Arginine Catabolic Enzymes Research Articles

UME6 is a key regulator of nitrogen repression and meiotic development.

This report describes the identification, cloning, and molecular analysis of UME6 (CAR80/CARGRI), a key transcriptional regulator of early meiotic gene expression. Loss of UME6 function results in the accumulation of fully derepressed levels (70- to 100-fold increase above basal level) of early meiotic transcripts during vegetative growth. In contrast, mutations in five previously identified UME loci (UME1 to UME5), result in low to moderate derepression (2- to 10-fold increase) of early meiotic genes. The behavior of insertion and deletion alleles indicates that UME6 is dispensable for mitotic division but is required for meiosis and spore germination. Despite the high level of meiotic gene expression during vegetative growth, the generation times of ume6 mutant haploid and diploid cells are only slightly reduced. However, both ascus formation and spore viability are affected more severely. The UME6 gene encodes a 91-kD protein that contains a C6 zinc cluster motif similar to the DNA-binding domain of GAL4. The integrity of this domain is required for UME6 function. It has been reported recently that a mutation in CAR80 fails to complement an insertion allele of UME6. CAR80 is a gene required for nitrogen repression of the arginine catabolic enzymes. Here, through sequence analysis, we demonstrate that UME6 and CAR80 are identical. Analyses of UME6 mRNA during both nitrogen starvation and meiotic development indicate that its transcription is constitutive, suggesting that regulation of UME6 activity occurs at a post-transcriptional level.

Map locations of some mutations conferring resistance to arginine hydroxamate in Bacillus subtilis 168.

Mutations conferring resistance to arginine hydroxamate in Bacillus subtilis 168 have been located on the genetic map by PBS1-mediated transduction. The majority of these mutations, belonging to classes 1, 2 and 4 of Harwood and Baumberg (1977) and affecting only expression of arginine catabolic enzymes, map at a locus designated ahr A cotransducible with cysA, purA and sacA. The order of markers in this region appears to be sacA-ahrA-purA-cysA. Certain anomalies were observed in the properties of Pur+ transductants from crosses with an Ahr donor and a purA recipient. A single ahr mutation (class 3), also affecting only arginine catabolism, maps between ctrA and sacA at a locus designated ahrB. Two others (class 6), affecting simultaneously enzymes of both arginine biosynthesis and catabolism, map between lys and aroD at a locus designated ahrC. Preliminary attempts to define the nature of functional products specified by these ahr loci suggest that a protein is encoded at ahrA.

L-ornithine transaminase synthesis in Saccharomyces cerevisiae: Regulation by inducer exclusion

The ornithine transaminase (EC.2.6.1.13) of Saccharomyces cerevisiae is induced by arginine, ornithine, and their analogs. Genetic regulatory elements which are involved in this induction process have been defined due to the isolation of specific mutants. Two classes of OTAse operator mutants have previously been described; three unlinked genes are presumed to code for a specific repressor, CARGR of both of the arginine catabolic enzymes, arginase, and ornithine transaminase. The level of transaminase of cells grown on ammonia plus arginine is much lower than it is when arginine is the sole nitrogen source. Ammonia thus seems to limit the amount of enzyme synthesized when arginine is present in the growth medium. Nevertheless, all attempts to disclose a nitrogen catabolite repression process in OTAse synthesis have failed; neither the action of mutations that release this regulation on arginase and other catabolic enzymes, nor the use of derepressing growth conditions, affect OTAse synthesis. A decrease of the cells' arginine pool when amonia or aminoacids (serine, glutamate) are added to arginine as a nitrogen nutrient results in a progressive reduction of transaminase synthesis. This suggests that arginine is the only physiological effector in those conditions: ammonia or some aminoacids would reduce the enzyme synthesis because of an inducer exclusion. The first stage of OTAse induction would then be operated by the CARGR repressor, and an additional regulatory element might take part in the full scale process. Preliminary data favoring the involvment of such an element are presented.

