Metabolism of valine and 3-methyl-2-oxobutanoate by the isolated perfused rat kidney.

Metabolism of branched-chain amino and 2-oxo acids was studied in the isolated perfused kidney. Significant amounts of 2-oxo acids were released by perfused kidney with all concentrations of amino acids tested (0.1-1.0 mM each), despite the high activity of branched-chain 2-oxo acid dehydrogenase in kidney. As perfusate valine concentration was increased from 0.2 to 1.0 mM, [1-14C]valine transamination (2-oxo acid oxidized + released) increased roughly linearly; [1-14C]valine oxidation, however, increased exponentially. Increasing perfusate concentration of 3-methyl-2-oxo[1-14C]butanoate from 0 to 1.0 mM resulted in a linear increase in the rate of its oxidation and a rise in perfusate valine concentration; at the same time significant decreases occurred in perfusate isoleucine and leucine concentrations, with corresponding increases in rates of release of their respective 2-oxo acids. Comparison of rates of oxidation of [1-14C]valine and 3-methyl-2-oxo[1-14C]butanoate suggests that 2-oxo acid arising from [1-14C]valine transamination has freer access to the 2-oxo acid dehydrogenase than has the 2-oxo acid from the perfusate. The observations indicate that, when branched-chain amino and 2-oxo acids are present in perfusate at near-physiological concentrations, rates of transamination of the amino and 2-oxo acids by isolated perfused kidney are greater than rates of oxidation.     

Feedback-Resistant Acetohydroxy Acid Synthase Increases Valine Production in Corynebacterium glutamicum

Acetohydroxy acid synthase (AHAS), which catalyzes the key reactions in the biosynthesis pathways of branched-chain amino acids (valine, isoleucine, and leucine), is regulated by the end products of these pathways. The whole Corynebacterium glutamicum ilvBNC operon, coding for acetohydroxy acid synthase (ilvBN) and aceto hydroxy acid isomeroreductase (ilvC), was cloned in the newly constructed Escherichia coli-C. glutamicum shuttle vector pECKA (5.4 kb, Km^r). By using site-directed mutagenesis, one to three amino acid alterations (mutations M8, M11, and M13) were introduced into the small (regulatory) AHAS subunit encoded by ilvN. The activity of AHAS and its inhibition by valine, isoleucine, and leucine were measured in strains carrying the ilvBNC operon with mutations on the plasmid or the ilvNM13 mutation within the chromosome. The enzyme containing the M13 mutation was feedback resistant to all three amino acids. Different combinations of branched-chain amino acids did not inhibit wild-type AHAS to a greater extent than was measured in the presence of 5 mM valine alone (about 57%). We infer from these results that there is a single binding (allosteric) site for all three amino acids in the enzyme molecule. The strains carrying the ilvNM13 mutation in the chromosome produced more valine than their wild-type counterparts. The plasmid-free C. glutamicum ΔilvA ΔpanB ilvNM13 strain formed 90 mM valine within 48 h of cultivation in minimal medium. The same strain harboring the plasmid pECKAilvBNC produced as much as 130 mM valine under the same conditions.     

Influence of Incubation Atmosphere on Growth and Amino Acid Requirements of Streptococcus mutans

The growth response of Streptococcus mutans representing antigenic type a or d in a chemically defined medium was influenced by the oxygen concentration of the growth atmosphere. Under controlled aerobic (1.5% O(2)) conditions these cultures attained a greater density than when the atmosphere contained 0.006% O(2) or less. The growth of S. mutans strains representing antigenic types b or c in the defined medium was independent of the oxygen concentration of the growth environment. Under the conditions used in this study, none of the strains tested could utilize ammonium ion as a sole source of nitrogen for growth. The requirement for certain amino acids and inhibition by other amino acids varied with antigenic type and relative oxygen concentration of the growth environment. Under conditions where the atmospheric oxygen was reduced to 0.0006% O(2) or less, the amino acid requirements of the cultures became either more numerous or more stringent. S. mutans strains of type c generally required the least number of amino acids, whereas cultures of type d had more numerous requirements. Nearly every culture tested under the anaerobic atmosphere was inhibited by one of the branched-chain amino acids, leucine, valine, or isoleucine. Methionine and lysine were also found to be inhibitory, particularly toward the type c strains.     

