Valine Synthesis Research Articles

Role of the leu-3 cistron in the regulation of the synthesis of isoleucine and valine biosynthetic enzymes of Neurospora.

The production by Neurospora of the enzymes of isoleucine and valine synthesis in response to specific end product-derived signals depends upon the presence of an effective leu-3 regulatory product and its effector alpha-isopropylmalate (alpha-IPM). In leu-3(+) strains, threonine deaminase production is repressed as a function of available isoleucine, acetohydroxy acid synthetase as a function of valine, and the isomeroreductase and dihydroxy acid dehydratase as a function of isoleucine and leucine. In the absence of an effective leu-3 regulatory product, alpha-isopropylmalate, or both, the production of isoleucine and valine biosynthetic enzymes is fixed at or near fully repressed levels even under conditions of severe end product limitation. Thus, in addition to its involvement in the regulation of expression of the three structural genes of leucine synthesis, the leu-3 alpha-IPM regulatory product is necessary for full expression of at least four genes specifying the structure of the enzymes of isoleucine and valine synthesis. It is suggested that the leu-3 alpha-IPM regulatory element may facilitate transcription of the genetically dispersed cistrons either by imposing specificity on ribonucleic acid polymerase for structurally similar promoters adjacent to each of the cistrons or by "opening" promoters after interaction with nearly identical stretches of deoxyribonucleic acid near each of the structural genes.

Changes in enzyme activity of germinating conidia of Neurospora crassa

The conidia of wild-type Neurospora crassa are shown to have a drastically lower activity for three enzymes of the isoleucine-valine pathway—acetohydroxy acid synthetase, dihydroxy acid dehydratase, and aminotransferase—than the actively growing mycelium. Lower activity was also found in the conidia for ornithine transcarbamylase and aspartate transcarbamylase. Lower activity (10- to 100-fold) was found for the overall synthesis of valine from pyruvate in the conidia as compared to the mycelium as expected. In addition it is also apparent that the distribution of the isoleucine-valine enzymes is different in conidia from the mycelium as regards activity in the mitochondria as compared to the cytosol. In conidia their activity in the mitochondria is lower than in the cytosol, but the opposite holds in the mycelium. These differences are also reflected in the overall activity. Cycloheximide inhibits the increase in total activity of the acetohydroxy acid synthetase and the dehydratase during germination of the conidia.

Inheritance of plasmodial valine requirement inPhysarum polycephalum

SUMMARYA defined medium forPhysarum polycephalumplasmodia has been devised containing glutamic acid, glycine, methionine, biotin, thiamine, glucose, salts and haematin, which supports good growth on agar plates of many different strains. Tests on this medium have revealed a requirement for valine in some plasmodia formed by homothallic progeny (mthapt-l+) from the crossa(mt1apt-l+) ×APT1(mthapt-l−). The valine requirement was also inherited among heterothallic progeny of this cross and its segregation was followed in several heterothallic crosses. To explain the results it is proposed that valine synthesis requires the presence of dominant alleles at either of two unlinked loci and that only plasmodia homozygous for recessive alleles at both loci are valine dependent. In some crosses studied only one pair of alleles is segregating and valine requirement thus provides a useful genetic marker, and the first reported nutritional marker inP. polycephalum. The value of crosses withapt−mutants for both the detection and analysis of plasmodial markers is demonstrated and discussed.

Branched-chain Amino Acid Biosynthesis in the Blue-green Alga Anabaena variabilis

SUMMARY: Four enzymes (threonine deaminase, a-acetohydroxyacid synthetase, iso-propylmalate synthetase and transaminase B) involved in leucine, isoleucine and valine synthesis have been detected in extracts of the blue-green alga Anabaena variabilis and the control of three of the enzymes examined. A new procedure for the determination of transaminase B is described. Threonine deaminase and a-acetohydroxyacid synthetase are subject to feedback control by their end-products. No evidence for the repression of enzyme synthesis in the pathway has been found, although the assimilation and incorporation of exogenous valine and leucine was demonstrated.

Effect of soil application of chlorinated insecticides on amino acid composition of maize (Zea mays)

Chlorinated insecticidesviz p,p′-DDT, aldrin, endrin and lindane stimulated the synthesis of arginine+histidine, leucine, lysine, proline, and tyrosine in maize but decreased the content of tryptophan. However, synthesis of methionine was stimulated by the application of aldrin, endrin, and lindane only. On the otherhand, synthesis of valine was inhibited by aldrin, endrin and DDT only.

Synthesis of amino acids from succinate by the mitochondria of Neurospora crassa.

