellular location of glucose transporter proteins and glycogen synthase (GS) activity. Oral administration of leucine or isoleucine without a glucose load induced GLUT4 and GLUT1 translocation to the plasma membrane. GS activity was augmented only in leucine-treated rats and was completely inhibited by rapamycin, an inhibitor of mammalian target of rapamycin. In summary, we found that leucine and isoleucine improved glucose metabolism in CCl(4) rats by promoting glucose uptake in skeletal muscle. This effect occurred as a result of upregulation of GLUT4 and GLUT1 and also by mammalian target of rapamycin-dependent activation of GS in skeletal muscle. From these results, we consider that BCAA treatment may have beneficial effects on glucose metabolism in cirrhotic patients.     

Pseudoauxotrophy of Methanococcus voltae for acetate, leucine, and isoleucine.

Methanococcus voltae is a methanogenic bacterium which requires leucine, isoleucine, and acetate for growth. However, it also can synthesize these amino acids, and it is capable of low levels of autotrophic acetyl coenzyme A (acetyl-CoA) biosynthesis. When cells were grown in the presence of 14CO2, as well as in the presence of compounds required for growth, the alanine found in the cellular protein was radiolabeled. The percentages of radiolabel in the C-1, C-2, and C-3 positions of alanine were 64, 24, and 16%, respectively. The incorporation of radiolabel into the C-2 and C-3 positions of alanine demonstrated the autotrophic acetyl-CoA biosynthetic pathway in this bacterium. Additional evidence was obtained in cell extracts in which autotrophically synthesized acetyl-CoA was trapped into lactate. In these extracts, both CO and CH2O stimulated acetyl-CoA synthesis. 14CH2O was specifically incorporated into the C-3 of lactate. Cell extracts of M. voltae also contained low levels of CO dehydrogenase, 13 nmol min-1 mg of protein-1. These results further confirmed the presence of the autotrophic acetyl-CoA biosynthetic pathway in M. voltae. Likewise, 14CO2 and [U-14C]acetate were also incorporated into leucine and isoleucine during growth. During growth with [U-14C]leucine or [U-14C]isoleucine, the specific radioactivity of these amino acids in the culture medium declined, and the specific radioactivities of these amino acids recovered from the cellular protein were 32 to 40% lower than the initial specific radioactivities in the medium.Cell extracts of M. voltae also contained levels of isopropyl malate synthase, an enzyme that is specific to the leucine biosynthetic pathway, of 0.8 nmol min-1 mg of protein-1. Thus, M. voltae is capable of autotrophic CO2 fixation and leucine and isoleucine biosynthesis.     

Localization of polypyrimidine-tract-binding protein is involved in the regulation of albumin synthesis by branched-chain amino acids in HepG2 cells

Long-term supplementation of branched-chain amino acids (BCAA) improves hypoalbuminemia in patients with cirrhosis. Our previous findings have suggested that the binding of polypyrimidine-tract-binding protein (PTB) to rat albumin mRNA attenuates its translation. The aim of the present study was to investigate the role of PTB in the regulation of albumin synthesis by BCAA in human hepatoma cells. HepG2 cells were cultured in a medium containing no amino acids (AA-free medium), a medium containing only 1 amino acid (a BCAA: valine, leucine or isoleucine) or a medium containing all 20 amino acids (AA-complete medium). HepG2 cells cultured in AA-complete medium secreted much more albumin than cells cultured in AA-free medium, with no difference in albumin mRNA levels. In cells cultured in AA-free medium, nuclear export of PTB was observed, and the level of the albumin mRNA-PTB complex was greater than in cells cultured in AA-complete medium. Addition of amino acids stimulated nuclear import of PTB. However, addition of amino acids with rapamycin inhibited the nuclear import of PTB. The addition of leucine, but not of valine or isoleucine, to AA-free medium increased albumin secretion and stimulated the nuclear import of PTB. These data indicate that the mammalian target of rapamycin is involved in the regulation of PTB localization and that leucine promotes albumin synthesis by inhibiting the formation of the albumin mRNA-PTB complex.