Biosynthesis of the Branched-Chain Amino Acids in Yeast: a Leucine-Binding Component and Regulation of Leucine Uptake

Use of an ion-exchange resin assay has shown that leucine is bound to a component of a dialyzed extract of yeast. Leucine binding may be related to in vivo uptake of the amino acid. A yeast strain with a 30-fold lower affinity for leucine uptake in vivo has a parallel reduction in affinity for in vitro leucine binding; the rate of leucine uptake in wild-type yeast can be increased four- to fivefold by growth on leucine as a sole nitrogen source. Under these conditions, the specific activity of the leucine-binding component also increases over threefold. Regulation of leucine uptake was studied by using wild-type strain 60615 and a mutant 60615/fl(2) with a constitutively elevated leucine uptake system. Leucine pool formation in the mutant was accompanied by an overshoot, leading to a loss of leucine from the pool. The phenomenon could be observed in the wild type under certain conditions. The mechanism of this process was examined. The leucine uptake system was found to be stable in the absence of protein synthesis. The rate of leucine uptake increased on reduction of the pool of amino acids, and in strain 60615/fl(2) the ability to overshoot was rapidly recovered on depletion of the leucine pool. The results suggest a control of leucine uptake by feedback inhibition, in which leucine or other amino acids, e.g., isoleucine, inhibit leucine uptake. The results do not exclude control by a rapidly activated-inactivated system.     

Biosynthesis of Branched-Chain Amino Acids in Yeast: Correlation of Biochemical Blocks and Genetic Lesions in Leucine Auxotrophs

The three enzymatic steps in the conversion of alpha-ketoisovalerate to alpha-ketoisocaproate were examined in wild-type and in leucine auxotrophic stocks of yeast. Procedures for the reliable assay of each of the enzymatic steps in crude extracts were devised. Crude extracts of the prototrophic haploid stock catalyzed all three enzymatic steps. Examination of a series of leucine auxotrophs permitted a correlation between the three enzymatic steps and the genetic lesions affecting 10 different loci. This examination revealed that a single locus (le-6) affected primarily alpha-isopropylmalate synthetase, the first step in the pathway. Lesions in six loci (le-1, le-4, le-5, le-7, le-8, and le-10) lead primarily to a deficiency in the activity of the second enzyme in the pathway, alpha-isopropylmalate isomerase. Stocks with lesions in three loci (le-2, le-3, and le-9) were primarily blocked in the third step of the pathway, catalyzed by beta-isopropylmalate dehydrogenase. The results with the mutants provide strong evidence that the pathway for leucine biosynthesis proposed by Strassman and his colleagues is the sole significant pathway in yeast.     

Biosynthesis of Branched-Chain Amino Acids in Yeast: Regulation of Synthesis of the Enzymes of Isoleucine and Valine Biosynthesis

Regulation of the levels of the five enzymes required for the biosynthesis of isoleucine and valine was studied in a Saccharomyces sp. When a mixture of isoleucine, valine, and leucine was added to the medium, the enzymes in the wild-type strain were repressed from about 30% (transaminase B) to about 90% (acetohydroxy acid synthetase) relative to the level in minimal medium-grown cells. Repression was also observed when threonine replaced isoleucine in the mixture but not when it replaced the other two amino acids. Significant derepression relative to the level in minimal-grown cells was not obtained by growing suitably blocked auxotrophs on medium containing limiting amounts of valine, isoleucine, or leucine.     

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.     

支链氨基酸合成调控机制及菌种选育策略研究进展

提高国内支链氨基酸产生菌的高产菌株选育水平有助于缩短与国外生产之间的差距,满足国内市场需求。根据支链氨基酸生物合成途径及代谢调节,重点阐述了合成过程中关键酶的代谢调控,介绍了诱变育种、代谢工程、基因组改组及全局转录机器工程四种育种策略的研究进展。在支链氨基酸选育方面,全局转录机器工程育种目前虽无成功实例,但具有很大的潜力,而其他育种策略在氨基酸的选育中均发挥重要作用,可供国内相关育种工作者参考使用。     

Escherichia coli K-12 Mutants Altered in the Transport of Branched-Chain Amino Acids