Carbon and nitrogen repression of arginine catabolic enzymes in Bacillus subtilis.

Specific activities of arginase and ornithine aminotransferase, inducible enzymes of arginine catabolism in Bacillus subtilis 168, were examined in cells grown with various carbon and nitrogen sources. Levels of these enzymes were similar in arginine-induced cultures whether glucose or citrate was the carbon source (in contrast to histidase), suggesting that carbon source catabolite repression has only limited effect. In media with combinations of nitrogen sources, glutamine strongly repressed induction of these enzymes by proline or arginine. Ammonium, however, only repressed induction by proline and had no effect on induction by arginine. These effects correlate with generation times in media containing these substances as sole nitrogen sources: growth rates decreased in the order glutamine-arginine-ammonium-proline. Similar phenomena were observed when glutamine or ammonium were added to arginine- or proline-grown cultures, or when arginine or proline were added to glutamine- or ammonium-grown cultures. In the latter cases, an additional feature was apparent, namely a surprisingly long transition between steady-state enzyme levels. The results are compared with those for other bacteria and for eucaryotic microorganisms.

Arginine Hydroxamate-resistant Mutants of Bacillus subtilis with Altered Control of Arginine Metabolism

Arginine hydroxamate inhibits the growth of Bacillus subtilis. From a large number of mutants isolated as resistant to this arginine analogue, 29 were chosen for further investigation. Most of these shared diminished ability to utilize arginine, citrulline and/or ornithine as sole nitrogen source. All 29 had reduced levels of the catabolic enzymes arginase and ornithine aminotransferase under various conditions in which these enzymes are induced in the parent. In some circumstances, five of the mutants also showed elevated levels of the biosynthetic enzyme ornithine carbamoyltransferase. On the basis of these data, the 29 mutants were divided into six phenotypic classes; in four of these, control of ornithine carbamoyltransferase was the same as in the wild type, while in the other two it was altered. It is suggested that the isolates carry regulatory mutations, and that certain of these may affect simultaneously the formation of arginine catabolic and biosynthetic enzymes. The implication of the latter is that in B. subtilis, as in yeast, controls of the catabolic and biosynthetic pathways are connected. Single representatives of five of the phenotypic classes carry mutations conferring arginine hydroxamate resistance linked to cysA by transduction with phage PBSI; this did not appear to be true for a representative of the sixth class.

Control of arginine utilization in Neurospora.

The response of Neurospora to changes in the availibility of exogenous arginine was investigated. Upon addition of arginine to the growth medium, catabolism is initiated within minutes. This occurs prior to expansion of the arginine pool or augmentation of catabolic enzyme levels. (Basal levels are approximately 25% of those found during growth in arginine-supplemented medium.) Catabolism of arginine is independent of protein synthesis, indicating that the catabolic enzymes are active but that arginine is not available for catabolism unless present in the medium. Upon exhaustion of the supply of exogenous arginine, catabolism ceases abruptly, despite an expanded arginine pool and induced levels of the catabolic enzymes. The arginine pool supports protein synthesis until the cells regain their normal capacity for endogenous arginine synthesis. These observations, combined with the known small level of induction of arginine catabolic enzymes, non-repressibility of most biosynthetic enzymes, and vesicular localization of the bulk of the arginine pool, suggest that compartmentation plays a significant role in controlling arginine metabolism in Neurospora.

Physiological consequences of starvation in Pseudomonas putida: degradation of intracellular protein and loss of activity of the inducible enzymes of L-arginine catabolism.

