Biosynthesis of branched-chain amino acids in yeast: regulation of leucine biosynthesis in prototrophic and leucine auxotrophic strains

The first enzyme in the biosynthesis of leucine in yeast, alpha-isopropylmalate synthetase, is inhibited by l-leucine. In a mutant resistant to the analogue 5',5',5'-trifluoroleucine, the enzyme is markedly resistant to inhibition by l-leucine. Growth ing the presence of exogenous l-leucine results in repression of the second and third enzymes of the pathway. The first enzyme is not repressed unless both l-leucine and l-threonine are supplied in the medium. Comparison of levels of the remaining two enzymes in leucine auxotrophs grown under conditions of leucine excess and leucine limitation reveals deviations from the wild-type derepression pattern in some mutants. In some, repression of the synthetase by leucine alone was observed. In others, the repressibility of the dehydrogenase was lost. It is unlikely that these deviations were due to the same primary mutational event that caused leucine auxotrophy. No mutants were found in which an altered gene was recognized to be clearly responsible for the level of the leucine-forming enzymes.     

Multivalent repression and genetic depression of isoleucine-valine biosynthetic enzymes in Serratia marcescens

The regulation of the formation of isoleucine-valine biosynthetic enzymes was examined to elucidate the mechanism of isoleucine-valine accumulation by alpha-aminobutyric acid-resistant (abu-r) mutants of Serratia marcescens. In the isoleucine-valine auxotroph, l-threonine dehydratase, acetohydroxy acid synthetase, and transaminase B were repressed when isoleucine, valine, and leucine were simultaneously added to minimal medium. These enzymes were derepressed at the limitation of any single branched-chain amino acid. Pantothenate, which stimulated growth of this auxotroph, had no effect on the enzyme levels. It became evident from these results that in S. marcescens isoleucine-valine biosynthetic enzymes are subject to multivalent repression by three branched-chain amino acids. The abu-r mutants had high enzyme levels in minimal medium, with or without three branched-chain amino acids. Therefore, in abu-r mutants, isoleucine-valine biosynthetic enzymes are genetically derepressed. This derepression was considered to be the primary cause for valine accumulation and increased isoleucine accumulation.     

Leucine biosynthesis in the blue-green bacterium Anacystis nidulans.

Leucine-requiring auxotrophs of the unicellular blue-green bacterium Anacystis nidulans have been isolated. Extracts of these mutants were deficient in alpha-isopropylmalate synthetase (EC 4.1.3.12). In wild-type cells, this enzyme was subject to feedback inhibition by leucine. However, formation of the enzymes of leucine biosynthesis was little affected by exogenous leucine in either wild-type or mutant strains. Cultures of the latter subjected to extreme leucine deprivation showed no change in specific activity of beta-isopropylmalate isomerase (EC 4.2.1.33) and at most a 50% increase in the specific activity of beta-isopropylmalate dehydrogenase (EC 1.1.1.85). These results are compared with others bearing on the evolution of the control of amino acid biosynthesis in blue-green bacteria.     

Phenylalanine and tyrosine biosynthesis in Escherichia coli K-12: mutants derepressed for 3-deoxy-D-arabinoheptulosonic acid 7-phosphate synthetase (phe), 3-deoxy-D-arabinoheptulosonic acid 7-phosphate synthetase (tyr), chorismate mutase T-prephenate dehydrogenase, and transaminase A

Mutant strains of Escherichia coli have been isolated in which the synthesis of 3-deoxy-d-arabinoheptulosonic acid 7-phosphate (DAHP) synthetase (phe) is derepressed, in addition to those enzymes of tyrosine biosynthesis previously shown to be controlled by the gene tyrR. The major enzyme of the terminal pathway of phenylalanine biosynthesis chorismate mutase-prephenate dehydratase is not derepressed in these strains. Genetic analysis of the mutants shows that the mutation or mutations causing derepression map close to previously reported tyrR mutations. A study of one of the mutations has shown it to be recessive to the wild-type allele in a diploid strain. It is proposed that the tyrR gene product is involved in the regulation of the synthesis of DAHP synthetase (phe) as well as the synthesis of DAHP synthetase (tyr), chorismate mutase-prephenate dehydrogenase, and transaminase A.     