The synthesis of valine by isolated Neurospora mitochondria in the presence of pyruvate is increased more than twofold when the cofactor assay mixture consisting of NADP, thiamine pyrophosphate (TPP), glucose-6-phosphate, and pyridoxal phosphate, and the respiring assay mixture containing ADP, orthophosphate, and succinate are combined. Although no single component of the cofactor mixture is completely responsible for the increase in valine synthesis, addition of TPP to the respiring assay mixture has the greatest effect.Neurospora mitochondria are able also to synthesize valine when pyruvate and glucose-6-phosphate are omitted from the combined mixtures. The succinate, a component of the respiring mixture, is the carbon source for the synthesis of valine, alanine, and glutamic acid. However, these syntheses require the addition of succinate, NADP, and TPP only. Addition of ADP and orthophosphate has no effect on the amounts of amino acids synthesized from succinate. Provisional evidence is presented that the source of pyruvate used in such syntheses is succinate-derived malate which is then converted to pyruvate by a mitochondrial decarboxylating malate dehydrogenase ("malic" enzyme).

Initiation of hemoglobin synthesis: comparison of model reactions that use artificial templates with those using natural messenger RNA.

PARTIAL REACTIONS DESIGNED TO STUDY THE INDIVIDUAL STEPS IN THE INITIATION OF PROTEIN SYNTHESIS MAY BE DIVIDED INTO TWO GROUPS: artificial models using artificial mRNA templates and natural models using naturally occurring mRNA. The requirements for each of the reticulocyte initiation factors, IF-M(1), IF-M(2), and IF-M(3), and for GTP are examined using these models in order to determine if either type of model is a valid representation of the events occurring in natural initiation. Binding of the initiator tRNA, Met-tRNA(F), to endogenous mRNA requires IF-M(1), IF-M(2), and GTP. A requirement for hydrolysis of GTP is not found when the artificial template ApUpG is used since GDPCP may be substituted for GTP. Met-tRNA(F) bound to the template ApUpG by IF-M(1) + IF-M(2) can form a peptide bond with the aminoacyl-tRNA analog, puromycin. Met-tRNA(F) bound by IF-M(1) + IF-M(2) to the initiator codon of natural globin mRNA, however, cannot form a peptide bond with either puromycin or valine-tRNA unless IF-M(3) is also present. The requirements for Met(F)-valine synthesis on exogenous globin mRNA are the same as the requirements on endogenous mRNA. Synthesis of the initial tripeptides of the alpha and beta chains of rabbit globin, Met(F)-Val-Leu and Met(F)-Val-His, requires, in addition, leucyl-tRNA and histidyl-tRNA. It appears, therefore, that model systems that use natural messenger RNA can duplicate the factor and energy requirements of natural initiation, but that the model systems thus far studied that use artificial messengers as templates do not.

In vitro mitochondrial complementation in Neurospora crassa.

Mitochondrial isoleucine-valine biosynthesis in strain 330a, an iv-1 mutant of Neurospora, is blocked at the dihydroxy acid dehydration step owing to a mutation in the nuclear structural gene for the specific enzyme dihydroxy acid dehydratase. Dehydratase purified from either the soluble or the mitochondrial fraction of wild-type Neurospora, and incubated in vitro with 330a mitochondria, restores valine synthesis from pyruvate-C 14 to wild type levels. Up to 29% of the restored synthesis could be attributed to the penetration of enzyme into the mitochondria. However, the bulk of the restored synthesis was found to be mediated via the secretion of dihydroxyvaline (DHV) by the mitochondria into the assay milieu, with subsequent enzymatic catalysis of this metabolite to ketovaline occurring outside the organelle. The ketovaline apparently diffuses back into mitochondria for final transamination to valine. This shunting of valine precursors in and out of mitochondria has been demonstrated to be the mechanism whereby two different populations of mitochondria isolated from mutants 330a and 305A (an iv-2 mutant lacking a functional reductoisomerase) can complement each other for the biosynthesis of valine, even when each population is enclosed in a separate dialysis bag. This observation provides the basis for a biochemical understanding of the growth complementation at the organismic level when these two iv-requiring mutants are cultured together in minimal medium.

Amino acid synthesis by the mitochondria of Neurospora crassa: I. Dependence on respiration of mitochondria

The synthesis of valine, isoleucine, alanine, glutamate, and aspartate by isolated mitochondria of Neurospora crassa was markedly enhanced by the addition of ADP, inorganic phosphate, and succinate to an incubation mixture containing the precursors, pyruvate, and α-ketobutyrate. The addition of either 2,4-dinitrophenol or oligomycin to the incubation mixture caused a significant reduction in the amount of amino acids produced. KCN caused an even more marked reduction. It is concluded that the synthesis of these amino acids by the mitochondria is in part dependent on respiration and associated oxidative phosphorylation. It was shown by using 14C-labeled succinate that carbon from succinate is not incorporated into either alanine or valine, but it is into aspartate and glutamate. The relative amount of incorporation into the latter two amino acids can be varied by adding or omitting exogenous pyruvate. While exogenous pyruvate is incorporated into valine, it is also apparent that a pool of endogenous source of precursor exists which contributes significantly to valine synthesis in respiring mitochondria.

Synthesis of valine and isoleucine in primitive earth conditions.