Two mutants of Escherichia coli K-12 are described which are resistant to the inhibition that valine exerts on the growth of E. coli. These mutants have lesions at two different loci on the chromosome. One of them, brnP, is linked to leu (87% cotransduction) and is located between leu and azi represented on the map at 1 min; the other, brnQ, is linked to phoA (96% cotransduction), probably between proC and phoA and represented at 10 min. These mutants are resistant to valine inhibition but are sensitive to dipeptides containing valine. Since it is known that dipeptides are taken up by E. coli through a transport system(s) different from those used by amino acids, this sensitivity to the peptides suggests an alteration in the active transport of valine. The mutants are resistant to valine only if leucine is present in the growth medium; the uptake of valine is less in both mutants than it is in wild-type E. coli, and it is reduced even further if leucine is present. Under these conditions the total uptake of valine is almost completely abolished in the brnQ mutant. The brnP mutant takes up about 60% as much valine as does the wild type, but no exogenous valine is incorporated into proteins. The apparent K(m) and V(max) of isoleucine, leucine, and valine for the transport system are reported; the brnP mutant, when compared to the wild type, has a sevenfold higher K(m) for isoleucine and a 17-fold lower K(m) for leucine; the V(max) for the three amino acids is reduced in the brnQ mutant, up to 20-fold for valine. The transport of arginine, aspartic acid, glycine, histidine, and threonine is not altered in the brnQ mutant under conditions in which that of the branched amino acids is. Evidence is reported that O-methyl-threonine enters E. coli through the transport system for branched amino acids, and that thiaisoleucine does not.     

Biosynthesis of Branched-Chain Amino Acids in Schizosaccharomyces pombe: Properties of Acetohydroxy Acid Synthetase1

The regulatory properties of acetohydroxy acid synthetase (AHAS), the first enzyme in the biosynthetic pathway to valine and the second in the isoleucine pathway, were investigated in the fission yeast Schizosaccharomyces pombe. The enzyme was partially purified from crude extracts by protamine sulfate treatment, ammonium sulfate fractionation, and gel filtration through Sephadex G-25. AHAS from S. pombe is unique in that its activity shows a single peak around pH 6.5; high sensitivity to feedback inhibition by valine at this pH (K(i) = 0.1 mM) indicates that the enzyme is involved in valine biosynthesis. Pyruvate saturation kinetics of AHAS extracted from cells grown on glycerol as sole carbon and energy source were normal and hyperbolic. In contrast, the enzyme from glucose-grown cells exhibited sigmoidal saturation kinetics, an effect which disappeared when the synthetase from such cells was partially purified. This phenomenon was shown to be due to competition for pyruvate between AHAS and pyruvate decarboxylase; the latter enzyme is present in large amounts in cells fermenting glucose. Valine inhibition is noncompetitive in nature, and this effector exhibits homotropic cooperative effects; isoleucine is a less-potent inhibitor of AHAS activity. Mercurial treatment reversibly desensitized the enzyme to valine inhibition. On the basis of these data, the S. pombe AHAS appears to be an allosteric regulatory enzyme with the properties of a negative V system.     

Astrocyte Leucine Metabolism: Significance of Branched-Chain Amino Acid Transamination

Abstract: We studied astrocytic metabolism of leucine, which in brain is a major donor of nitrogen for the synthesis of glutamate and glutamine. The uptake of leucine into glia was rapid, with a V _max of 53.6 ± 3.2 nmol/mg of protein/min and a K _m of 449.2 ± 94.9 µ M . Virtually all leucine transport was found to be Na⁺ independent. Astrocytic accumulation of leucine was much greater (3×) in the presence of α-aminooxyacetic acid (5 m M ), an inhibitor of transamination reactions, suggesting that the glia rapidly transaminate leucine to α-ketoisocaproic acid (KIC), which they then release into the extracellular fluid. This inference was confirmed by the direct measurement of KIC release to the medium when astrocytes were incubated with leucine. Approximately 70% of the leucine that the glia cleared from the medium was released as the keto acid. The apparent K _m for leucine conversion to extracellular KIC was a medium [leucine] of 58 µ M with a V _max of ∼2.0 nmol/mg of protein/min. The transamination of leucine is bidirectional (leucine + α-ketoglutarate ↮ KIC + glutamate) in astrocytes, but flux from leucine → glutamate is more active than that from glutamate → leucine. These data underscore the significance of leucine handling to overall brain nitrogen metabolism. The release of KIC from glia to the extracellular fluid may afford a mechanism for the "buffering" of glutamate in neurons, which would consume this neurotransmitter in the course of reaminating KIC to leucine.     