Toxicity of the pyrimidine biosynthetic pathway intermediate carbamyl aspartate in Salmonella typhimurium

Growth of Salmonella typhimurium pyrC or pyrD auxotrophs was severely inhibited in media that caused derepressed pyr gene expression. No such inhibition was observed with derepressed pyrA and pyrB auxotrophs. Growth inhibition was not due to the depletion of essential pyrimidine biosynthetic pathway intermediates or substrates. This result and the pattern of inhibition indicated that the accumulation of the pyrimidine biosynthetic pathway intermediate carbamyl aspartate was toxic. This intermediate is synthesized by the sequential action of the first two enzymes of the pathway encoded by pyrA and pyrB and is a substrate for the pyrC gene product. It should accumulate to high levels in pyrC or pyrD mutants when expression of the pyrA and pyrB genes is elevated. The introduction of either a pyrA or pyrB mutation into a pyrC strain eliminated the observed growth inhibition. Additionally, a direct correlation was shown between the severity of growth inhibition of a pyrC auxotroph and the levels of the enzymes that synthesize carbamyl aspartate. The mechanism of carbamyl aspartate toxicity was not identified, but many potential sites of growth inhibition were excluded. Carbamyl aspartate toxicity was shown to be useful as a phenotypic trait for classifying pyrimidine auxotrophs and may also be useful for positive selection of pyrA or pyrB mutants. Finally, we discuss ways of overcoming growth inhibition of pyrC and pyrD mutants under derepressing conditions.     

Regulation of the carbamoylphosphate synthetase belonging to the arginine biosynthetic pathway of Saccharomyces cerevisiae

Two classes of regulatory mutations affecting the synthesis of the carbamoylphosphate synthetase belonging to the arginine biosynthetic pathway have been selected in Saccharomyces cerevisiae. Together, they delineate a negative type of control. The cpaI0 mutations, closely linked with one of the two genes coding for the enzyme and cis dominant, meet properties of operator mutations. The cpaR mutations can be interpreted as mutations impairing the formation of an active repressor of carbamoylphosphate synthetase which is distinct from the one acting on the synthesis of the other enzymes of the arginine biosynthetic pathway.     

Mutants of Escherichia coli with an altered tyrosyl-transfer ribonucleic acid synthetase

We have isolated several mutants defective in the gene for tyrosyl-transfer ribonucleic acid (tRNA) synthetase (tyrS). One of these mutants is described in detail. It was isolated as a tyrosine auxotroph with defects both in the tyrosyl-tRNA synthetase and in the tyrosine biosynthetic enzyme, prephenate dehydrogenase. It also had derepressed levels of the tyrosine-specific 3-deoxy-d-arabinoheptulosonic acid-7-phosphate (DAHP) synthetase. The latter finding suggested that a wild-type tyrS gene was required for repression of the tyrosine biosynthetic enzymes. The following results demonstrated that this hypothesis was not correct. (i) When the defective tyrS gene was transferred to another strain, the tyrosine-specific DAHP synthetase in that strain was not derepressed, and (ii) two other mutants with defective tyrosyl-tRNA synthetases had repressed levels of the tyrosine biosynthetic enzymes. The tyrS gene was located near minute 32 on the Escherichia coli chromosome by interrupted mating experiments.     

Minor threonine dehydratase encoded within the threonine synthetic region of Bacillus subtilis

Challenging auxotrophs on metabolites that are precursors of a biosynthetic step involving a mutated enzyme has revealed a new class of suppressor mutations which act by derepressing a minor enzyme activity not normally detected in the wild-type strain. These indirect, partial suppressor mutations which allow isoleucine auxotrophs to grow on homoserine or threonine have been analyzed to determine their effect on enzymes involved in the biosynthesis of these amino acids. It has been found that one class of these suppressor mutations (sprA) leads to the derepression of homoserine kinase, homoserine dehydrogenase, and a minor threonine dehydratase that is not sufficiently active to be detected in the wild-type strain. The gene encoding this second threonine dehydratase activity has been found to be located between the structural genes for homoserine kinase and homoserine dehydrogenase. The results of these experiments indicate that plating of auxotrophs on precursors of a biosynthetic step involving mutated enzymes could prove to be a valuable method for the detection of regulatory mutants as well as a possible tool in studying the evolution of biochemical pathways.     