We investigated the degradation of radioisotopically labeled intracellular protein in starved, intact cells of Pseudomonas putida P2 (ATCC 25571) and the regulation of this process. Intracellular protein isotopically labeled with L-[4,5-3H]leucine during log-phase growth at 30 C is degraded at rates of 1 to 2%/h in log-phase cells and 7 to 9%/h in starved cells. Rifampin, chloramphenicol, and tosyllysine chloromethylketone lower the rate of protein degradation by starved cells. Addition to starved cells of a nutrient upon which the culture is induced for growth rapidly lowers the rate of protein degradation from 7 to 9%/h to less than 1.5%/h. A nutrient that is oxidized but that cannot immediately support growth also lowers the rate of starvation-induced protein degradation. Proteolytic activity of cell extracts requires a divalent metal ion and may be inhibited up to 60% by tosyllysine chloromethylketone or p-toluenesulfonyl fluoride. Rifampin and chloramphenicol have no effect. In contrast to intact cells, extracts of growing or starving cells degrade protein at equivalent rates. We also investigated the stabilities of the inducible transport system and of four inducible intracellular enzymes of L-arginine catabolism. These include: the membrane-associated, L-arginine-specific transport system; L-arginine oxidase (oxidase); alpha-ketoarginine decarboxylase (decarboxylase); gamma-guanidinobutyraldehyde dehydrogenase ( dehydrogenase); and gamma-guanidinobutyrate amidinohydrolase (hydrolase). In starved cells, the rates of loss of activities were: transport and dehydrogenase activities, stable; oxidase and decarboxylase activities, 20 to 30%/h; hydrolase activity, 5 to 8%/h. Chloramphenicol decreases the rate of loss of oxidase, decarboxylase, and hydrolase activity, whereas p-toluenesulfonyl fluoride lowers the rate of loss of decarboxylase but not of oxidase or hydrolase activity. Addition to starved cells of a nutrient for which they are already induced for growth (e.g., malate, a noninducer of arginine catabolic enzymes) decreases the rate of loss of oxidase and decarboxylase activity but not that of the hydrolase.

Chapter 14 Isolation of Regulatory Mutants in Saccharomyces cerevisiae

This chapter describes isolation of regulatory mutants in Saccharonyces cerevisiae. Regulatory mutations have been invaluable in the study of genetic control in Escherichia coli . Operator and repressor mutations have defined these regulatory genes and given insights into the physiology of regulation. By studying double mutants and dominance in heterozygotes, it is possible to obtain a picture of the regulatory circuit and regulatory interactions. A difficult technical problem exists in the isolation of mutations that affect controlling elements or regulatory genes in most systems. Regulatory mutations do not usually alter the growth characteristics of the wild type, and therefore have no distinguishing phenotype. To identify regulatory mutants, one has to devise conditions under which regulatory mutations have a clear-cut phenotype. The chapter reviews studies on gene regulation in yeast to identify those techniques that have been most successful in selecting regulatory mutants. Arginine biosynthesis and arginine degradation is discussed and controls linking arginine catabolic and anabolic enzymes are explained in the chapter. The chapter describes acid phosphatase biosynthesis, pyrimidine biosynthesis, methionine biosynthesis, and histidine biosynthesis.

Regulation of arginine catabolism in Aspergillus nidulans.

IN Aspergillus nidulans, mutations at more than 20 loci result in increased levels of two arginine catabolic enzymes, arginase and ornithine δ-transaminase (OTAse)1. The large number of genes involved either directly or indirectly in regulation of arginine catabolism makes interpretation of the regulatory mechanism(s) complicated. Some of these genes at least are concerned with catabolite repression rather than with control of specific induction and repression processes2. Here we present evidence that the synthesis of arginase and OTAse in A. nidulans is regulated in a positive fashion. Interrelations between the gene responsible for positive control and several other genes involved in the regulation of these enzymes were established. This enabled us to propose a model of the regulatory mechanism involving interaction between a specific positive and a nonspecific negative mode of regulation and providing a possible explanation of ammonium repression.

Ammonium and glucose repression of the arginine catabolic enzymes in Aspergillus nidulans.