Acute Effect of Ammonia on Branched-Chain Amino Acid Oxidation and Incorporation into Proteins in Astrocytes and in Neurons in Primary Cultures

14CO2 production and incorporation of label into proteins from the labeled branched-chain amino acids, leucine, valine, and isoleucine, were determined in primary cultures of neurons and of undifferentiated and differentiated astrocytes from mouse cerebral cortex in the absence and presence of 3 mM ammonium chloride. Production of 14CO2 from [1-14C]leucine and [1-14C]valine was larger than 14CO2 production from [U-14C]leucine and [U-14C]valine in both astrocytes and neurons. In most cases more 14CO2 was produced in astrocytes than in neurons. Incorporation of labeled branched-chain amino acids into proteins varied with the cell type and with the amino acid. Addition of 3 mM ammonium chloride greatly suppressed 14CO2 production from [1-14C]-labeled branched chain amino acids but had little effect on 14CO2 production from [U-14C]-labeled branched-chain amino acids in astrocytes. Ammonium ion, at this concentration, suppressed the incorporation of label from all three branched-chain amino acids into proteins of astrocytes. In contrast, ammonium ion had very little effect on the metabolism (oxidation and incorporation into proteins) of these amino acids in neurons. The possible implications of these findings are discussed, especially regarding whether they signify variations in metabolic fluxes and/or in magnitudes of precursor pools.     

Regulation of Transaminase C Synthesis in Escherichia coli: Conditional Leucine Auxotrophy

The regulation of synthesis of the valine-alanine-alpha-aminobutyrate transaminase (transaminase C) was studied in Escherichia coli mutants lacking the branched-chain amino acid transaminase (transaminase B). An investigation was made of two strains, CU2 and CU2002, each carrying the same transaminase B lesion but exhibiting different growth responses on a medium supplemented with branched-chain amino acids. Both had the absolute isoleucine requirement characteristic of ilvE auxotrophs, but growth of strain CU2 was stimulated by valine, whereas that of strain CU2002 was markedly inhibited by valine. Strain CU2002 behaved like a conditional leucine auxotroph in that the inhibition by valine was reversed by leucine. Results of enzymatic studies showed that synthesis of transaminase C was repressed by valine in strain CU2002 but not in strain CU2. Inhibition by valine in strain CU2002 appears to be the combined effect of repression on transaminase C synthesis and valine-dependent feedback inhibition of alpha-acetohydroxy acid synthase activity, causing alpha-ketoisovalerate (and hence leucine) limitation. The ilvE markers of strains CU2 and CU2002 were each transferred by transduction to a wild-type genetical background. All ilvE recombinants from both crosses resembled strain CU2002 and were inhibited by valine in the presence of isoleucine. Thus, strain CU2 carries an additional lesion that allows it to grow on a medium containing isoleucine plus valine. It is concluded that conditional leucine auxotrophy is characteristic of mutants carrying an ilvE lesion alone.     

Uncovering the role of branched-chain amino acid transaminases in Saccharomyces cerevisiae isobutanol biosynthesis

