The Product of the LEU-3 Cistron as a Regulatory Element for the Production of the Leucine Biosynthetic Enzymes of Neurospora

A class of intracistronic (or closely linked) partial reversions of leu-3 mutations has been found to be conditionally constitutive with respect to the synthesis of isopropylmalate isomerase (specified by the leu-2 cistron) and beta-isopropylmalate dehydrogenase (specified by the leu-1 cistron), two of the enzymes of leucine biosynthesis in Neurospora. The intermediate level of enzyme production by these leu-3(cc) mutants is independent of the obligatory inducer effector, alpha-isopropylmalate, but dependent upon the presence of the branched-chain amino acids, isoleucine, valine and leucine. The properties of leu-3+, leu-3 and leu-3(cc) in heterokaryons indicate that the transnuclear regulatory activity of the leu-3 product varies specifically as a function of available effector molecules. The information presented suggests that the leu-3 cistron is responsible not only for the production of a "positive" regulatory substance necessary for the expression of the leu-1 and leu-2 cistrons, but that it probably serves also a coordinating role in the expression of many of the genes involved in branched-chain amino acid metabolism.     

Biosynthesis of branched-chain amino acids in yeast: effect of carbon source on leucine biosynthetic enzymes.

The three enzymes in the leucine biosynthetic pathway of yeast do not exhibit coordinate repression and derepression in response to the carbon source available in the culture medium. Growth in an acetate medium results in derepression of the first enzyme in the pathway, alpha-isopropylmalate synthase, and repression of the second two enzymes, alpha-isopropylmalate isomerase and beta-isopropylmalate dehydrogenase, relative to the levels found in glucose-grown cells. The role of endogenous leucine pools as a mediator of these differences was investigated. The leucine pools did not differ significantly between acetate-grown and glucose-grown cells. However, an elevated endogenous leucine pool, caused by exogenous leucine in the growth medium, did decrease the rate of decay of alpha-isopropylmalate synthase activity observed when acetate-grown cells were shifted to glucose. Evidence is provided suggesting that an elevated endogenous leucine pool may increase the in vivo stability of alpha-isopropylmalate synthase under several different conditions. Studies on the kinetics of alpha-isopropylmalate synthase decay in vivo and sensitivity to leucine inhibition indicate that there are two classes of the enzyme in acetate-grown yeast cells.     

Regulation of isopropylmalate isomerase synthesis in Neurospora crassa.

The capacity to synthetize isopropylmalate isomerase (EC 4.2.1.33) by Neurospora crassa increased during induction in the presence of cycloheximide but was inhibited by proflavine and other inhibitors of RNA synthesis. Turnover of the enzyme once formed appeared negligible, but the message (measured as enzyme-forming capacity) had a half-life of 4 to 8 min. A comparison of the kinetics of induction in the wild type and a newly isolated alpha-isopropylmalate-permeable strain suggested strongly that feedback control by leucine of alpha-isopropylmalate production can adequately serve as the primary physiological regulator of endogenous inducer concentration. Genetic data are presented which implicate the involvement of two unlinked genes, ipm-1 and ipm-2, in determining permeation of alpha-isopropylmalate.     

A comprehensive survey on isoleucine biosynthesis pathways in seven epidemic Leptospira interrogans reference strains of China

Previous studies have indicated that different species of Leptospira synthesize isoleucine via either pyruvate and/or threonine pathways. Seven epidemic Leptospira interrogans reference strains from China belonging to different serovars, together with three saprophytic strains of Leptospira biflexa and Leptospira meyeri, were analysed. The isoleucine biosynthesis properties were studied firstly by measuring the key enzymes of the two pathways, citramalate synthase (CimA, CE4.1.3.-) and threonine deaminase (IlvA, CE4.2.1.16), from cell extracts of the bacteria. Meanwhile, alpha-isopropylmalate synthase (LeuA, CE4.2.1.12), the key enzyme of leucine biosynthesis, was also measured as a control. It was found that all L. interrogans strains synthesized isoleucine via the pyruvate pathway exclusively, but L. biflexa and L. meyeri used both pathways. Dot-Blot and PCR amplification of both cimA and ilvA genes in the corresponding strains provided additional evidence consistent with the data of enzymatic assays. Although it is evident that leptospires' isoleucine biosynthesis may preferentially adapt either to the pyruvate pathway exclusively for pathogens or to the combination of both pyruvate and threonine pathways for saprophytes, broader sampling with careful genomospecies identification is needed for a solid conclusion.     