SYNTHESIS of small amounts of valine, isoleucine and leucine from the polymerization of ammonium cyanide solutions has been reported1,2 in addition to the substantial yields of glycine, alanine, aspartic acid and serine3. We have been unable to find any valine, isoleucine or leucine synthesized by the polymerization reaction of NH4CN alone. On adding acetone or methyl ethyl ketone to the NH4CN solutions, significant yields of valine, alloisoleucine and isoleucine are, however, obtained. The previous identifications were based on the elution pattern from a Beckman1 automatic amino-acid column4. Because several non-protein amino-acids appear at the same elution positions as valine, isoleucine and leucine5,6 (for example, isoasparagine elutes with isoleucine), an error of identification is possible, although the specific amino-acids in this case are not known. We have used a more elaborate separation scheme to avoid the problem of overlapping peaks.

Isoleucine and valine metabolism of Escherichia coli. XVI. Pattern of multivalent repression in strain K-12.

The levels of the five enzymes required for isoleucine and valine synthesis were examined under several growth conditions in strain K-12 of Escherichia coli and mutants derived from it. In strains with wild type repressibility, the same pattern of derepression was found on limiting isoleucine as is found to be constitutive in strain Tir-8, which has an altered isoleucine-activating enzyme. Homoserine dehydrogenase, which is essential for the biosynthesis of threonine and is normally derepressed on limiting isoleucine or threonine, is also derepressed in strain Tir-8. Threonine deaminase and homoserine dehydrogenase were partially repressed in strain Tir-8 by very high levels of isoleucine, but were not further derepressed over levels in minimal medium by limiting isoleucine.

Pyruvate metabolism by aminopterin-inhibited Aerobacter aerogenes.

1. The synthesis and utilization of both alanine (by reductive amination, oxidative deamination and transamination) and valine (by transamination only) in Aerobacter aerogenes are unaffected by aminopterin. These amino acids, which accumulate in aminopterin-treated cultures of this organism, are therefore considered to be formed as secondary products from the excess of pyruvate that also accumulates. 2. Oxidative metabolism of pyruvate and the synthesis of acetylmethylcarbinol by A. aerogenes cells are unaltered by growth in the presence of aminopterin. 3. Cells from static and anaerobic cultures that have been treated with the folic acid antagonist in the early exponential phase have a decreased ability to cleave pyruvate to acetate and formate, and to effect the exchange of formate with the carboxyl group of pyruvate. 4. 3-Methyl-2-oxobutanoate, the keto acid precursor of valine, cannot replace pyruvate as substrate in either the phosphoroclastic or the exchange reaction.

The overall in vitro synthesis of valine from pyruvate by neurospora homogenates

1. (1) The conditions for the synthesis in vitro of valine from pyruvate by the 39 000 × g pellet fraction of Neurospora homogenate are described. This activity for valine synthesis was also obtained in the supernatant fraction by appropriate means of preparation of the homogenate. 2. (2) Evidence is presented which indicates that the overall synthesis of valine from pyruvate occurs in the presence of a complex of at least four enzymes which is bound in vivo to the particulate part of the cell.

CONTROL OF VALINE AND ISOLEUCINE METABOLISM IN PSEUDOMONAS AERUGINOSA AND ESCHERICHIA COLI.

SUMMARY: Isoleucine increased the quantity of α-acetolactic acid-forming enzyme which takes part in the synthesis of valine and isoleucine in Pseudomonas aeruginosa. Threonine and α-ketobutyric acid, the precursors of isoleucine, had a similar effect. In P. aeruginosa during the initial stage of growth after inoculation, a temporary substantial increase of α-acetolactic acid-forming enzyme can be observed. This increase was not so great when valine was present, and was enhanced by isoleucine, which was effective during the whole period of growth. The repressive effect of valine can be counteracted with isoleucine and the end product inductive effect of isoleucine can be inhibited by valine. In extracts of disrupted organisms the activity of the α,β-dihydroxy acid dehydrase changes parallel with the concentration of the α-acetolactic acid-forming enzyme. In the case of Escherichia coli the repressive effect of valine on the α-acetolactic acid-forming enzyme can be counteracted with isoleucine.

Studies of higher alcohol formation by yeasts through gas chromatography

The methods of gas chromatography have made possible significant advances in understanding the pathways of formation of higher alcohols during alcoholic fermentation. The higher alcohols in wines of fermented media were quantitatively concentrated by close fractional distillation coupled with drying and extraction with diethyl ether. Gas-liquid chromatography was employed for determining the percentage of the individual alcohol components as well as for separation and collection of specific alcohols by condensation of the effluent gases in order to measure radioactive carbon-14 activities incorporated from C-14 intermediates and for degradation studies. The principal results of recent studies of the pathways of higher alcohol formation indicate that they are formed by reactions closely related to the synthesis of valine, leucine and isoleucine. However, the amounts of higher alcohols formed by the Ehrlich pathway of direct breakdown of endogenous amino acids are often negliglible or non-existent. Evidence was obtained to support the hypothesis that 2-ketobutyric acid is the intermediate from which bothn-propanol andn-butanol are found in fermentations by mutant strains of yeast.