Isobutanol and other branched-chain higher alcohols (BCHAs) are promising advanced biofuels derived from the degradation of branched-chain amino acids (BCAAs). The yeast Saccharomyces cerevisiae is a particularly attractive host for the production of BCHAs due to its high tolerance to alcohols and prevalent use in the bioethanol industry. Degradation of BCAAs begins with transamination reactions, catalyzed by branched-chain amino acid transaminases (BCATs) located in the mitochondria (Bat1p) and cytosol (Bat2p). However, the roles that these transaminases play in isobutanol production remain poorly understood and obscured by conflicting reports in the literature. In this work, we elucidate the influence of BCATs on isobutanol production in two genetic backgrounds (CEN.PK2-1C and BY4741). In the process, we uncover and characterize two competing isobutanol pathways, which can be manipulated by overexpressing or deleting BAT1 or BAT2, and adding or removing valine from the fermentation media. We show that deletion of BAT1 alone increases isobutanol production by 14.2-fold over wild type strains in media lacking valine, and examine how interactions between valine and the regulatory protein Ilv6p affect isobutanol production. Compartmentalizing the five-gene isobutanol biosynthetic pathway in mitochondria of BAT1 deletion strains results in an additional 2.1-fold increase in isobutanol production in the absence of valine. While valine inhibits isobutanol production, it boosts 2-methyl-1-butanol production. This work clarifies the role of transamination activity in BCHA biosynthesis, and develops valuable strategies and strains for future optimization of isobutanol production.     

Pyruvate Decarboxylase Catalyzes Decarboxylation of Branched-Chain 2-Oxo Acids but Is Not Essential for Fusel Alcohol Production by Saccharomyces cerevisiae

The fusel alcohols 3-methyl-1-butanol, 2-methyl-1-butanol, and 2-methyl-propanol are important flavor compounds in yeast-derived food products and beverages. The formation of these compounds from branched-chain amino acids is generally assumed to occur via the Ehrlich pathway, which involves the concerted action of a branched-chain transaminase, a decarboxylase, and an alcohol dehydrogenase. Partially purified preparations of pyruvate decarboxylase (EC 4.1.1.1) have been reported to catalyze the decarboxylation of the branched-chain 2-oxo acids formed upon transamination of leucine, isoleucine, and valine. Indeed, in a coupled enzymatic assay with horse liver alcohol dehydrogenase, cell extracts of a wild-type Saccharomyces cerevisiae strain exhibited significant decarboxylation rates with these branched-chain 2-oxo acids. Decarboxylation of branched-chain 2-oxo acids was not detectable in cell extracts of an isogenic strain in which all three PDC genes had been disrupted. Experiments with cell extracts from S. cerevisiae mutants expressing a single PDC gene demonstrated that both PDC1- and PDC5-encoded isoenzymes can decarboxylate branched-chain 2-oxo acids. To investigate whether pyruvate decarboxylase is essential for fusel alcohol production by whole cells, wild-type S. cerevisiae and an isogenic pyruvate decarboxylase-negative strain were grown on ethanol with a mixture of leucine, isoleucine, and valine as the nitrogen source. Surprisingly, the three corresponding fusel alcohols were produced in both strains. This result proves that decarboxylation of branched-chain 2-oxo acids via pyruvate decarboxylase is not an essential step in fusel alcohol production.Saccharomyces cerevisiae has been used for centuries in the production of bread and alcoholic beverages. Along with ethanol and carbon dioxide, fermenting cultures of this yeast produce a variety of low-molecular-weight flavor compounds (including alcohols, diacetyl, esters, organic acids, organic sulfides, and carbonyl compounds). The compounds 3-methyl-1-butanol, 2-methyl-1-butanol, and 2-methyl-1-propanol, commonly known as fusel alcohols, and their esters make an important contribution to the flavor of alcoholic beverages and bread (1, 14).A metabolic pathway for production of fusel alcohols by yeast was first proposed by Ehrlich (6). The Ehrlich pathway starts with the enzyme-catalyzed decarboxylation of branched-chain 2-oxo acids to the corresponding aldehydes. Subsequently, the aldehyde is reduced to the corresponding fusel alcohol by an alcohol dehydrogenase (11, 16, 24). The branched-chain 2-oxo acid substrates for the Ehrlich pathway can be produced by the deamination of l-leucine, l-isoleucine, or l-valine. Growth of S. cerevisiae with any of these three amino acids as the nitrogen source results in the accumulation of the corresponding fusel alcohol (2, 3, 21). Alternatively, branched-chain 2-oxo acids may be synthesized de novo from carbohydrates as intermediates of branched-chain amino acid synthesis (13).The conversion of branched-chain oxo acids into their respective aldehydes and alcohols via the Ehrlich pathway resembles the fermentative metabolism of pyruvate, which yields ethanol and carbon dioxide. In both cases, the decarboxylation of a 2-oxo acid is followed by the reduction of the resulting aldehyde. Partially purified preparations of yeast pyruvate decarboxylase have been shown to catalyze the decarboxylation of various 2-oxo acids, including the putative intermediates of the Ehrlich pathway (8, 12, 16, 21). However, it has not been conclusively proven that pyruvate decarboxylase is essential for or even involved in fusel alcohol production by S. cerevisiae.Dickinson and Dawes (4) have reported that, at least under some conditions, oxidative decarboxylation by a mitochondrial branched-chain oxo acid dehydrogenase complex (17) is involved in the catabolism of branched-chain 2-oxo acids. Mutants that did not express the lipoamide dehydrogenase subunit of this enzyme complex accumulated branched-chain oxo acids in batch cultures grown on media containing leucine, isoleucine, or valine (4), thus casting some doubt on the exclusive role of pyruvate decarboxylase in the decarboxylation of branched-chain oxo acids.The aim of this study was to reinvestigate the role of pyruvate decarboxylase in the production of fusel alcohols by S. cerevisiae. The S. cerevisiae genome harbors three structural genes (PDC1, PDC5, and PDC6) that can each encode an active pyruvate decarboxylase (9). In wild-type yeast strains, PDC6 expression is either very low or absent (7, 9). However, revertants of pdc1-pdc5 double mutants, in which a recombination event has caused a fusion of the PDC1 promoter and the PDC6 open reading frame, express a functional enzyme (10). Therefore, studies on the physiological effects of pyruvate decarboxylase deficiency are most easily interpreted when they are performed with strains in which all three PDC genes are disrupted.In the present study, the decarboxylation of branched-chain 2-oxo acids was studied in cell extracts of wild-type S. cerevisiae and in extracts of an isogenic pyruvate decarboxylase-negative mutant. Furthermore, conversion of branched-chain amino acids to the corresponding fusel alcohols by intact cells was analyzed in ethanol-grown cultures of a wild-type S. cerevisiae strain and in those of the Pdc⁻ mutant.     