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.     

Genetic Studies of Leucine Biosynthesis in Bacillus subtilis

The mutations in a series of leucine auxotrophs isolated after treatment with nitrosoguanidine, ultraviolet light, and ICR-191 have been mapped between ilvC and pheA on the Bacillus subtilis chromosome. A fine structure map of the region was constructed by transformation. Analysis of several strains by assaying levels of their leucine bioysnthetic enzymes has shown that the region encodes three enzymes. The order of the genes with respect to the biosynthetic steps catalyzed by the gene products is 1–3–2.     

Analysis of Polar and Nonpolar Tryptophan Mutants by Derepression Kinetics

A comparison of the rates of synthesis of the tryptophan biosynthetic enzymes of Salmonella typhimurium under derepression showed that the genes of the trp operon can be expressed in a coordinate fashion in auxotrophs carrying nonpolar mutations. This coordination disappeared in trpA polar mutants. The loss of coordination affected only trpB, the second gene in the operon, which was always more drastically affected than the three distal genes. Polar mutations in trpA, the first gene of the trp operon, reduced the rates of synthesis of the tryptophan biosynthetic enzymes under conditions of derepression. When these rates were measured and correlated with the map position of each polar mutation, a polarity gradient of decreasing intensity (moving distally from the operator end of the gene) was obtained. Certain mutations ("unusual mutations") mapping at the operator distal end of trpA, and considered by other workers to correspond to the operator proximal end of trpB, were found to be polar. The bearing of our observations on the question of coordinate versus semicoordinate expression of the trp genes and the status of the "unusual mutations" is discussed.     

Tryptophan biosynthesis in the marine luminous bacterium Vibrio harveyi.

Tryptophan biosynthetic enzyme levels in wild-type Vibrio harveyi and a number of tryptophan auxotrophs of this species were coordinately regulated over a 100-fold range of specific activities. The tryptophan analog indoleacrylic acid evoked substantial derepression of the enzymes in wild-type cells. Even higher enzyme levels were attained in auxotrophs starved for tryptophan, regardless of the location of the block in the pathway. A derepressed mutant selected by resistance to 5-fluorotryptophan was found to have elevated basal levels of trp gene expression; these basal levels were increased only two- to threefold by tryptophan limitation. The taxonomic implications of these and other biochemical results support previous suggestions that the marine luminous bacteria are more closely related to enteric bacteria than to other gram-negative taxa.     

Evidence for an Altered Operator Specificity: Catabolite Repression Control of the Leucine Operon in Salmonella typhimurium

A mutation, GD-1, in the leucine operon imposed unusual growth characteristics upon a leucine auxotrophic strain bearing the leucine operator mutation, leu-500. The strain with the GD-1 mutation was able to grow on a minimal salts medium when citrate was the sole carbon source, but required leucine when glucose was present. Tests with a large number of carbohydrates suggest that in the strain bearing the GD-1 mutation the leucine biosynthetic enzymes are under catabolite repressor control. Recombination studies indicate that the GD-1 mutation is a secondary alteration of the leucine operator at or very close to the site of the leu-500 mutation. Mutations at the supX locus (previously termed su leu 500 and located on the chromosome between the cysteine B and tryptophan gene clusters) result in elimination of the catabolite repression effect. The data are interpreted as an indication that the GD-1 and leu-500 mutations alter the leucine operator with respect to its specificity of response to repressors.     