The synthesis of two enzymes of the arginine catabolic pathway, arginase and ornithine δ-transaminase (OTAse), in Aspergillus nidulans was found to be sensitive to both glucose and ammonium repression. The glucose and nitrogen starvation result in the identical derepression of OTAse synthesis and have no effects on arginase synthesis. Glucose and ammonium affect the kinetics of induction of both enzymes, however, the effect of ammonium is much stronger. Evidence was obtained for the direct involvement of ammonium in the repression phenomenon. The relations between glucose and ammonium repression are discussed.

Mutations simultaneously affecting ammonium and glucose repression of the arginine catabolic enzymes in Aspergillus nidulans

Two kinds of mutants of Aspergillus nidulans with altered response of arginine catabolic enzymes to glucose and ammonium repression were obtained. Mutations in the suF locus result in the insensitivity of these enzymes to glucose and to one type of ammonium repression. Mutations in the AniA locus result in hypersensitivity to both types of repression. The enzymes studied can be induced by arginine in AniA mutants only when glucose or the nitrogen source is removed from the medium. The suF mutations are recessive while AniA are dominant. Double suF AniA mutants retain only the suF properties. The functions of both genes and their interrelations are discussed.

The regulation of the synthesis of arginine catabolizing enzymes during the cell cycle in Saccharomyces cerevisiae

Summary In ammonia grown cells of S. cerevisiae we observed that arginase and ornithine transaminase, two enzymes catabolizing arginine and susceptible to coordinate induction by arginine, are synthesized once per cell cycle. However, although these enzymes are members of a common metabolic pathway the synthesis of arginase occurs earlier in the cell cycle than ornithine transaminase. The internal concentration of the arginine pool does not show any significant fluctuation during the cell cycle whether S. cerevisiae is grown on arginine or ammonia. The synthesis of arginase and ornithine transaminase occurs at the same stages in the cell cycle in both media although a higher internal arginine concentration is found in cells growing in this amino acid. Therefore, although arginine regulates the amount of enzymes synthesized, the timing of arginase and ornithine transaminase synthesis appears to be independent of the internal concentration of arginine and the presence or absence of this amino acid in the medium. These results do not support the hypothesis that the regulation of enzyme synthesis during the cell cycle is controlled by oscillatory repression or induction.

Effects of lysine on the synthesis and activity of arginase and ornithine transaminase in Saccharomyces cerevisiae

Recently evidence has been presented of an inhibitory effect of l-lysine upon post-exponential growth of Saccharomyces cerevisiae, which seems to be related to a very low arginine pool (ten to twenty times lower than usual). Explanation of this phenomenon has been sought in a study of the effects of l-lysine upon arginine catabolic enzymes. It has been shown that the presence of 5 mM l-lysine in a medium containing 10 mM ammonium sulfate results in an important increase of arginase and ornithine transaminase activities during exponential growth: arginase activity increases roughly from 6 to 40 milliunits; ornithine transaminase activity is four times higher than the usual maximum, even during exponential growth, but shows then no starvation derepression. The addition of l-lysine in the course of exponential growth results immediately in a derepression of both enzymes, which is then lengthened by the starvation derepression. Properties of both enzymes have been studied with crude extracts after Sephadex filtration. The Km of arginase towards arginine has been found to be equal to 5.1 mM; study of lysine effects at two concentrations of substrate (50 mM, 5 mM) has shown that l-lysine exerts a competitive inhibition with a Ki equal to 1.9 mM. On the other hand l-lysine has no effect upon ornithine transaminase activity. Growth studies of an arginine-auxotrophic mutant, with or without lysine, have shown that 1 mM l-lysine inhibits post-exponential growth if arginine concentration is low, but does not inhibit it if arginine concentration is higher than 0.6 mM; this is in accordance with the explanation of growth inhibition by the effect upon arginine metabolism. Since an arginase-deficient mutant remains as sensitive as the wild type to lysine effect upon growth, this effect is more likely to be attributed to negative influence on biosynthesis than to positive influence on catabolism.