Feedback-resistant acetohydroxy acid synthase increases valine production in Corynebacterium glutamicum

Acetohydroxy acid synthase (AHAS), which catalyzes the key reactions in the biosynthesis pathways of branched-chain amino acids (valine, isoleucine, and leucine), is regulated by the end products of these pathways. The whole Corynebacterium glutamicum ilvBNC operon, coding for acetohydroxy acid synthase (ilvBN) and aceto hydroxy acid isomeroreductase (ilvC), was cloned in the newly constructed Escherichia coli-C. glutamicum shuttle vector pECKA (5.4 kb, Km(r)). By using site-directed mutagenesis, one to three amino acid alterations (mutations M8, M11, and M13) were introduced into the small (regulatory) AHAS subunit encoded by ilvN. The activity of AHAS and its inhibition by valine, isoleucine, and leucine were measured in strains carrying the ilvBNC operon with mutations on the plasmid or the ilvNM13 mutation within the chromosome. The enzyme containing the M13 mutation was feedback resistant to all three amino acids. Different combinations of branched-chain amino acids did not inhibit wild-type AHAS to a greater extent than was measured in the presence of 5 mM valine alone (about 57%). We infer from these results that there is a single binding (allosteric) site for all three amino acids in the enzyme molecule. The strains carrying the ilvNM13 mutation in the chromosome produced more valine than their wild-type counterparts. The plasmid-free C. glutamicum DeltailvA DeltapanB ilvNM13 strain formed 90 mM valine within 48 h of cultivation in minimal medium. The same strain harboring the plasmid pECKAilvBNC produced as much as 130 mM valine under the same conditions.     