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 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.     

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.     

alpha-Isopropylmalate, a leucine biosynthesis intermediate in yeast, is transported by the mitochondrial oxalacetate carrier

In Saccharomyces cerevisiae, alpha-isopropylmalate (alpha-IPM), which is produced in mitochondria, must be exported to the cytosol where it is required for leucine biosynthesis. Recombinant and reconstituted mitochondrial oxalacetate carrier (Oac1p) efficiently transported alpha-IPM in addition to its known substrates oxalacetate, sulfate, and malonate and in contrast to other di- and tricarboxylate transporters as well as the previously proposed alpha-IPM transporter. Transport was saturable with a half-saturation constant of 75 +/- 4 microm for alpha-IPM and 0.31 +/- 0.04 mm for beta-IPM and was inhibited by the substrates of Oac1p. Though not transported, alpha-ketoisocaproate, the immediate precursor of leucine in the biosynthetic pathway, inhibited Oac1p activity competitively. In contrast, leucine, alpha-ketoisovalerate, valine, and isoleucine neither inhibited nor were transported by Oac1p. Consistent with the function of Oac1p as an alpha-IPM transporter, cells lacking the gene for this carrier required leucine for optimal growth on fermentable carbon sources. Single deletions of other mitochondrial carrier genes or of LEU4, which is the only other enzyme that can provide the cytosol with alpha-IPM (in addition to Oac1p) exhibited no growth defect, whereas the double mutant DeltaOAC1DeltaLEU4 did not grow at all on fermentable substrates in the absence of leucine. The lack of growth of DeltaOAC1DeltaLEU4 cells was partially restored by adding the leucine biosynthetic cytosolic intermediates alpha-ketoisocaproate and alpha-IPM to these cells as well as by complementing them with one of the two unknown human mitochondrial carriers SLC25A34 and SLC25A35. Oac1p is important for leucine biosynthesis on fermentable carbon sources catalyzing the export of alpha-IPM, probably in exchange for oxalacetate.     

Subcellular localization of the leucine biosynthetic enzymes in yeast

When baker's yeast spheroplasts were lysed by mild osmotic shock, practically all of the isopropylmalate isomerase and the beta-isopropylmalate dehydrogenase was released into the 30,000 x g supernatant fraction, as was the cytosol marker enzyme, glucose-6-phosphate dehydrogenase. alpha-Isopropylmalate synthase, however, was not detected in the initial supernatant, but could be progressively solubilized by homogenization, appearing more slowly than citrate synthase but faster than cytochrome oxidase. Of the total glutamate-alpha-ketoisocaproate transaminase activity, approximately 20% was in the initial soluble fraction, whereas solubilization of the remainder again required homogenization of the spheroplast lysate. Results from sucrose density gradient centrifugation of a cell-free particulate fraction and comparison with marker enzymes suggested that alpha-isopropylmalate synthase was located in the mitochondria. It thus appears that, in yeast, the first specific enzyme in the leucine biosynthetic pathway (alpha-isopropylmalate synthase) is particulate, whereas the next two enzymes in the pathway (isopropylmalate isomerase and beta-isopropylmalate dehydrogenase) are "soluble," with glutamate-alpha-ketoisocaproate transaminase activity being located in both the cytosol and particulate cell fractions.     

Branched chain amino acid regulation of the ILV2 locus in Saccharomyces cerevisiae

Mutant regulatory loci of the branched pathway for the biosynthesis of isoleucine-valine and leucine were identified with the unusual phenotype of an amino acid dependent auxotrophy. Two mutant loci, bcs1 and bcs2, conferred branched chain amino acid sensitivity and showed independent segregation. Linkage studies defined bcs1 as a cis-acting regulatory site of ILV2 (SMR1). ILV2 upstream deletion analyses and high-copy transformation of the positive regulatory locus LEU3 ruled out the possibility of LEU3 protein binding palindromes mediating the branched chain amino acid dependent auxotrophy. In the presence of leucine and valine, the general amino acid control system (GCN4) was epistatic to bcs1 and bcs2, and under nonstarvation conditions GCN4 strains showed an increased acetolactate synthase activity over gcn4 strains. Thus in addition to general regulation of ILV2, GCN4 functions in basal level expression when the locus is subject to specific repression by pathway end product.