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

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

支链氨基酸的抗疲劳作用

支链氨基酸作为必需氨基酸,不仅是合成机体蛋白质的原料,而且具有特殊的生理、生物学功能。其代谢与抗疲劳作用的机理在本文中进行了详细阐述。     

支链氨基酸对运动大鼠氨基酸代谢和运动能力的影响

观察了支链氨基酸(BCAA)对大鼠运动能力和血清游离氨基酸代谢的影响。实验用21只雄性wistar大鼠,随机分为3组:正常组、游泳对照组和游泳补充BCAA组。2个运动组每天游泳训练1h,10天后游泳6h,观察补充BCAA对大鼠游泳运动能力和血清游离氨基酸水平的影响。实验结果表明,补充BCAA可明显提高大鼠游泳存活率,抑制血清中必需氨基酸、非必需氨基酸和总氨基酸水平升高,游泳运动后大鼠的血清中乳酸和LDH的升高幅度有所降低,抑制骨骼肌LDH活力的下降。说明补充BCAA可明显提高大鼠的运动能力,减少运动造成的蛋白质分解     

肝硬化疾病与支链氨基酸应用研究进展

蛋白质-营养不良是肝硬化病人最常见的并发症之一。肝脏作为蛋白质、脂类和糖代谢的主要器官,病变后的代谢紊乱随之而来。不适宜的蛋白质-能量摄入只会加重病情最后发生肝性脑病等危及生命的严重后果。因此,肝硬化病人的营养管理显得尤为重要,氨基酸的适宜供给无疑是营养治疗的重中之重。已知支链氨基酸能通过刺激肝细胞合成、减少肝损伤后的分解代谢等诸多方式改善营养状况,但是各种试验结果仍存在争议。最佳适宜量究竟多大,安全性范围的设定以及确切的保护机理等问题仍待进一步深入研究。     

Regulation of Synthesis of the Aminoacyl-Transfer Ribonucleic Acid Synthetases for the Branched-Chain Amino Acids of Escherichia coli

The regulation of synthesis of valyl-, leucyl-, and isoleucyl-transfer ribonucleic acid (tRNA) synthetases was examined in strains of Escherichia coli and Salmonella typhimurium. When valine and isoleucine were limiting growth, the rate of formation of valyl-tRNA synthetase was derepressed about sixfold; addition of these amino acids caused repression of synthesis of this enzyme. The rate of synthesis of the isoleucyl- and leucyl-tRNA synthetases was derepressed only during growth restriction by the cognate amino acid. Restoration of the respective amino acid to these derepressed cultures caused repression of synthesis of the aminoacyl-tRNA synthetase, despite the resumption of the wild-type growth rate.     

Biosynthesis of Vitamin B6 by a Yeast Mutant

The gradient-plate technique was employed to isolate mutants of Saccharomyces marxianus (NRRL-Y-1550) which, when grown in a synthetic culture medium, excreted about 2 mug/ml of vitamin B(6) as ascertained by microbiological assay. The major component that possessed vitamin B(6) activity was isolated by ion-exchange column chromatography and identified as pyridoxol by ultraviolet and fluorescence spectroscopy, as well as by paper chromatography and various chemical tests. Pyridoxal was also identified as one of the excreted compounds. Two other compounds that possessed vitamin B(6) activity were excreted in smaller quantities in the growth medium and have not yet been identified; they are not phosphates of vitamin B(6). The amount of vitamin B(6) excreted was not increased when the mutant was grown in the presence of various oxidation products of this vitamin. The methods and results reported here may be helpful in future studies on the biosynthesis of vitamin B(6).     

Neuronal Metabolism of Branched-Chain Amino Acids: Flux Through the Aminotransferase Pathway in Synaptosomes

Abstract: The metabolism of branched-chain amino acids (BCAAs) was studied in cortical synaptosomes. With [¹⁵N]leucine (1 mM) as precursor, the cumulative appearance of ¹⁵N in [¹⁵N]glutamate and [¹⁵N]aspartate was 0.2 nmol/min/mg of protein without supplemental α-ketoglutarate and 0.3 nmol/min/mg of protein in the presence of α-ketoglutarate (0.5 mM). The BCAA aminotransferase reaction also proceeded in the “reverse” direction [α-ketoisocaproate (KIC) + glutamate → leucine + α-ketoglutarate]. This was documented by incubating synaptosomes with [¹⁵N]glutamate and measuring the formation of [¹⁵N]leucine. Without KIC in the medium, the rate of [¹⁵N]leucine production was 0.13 nmol/min/mg of protein. In the presence of 25 µM KIC the rate was 0.79 nmol/min/mg of protein and even greater (1.0 nmol/min/mg of protein) in the presence of 500 µM KIC. The reamination of KIC was two- to threefold faster with [2-¹⁵N]glutamine as precursor compared with [¹⁵N]glutamate. The ketoacid of valine, α-ketoisovalerate (KIV), was reaminated to [¹⁵N]valine at a rate comparable to that observed with respect to KIC. The BCAA transaminase mediated not only the bidirectional transfer of amino groups between leucine or valine and glutamate, but also the direct transfer of nitrogen between leucine and valine. This was ascertained in studies in which the incubation medium was supplemented with either [¹⁵N]leucine and KIV or [¹⁵N]valine and KIC (amino acids at 1 mM and ketoacids at 25 or 500 µM). The rate was faster in the direction of leucine formation at both the lower (6.1-fold) and higher (1.7-fold) KIC concentration. It is suggested that in synaptosomes the BCAA transaminase (a) functions predominantly in the direction of leucine formation and (b) maintains a constant ratio of BCAAs and ketoacids to one other.     