Catabolism of leucine to branched-chain fatty acids in Staphylococcus xylosus

AIMS: Staphylococcus xylosus is an important starter culture in the production of flavours from the branched-chain amino acids leucine, valine and isoleucine in fermented meat products. The sensorially most important flavour compounds are the branched-chain aldehydes and acids derived from the corresponding amino acids and this paper intends to perspectivate these flavour compounds in the context of leucine metabolism. METHODS AND RESULTS: GC and GC/MS analysis combined with stable isotope labelling was used to study leucine catabolism. This amino acid together with valine and isoleucine was used as precursors for the production of branched-chain fatty acids for cell membrane biosynthesis during growth. A 83.3% of the cellular fatty acids were branched. The dominating fatty acid was anteiso-C(15:0) that constituted 55% of the fatty acids. A pyridoxal 5'-phosphate and alpha-ketoacid dependent reaction catalysed the deamination of leucine, valine and isoleucine into their corresponding alpha-ketoacids. As alpha-amino group acceptor alpha-keto-beta-methylvaleric acid and alpha-ketoisovaleric acid was much more efficient than alpha-ketoglutarate. The sensorially and metabolic key intermediate on the pathway to the branched-chain fatty acids, 3-methylbutanoic acid was produced from leucine at the onset of the stationary growth phase and then, when the growth medium became scarce in leucine, from the oxidation of glucose via pyruvate. CONCLUSIONS: This paper demonstrates that the sensorially important branched-chain aldehydes and acids are important intermediates on the metabolic route leading to branched-chain fatty acids for cell membrane biosynthesis. SIGNIFICANCE AND IMPACT OF THE STUDY: The metabolic information obtained is extremely important in connection with a future biotechnological design of starter cultures for production of fermented meat.     

Production of 2-methyl-1-butanol and 3-methyl-1-butanol in engineered Corynebacterium glutamicum

The pentanol isomers 2-methyl-1-butanol and 3-methyl-1-butanol represent commercially interesting alcohols due to their potential application as biofuels. For a sustainable microbial production of these compounds, Corynebacterium glutamicum was engineered for producing 2-methyl-1-butanol and 3-methyl-1-butanol via the Ehrlich pathway from 2-keto-3-methylvalerate and 2-ketoisocaproate, respectively. In addition to an already available 2-ketoisocaproate producer, a 2-keto-3-methylvalerate accumulating C. glutamicum strain was also constructed. For this purpose, we reduced the activity of the branched-chain amino acid transaminase in an available C. glutamicum l-isoleucine producer (K2P55) via a start codon exchange in the ilvE gene enabling accumulation of up to 3.67 g/l 2-keto-3-methylvalerate. Subsequently, nine strains expressing different gene combinations for three 2-keto acid decarboxylases and three alcohol dehydrogenases were constructed and characterized. The best strains accumulated 0.37 g/l 2-methyl-1-butanol and 2.76 g/l 3-methyl-1-butanol in defined medium within 48 h under oxygen deprivation conditions, making these strains ideal candidates for additional strain and process optimization.     

Fatty Acid Production from Amino Acids and α-Keto Acids by Brevibacterium linens BL2

Low concentrations of branched-chain fatty acids, such as isobutyric and isovaleric acids, develop during the ripening of hard cheeses and contribute to the beneficial flavor profile. Catabolism of amino acids, such as branched-chain amino acids, by bacteria via aminotransferase reactions and α-keto acids is one mechanism to generate these flavorful compounds; however, metabolism of α-keto acids to flavor-associated compounds is controversial. The objective of this study was to determine the ability of Brevibacterium linens BL2 to produce fatty acids from amino acids and α-keto acids and determine the occurrence of the likely genes in the draft genome sequence. BL2 catabolized amino acids to fatty acids only under carbohydrate starvation conditions. The primary fatty acid end products from leucine were isovaleric acid, acetic acid, and propionic acid. In contrast, logarithmic-phase cells of BL2 produced fatty acids from α-keto acids only. BL2 also converted α-keto acids to branched-chain fatty acids after carbohydrate starvation was achieved. At least 100 genes are potentially involved in five different metabolic pathways. The genome of B. linens ATCC 9174 contained these genes for production and degradation of fatty acids. These data indicate that brevibacteria have the ability to produce fatty acids from amino and α-keto acids and that carbon metabolism is important in regulating this event.     