Genetic and physiological control of serine and glycine biosynthesis in Saccharomyces

Two of the three known metabolic pathways to serine and glycine have been shown to be present in prototrophic yeast strains, i.e., the phosphorylated pathway from glycolytic intermediates and the glyoxylate pathway from tricarboxylic acid cycle intermediates. Two serine-glycine auxotrophs (ser1 and ser2) were found to be blocked in the phosphoglycerate pathway. The ser1 gene controls l-glutamate:phosphohydroxypyruvate transaminase biosynthesis, and the ser2 gene controls phosphoserine phosphatase biosynthesis. The other pathway to glycine, from isocitrate, is repressed by growth in glucose media, specifically, at isocitrate lyase and at the alanine:glyoxylate transaminase. This pathway is derepressed by growth to stationary phase in glucose media yielding high activity of these enzymes. The phosphorylated pathway appears to be the principal biosynthetic pathway to serine and glycine during growth on sugar media. Strains which are serine-glycine dependent in glucose media became capable of serine-glycine independent growth on acetate media. These results describe a method of physiological control of a secondary metabolic pathway allowing a single lesion in the principal biosynthetic pathway to produce auxotrophy. This may be termed conditional auxotrophy.     

Molecular characterization of Arabidopsis thaliana cDNAs encoding three purine biosynthetic enzymes

Glycinamide ribonucleotide (GAR) synthetase, GAR transformylase and aminoimidazole ribonucleotide (AIR) synthetase are the second, third and fifth enzymes in the 10-step de novo purine biosynthetic pathway. From a cDNA library of Arabidopsis thaliana, cDNAs encoding the above three enzymes were cloned by functional complementation of corresponding Escherichia coli mutants. Each of the cDNAs encode peptides comprising the complete enzymatic domain of either GAR synthetase, GAR transformylase or AIR synthetase. Comparisons of the three Arabidopsis purine biosynthetic enzymes with corresponding enzymes/polypeptide-fragments from procaryotic and eucaryotic sources indicate a high degree of conserved homology at the amino acid level, in particular with procaryotic enzymes. Assays from extracts of E. coli expressing the complementing clones verified the specific enzymatic activity of Arabidopsis GAR synthetase and GAR transformylase. Sequence analysis, as well as Northern blot analysis indicate that Arabidopsis has single and monofunctional enzymes. In this respect the organization of these three plant purine biosynthesis genes is fundamentally different from the multifunctional purine biosynthesis enzymes characteristic of other eucaryotes and instead resembles the one gene, one enzyme relationship found in procaryotes.     

Immunochemical characterization of glutamine synthetase from Neurospora crassa glutamine auxotrophs.

Glutamine synthetase derived from two Neurospora crassa glutamine auxotrophs was characterized. Previous genetic studies indicated that the mutations responsible for the glutamine auxotrophy are allelic and map in chromosome V. When measured in crude extracts, both mutant strains had lower glutamine synthetase specific activity than that found in the wild-type strain. The enzyme from both auxotrophs and the wild-type strain was partially purified from cultures grown on glutamine as the sole nitrogen source, and immunochemical studies were performed in crude extracts and purified fractions. Quantitative rocket immunoelectrophoresis indicated that the activity per enzyme molecule is lower in the mutants than in the wild-type strain; immunoelectrophoresis and immunochemical titration of enzyme activity demonstrated structural differences between the enzymes from both auxotrophs. On the other hand, the monomer of glutamine synthetase of both mutants was found to be of a molecular weight similar to that of the wild-type strain. These data indicate that the mutations are located in the structural gene of N. crassa glutamine synthetase.     

Enzymes of the Isoleucine-Valine Pathway in Acinetobacter

Regulation of four of the enzymes required for isoleucine and valine biosynthesis in Acinetobacter was studied. A three- to fourfold derepression of acetohydroxyacid synthetase was routinely observed in two different wild-type strains when grown in minimal medium relative to cells grown in minimal medium supplemented with leucine, valine, and isoleucine. A similar degree of synthetase derepression was observed in appropriately grown isoleucine or leucine auxotrophs. No significant derepression of threonine deaminase or transaminase B occurred in either wild-type or mutant cells grown under a variety of conditions. Three amino acid analogues were tested with wild-type cells; except for a two- to threefold derepression of dihydroxyacid dehydrase when high concentrations of aminobutyric acid were added to the medium, essentially the same results were obtained. Experiments showed that threonine deaminase is subject to feedback inhibition by isoleucine and that valine reverses this inhibition. Cooperative effects in threonine deaminase were demonstrated with crude extracts. The data indicate that the synthesis of isoleucine and valine in Acinetobacter is regulated by repression control of acetohydroxyacid synthetase and feedback inhibition of threonine deaminase and acetohydroxyacid synthetase.