flrB, a Regulatory Locus Controlling Branched-Chain Amino Acid Biosynthesis in Salmonella typhimurium

Salmonella typhimurium strain CV123 (ara-9 gal-205 flrB1), isolated as a mutant resistant to trifluoroleucine, has derepressed and constitutive levels of enzymes forming branched-chain amino acids. This strain grows more slowly than the parent at several temperatures, both in minimal medium and nutrient broth. It overproduces and excretes sizeable amounts of leucine, valine, and isoleucine in comparison with the parental strain. Both leuS (coding for leucyl-transfer ribonucleic acid [tRNA]synthetase) and flrB are linked to lip (min 20 to 25) by P1 transduction, whereas only leuS is linked to lip by P22 transduction. Strain CV123 containing an F' lip(+) episome from Escherichia coli has repressed levels of leucine-forming enzymes, indicating that flrB(+) is dominant to flrB. Leucyl-tRNA synthetase from strain CV123 appears to be identical to the leucyl-tRNA synthetase in the parent. No differences were detected between strain CV123 and the parent with respect to tRNA acceptor activity for a number of amino acids. Furthermore, there was no large difference between the two strains in the patterns of leucine tRNA isoaccepting species after fractionation on several different columns. Several other flrB strains exhibited temperature-sensitive excretion of leucine, i.e., they excreted leucine at 37 C but not 25 C. In one such strain, excretion at 37 C was correlated with derepression of some enzymes specified by ilv and leu. These latter results suggest that flrB codes for a protein.     

Quantitative Analysis of the Whole-Body Metabolic Fate of Branched-Chain Amino Acids

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Biosynthesis of Amino Acids from Hydrocarbons

Nine strains of bacteria, capable of synthesising amino acids from hydrocarbons, have been identified. Pseudomonas arvilla, Ps. fluorescens and Micrococcus ureae, previously reported as hydrocarbons assimilators, were found capable of synthesising amino acids. Pseudomonas taetrolens, Bacillus cereus var. mycoides, Bacillus subtilis and Bacillus sphaericus also synthesised amino acids from hydrocarbons.     

YjeH Is a Novel Exporter of l-Methionine and Branched-Chain Amino Acids in Escherichia coli

Amino acid efflux transport systems have important physiological functions and play vital roles in the fermentative production of amino acids. However, no methionine exporter has yet been identified in Escherichia coli. In this study, we identified a novel amino acid exporter, YjeH, in E. coli. The yjeH overexpression strain exhibited high tolerance to the structural analogues of l-methionine and branched-chain amino acids, decreased intracellular amino acid levels, and enhanced export rates in the presence of a Met-Met, Leu-Leu, Ile-Ile, or Val-Val dipeptide, suggesting that YjeH functions as an exporter of l-methionine and the three branched-chain amino acids. The export of the four amino acids in the yjeH overexpression strain was competitively inhibited in relation to each other. The expression of yjeH was strongly induced by increasing cytoplasmic concentrations of substrate amino acids. Green fluorescent protein (GFP)-tagged YjeH was visualized by total internal reflection fluorescence microscopy to confirm the plasma membrane localization of YjeH. Phylogenetic analysis of transporters indicated that YjeH belongs to the amino acid efflux family of the amino acid/polyamine/organocation (APC) superfamily. Structural modeling revealed that YjeH has the typical “5 + 5” transmembrane α-helical segment (TMS) inverted-repeat fold of APC superfamily transporters, and its binding sites are strictly conserved. The enhanced capacity of l-methionine export by the overexpression of yjeH in an l-methionine-producing strain resulted in a 70% improvement in titer. This study supplements the transporter classification and provides a substantial basis for the application of the methionine exporter in metabolic engineering.