Regulation of branched-chain amino acid oxidation in isolated muscles, nerves and aortas of rats.

1. The oxidation of the three branched-chain amino acids was regulated in parallel fashion in rat tissues studied in vitro. 2. With 0.1 mM-[1-14C]isoleucine as substrate in the presence of 5.5 mM-glucose, 14CO2 production was 0.6 mumol/2 h per g in the aorta, 0.3 in peripheral nerve, 0.2 in muscle and 0.13 in spinal cord. 3. The ratio 14C oxidized/14C incorporated into proteins with 0.1 mM-[1-14C]leucine was 1.3 in hemidiaphragms, 3.3 in sciatic nerve and 1.0 in nerves undergoing Wallerian degeneration. Leucine oxidation decreased only slightly during degeneration, but protein synthesis doubled. 4. Hemidiaphragms incubated with [1-14C]leucine or 4-methyl-2-oxo[1-14C]pentanoate increased 14CO2 production 7-9-fold as substrate concentration was increased from 0.1 to 0.5 mM; under the same conditions 14CO2 production by nerves increased only 2-3-fold. 5. 2-Oxoglutarate stimulated the oxidation of the branched-chain amino acids by muscles and peripheral nerves and the oxidation of 4-methyl-2-oxopentanoate by hemidiaphragms but not by nerves. 6. Octanoate (0.1-1.0 mM) markedly stimulated the oxidation of branched-chain amino acids and of 4-methyl-2-oxopentanoate in hemidiaphragms, but inhibited oxidation of both by peripheral nerves and spinal cord. In aortas, oxidation of isoleucine (the only substance tested) was inhibited by octanoate. 7. The effects of octanoate and 2-oxoglutarate on leucine oxidation by hemidiaphragms were additive at low concentrations. When maximally stimulating concentrations of either agent were used, addition of the other was ineffective. 8. Pyruvate inhibited the oxidation of branched-chain amino acids and 4-methyl-2-oxopentanoate in all tissues tested. 9. Insulin did not affect the oxidation of 4-methyl-2-oxopentanoate by muscles or nerves. 10. The oxidative decarboxylation of the branched-chain alpha-oxo acids is suggested as a regulatory site of branched-chain amino acid oxidation. Differences in regulation between muscle on the one hand, and nerve and aorta on the other, are discussed.     

Separate regulation of transport and biosynthesis of leucine, isoleucine, and valine in bacteria.

Since both transport activity and the leucine biosynthetic enzymes are repressed by growth on leucine, the regulation of leucine, isoleucine, and valine biosynthetic enzymes was examined in Escherichia coli K-12 strain EO312, a constitutively derepressed branched-chain amino acid transport mutant, to determine if the transport derepression affected the biosynthetic enzymes. Neither the iluB gene product, acetohydroxy acid synthetase (acetolactate synthetase, EC 4.1.3.18), NOR THE LEUB gene product, 3-isopropylmalate dehydrogenase (2-hydroxy-4-methyl-3-carboxyvalerate-nicotinamide adenine dinucleotide oxido-reductase, EC 1.1.1.85), were significantly affected in their level of derepression or repression compared to the parental strain. A number of strains with alterations in the regulation of the branched-chain amino acid biosynthetic enzymes were examined for the regulation of the shock-sensitive transport system for these amino acids (LIV-I). When transport activity was examined in strains with mutations leading to derepression of the iluB, iluADE, and leuABCD gene clusters, the regulation of the LIV-I transport system was found to be normal. The regulation of transport in an E. coli strain B/r with a deletion of the entire leucine biosynthetic operon was normal, indicating none of the gene products of this operon are required for regulation of transport. Salmonella typhimurium LT2 strain leu-500, a single-site mutation affecting both promotor-like and operator-like function of the leuABCD gene cluster, also had normal regulation of the LIV-I transport system. All of the strains contained leucine-specific transport activity, which was also repressed by growth in media containing leucine, isoleucine and valine. The concentrated shock fluids from these strains grown in minimal medium or with excess leucine, isoleucine, and valine were examined for proteins with leucine-binding activity, and the levels of these proteins were found to be regulated normally. It appears that the branched-chain amino acid transport systems and biosynthetic enzymes in E. coli strains K-12 and B/r and in S. typhimurium strain LT2 are not regulated together by a cis-dominate type of mechanism, although both systems may have components in common.     

Amino Acid Metabolism of Lemna minor L. : II. Responses to Chlorsulfuron

Chlorsulfuron, an inhibitor of acetolactate synthase (EC 4.1.3.18) (TB Ray 1984 Plant Physiol 75: 827-831), markedly inhibited the growth of Lemna minor at concentrations of 10⁻⁸ molar and above, but had no inhibitory effects on growth at 10⁻⁹ molar. At growth inhibitory concentrations, chlorsulfuron caused a pronounced increase in total free amino acid levels within 24 hours. Valine, leucine, and isoleucine, however, became smaller percentages of the total free amino acid pool as the concentration of chlorsulfuron was increased. At concentrations of chlorsulfuron of 10⁻⁸ molar and above, a new amino acid was accumulated in the free pool. This amino acid was identified as α-amino-n-butyrate by chemical ionization and electron impact gas chromatography-mass spectrometry. The amount of α-amino-n-butyrate increased from undetectable levels in untreated plants, to as high as 840 nanomoles per gram fresh weight (2.44% of the total free pool) in plants treated with 10⁻⁴ molar chlorsulfuron for 24 hours. The accumulation of this amino acid was completely inhibited by methionine sulfoximine. Chlorsulfuron did not inhibit the methionine sulfoximine induced accumulations of valine, leucine, and isoleucine, supporting the idea that the accumulation of the branched-chain amino acids in methionine sulfoximine treated plants is the result of protein turnover rather than enhanced synthesis. Protein turnover may be primarily responsible for the failure to achieve complete depletion of valine, leucine, and isoleucine even at concentrations of chlorsulfuron some 10⁴ times greater than that required to inhibit growth. Tracer studies with ¹⁵N demonstrate that chlorsulfuron inhibits the incorporation of ¹⁵N into valine, leucine, and isoleucine. The α-amino-n-butyrate accumulated in the presence of chlorsulfuron and [¹⁵N]H₄⁺ was heavily labeled with ¹⁵N at early time points and appeared to be derived by transamination from a rapidly labeled amino acid such as glutamate or alanine. We propose that chlorsulfuron inhibition of acetolactate synthase may lead to accumulation of 2-oxobutyrate in the isoleucine branch of the pathway, and transamination of 2-oxobutyrate to α-amino-n-butyrate by a constitutive transaminase utilizing either glutamate or alanine as α-amino-N donors.     

Interrelationships of Leucine and Glutamate Metabolism in Cultured Astrocytes

Abstract: The aim was to study the extent to which leu-cine furnishes α-NH₂ groups for glutamate synthesis via branched-chain amino acid aminotransferase. The transfer of N from leucine to glutamate was determined by incubating astrocytes in a medium containing [¹⁵N]leucine and 15 unlabeled amino acids; isotopic abundance was measured with gas chromatography-mass spectrometry. The ratio of labeling in both [¹⁵N]glutamate/[¹⁵N]leucine and [2-¹⁵N]glutamine/[¹⁵N]leucine suggested that at least one-fifth of all glutamate N had been derived from leucine nitrogen. At the same time, enrichment in [¹⁵N]leucine declined, reflecting dilution of the ¹⁶N label by the unlabeled amino acids that were in the medium. Isotopic abundance in [¹⁶N]-isoleucine increased very quickly, suggesting the rapidity of transamination between these amino acids. The appearance of ¹⁵N in valine was more gradual. Measurement of branched-chain amino acid transaminase showed that the reaction from leucine to glutamate was approximately six times more active than from glutamate to leucine (8.72 vs. 1.46 nmol/min/mg of protein). However, when the medium was supplemented with α-ketoisocaproate (1 mM), the ketoacid of leucine, the reaction readily ran in the “reverse” direction and intraastrocytic [glutamate] was reduced by ～50% in only 5 min. Extracellular concentrations of α-ketoisocaproate as low as 0.05 mM significantly lowered intracellular [glutamate]. The relative efficiency of branched-chain amino acid transamination was studied by incubating astrocytes with 15 unlabeled amino acids (0.1 mM each) and [¹⁵N]glutamate. After 45 min, the most highly labeled amino acid was [¹⁵N]alanine, which was closely followed by [¹⁵N]leucine and [¹⁵N]isoleucine. Relatively little ¹⁵N was detected in any other amino acids, except for [¹⁵N]serine. The transamination of leucine was ～17 times greater than the rate of [1-¹⁴C]leucine oxidation. These data indicate that leucine is a major source of glutamate nitrogen. Conversely, reamination of a-ketoisocaproate, the ketoacid of leucine, affords a mechanism for the temporary “buffering” of intracellular glutamate.     

Nutritional requirements for growth of Helicobacter pylori.

A chemically defined medium consisting of a buffered mineral base supplemented with amino acids, a purine, and thiamine supported growth of 23 clinical isolates and the type strain of Helicobacter pylori. The growth of four strains was inhibited by the presence of certain amino acids. All but one strain required alanine for growth. The amino acids leucine, valine, phenylalanine, methionine, arginine, and histidine were generally required. Isoleucine either was required or stimulated growth. Strains could be differentiated into groups on the basis of a requirement for one or more of the amino acids cysteine, serine, and proline. Only one strain however, demonstrated a requirement for all three of these amino acids.     

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.     

Uptake of Branched-Chain Amino Acids by Streptococcus thermophilus

The transport of branched-chain amino acids in Streptococcus thermophilus was energy dependent. The metabolic inhibitors of glycolysis and ATPase enzymes were active, but the proton-conducting uncouplers were not. Transport was optimal at temperatures of between 30 and 45°C and at pH 7.0 for the three amino acids leucine, valine, and isoleucine; a second peak existed at pH 5.0 with valine and isoleucine. By competition and kinetics studies, the branched-chain amino acids were found to share at least a common transport system.     

Leucine biosynthesis: effect of branched-chain amino acids and threonine on a-isopropylmalate synthase activity from aerobic and anaerobic microorganisms

$\text{a-}\text{Isopropylmalate}$ synthase activity was demonstrated in the Sephadex G 25 gel filtrated crude extracts of one yeast and 43 bacterial strains belonging to 14 families. The enzyme was inhibited by leucine from all strains Bacteroides fragilis, Clostridia and several phototropic bacteria. The enzyme was inhibited by leucine from all strains investigated. In crude extracts of 17 species (8 genera) the leucine-mediated inhibition could be relieved by the addition of valine or isoleucine , but not by the addition of threonine or alanine. The enzymes from 11 species (7 genera) were inhibited by 1 mM valine and isoleucine, whereas the enzyme activity from 5 bacteria (4genera) were not so affected. These results suggest that valine and isoleucine are specifically involved in the regulation of leucine biosynthesis in several bacteria. The affect of valine and isoleucine on the IPM-synthase activity from mycobacteria and Corynebacterium autotrophicum lends support to the reclassification of Mycobacterium flavum 301 to C. autotrophicum. The antagonism between 5′,5′,5′-trifluoroleucine and amino acids and $\text{a-}\text{ketoisovalerate}$ was $\text{a-}\text{isopropylmalate}$ synthase in the presence or abssence of leucine and the reversal of the 5′,5′,5′-trifluoroleucine-mediated growth inhibition by these amino acids.