Specific induction of catabolism and its relation to repression of biosynthesis in arginine metabolism of Saccharomyces cerevisiae.

Experimental results are presented in support of the model previously proposed for specific induction of the synthesis of enzymes for arginine catabolism in Saccharomyces cerevisiae (Wiame, 1971a,b), and its connection with end-product repression of arginine biosynthetic enzymes. The data support the occurrence of negative regulation of metabolism in a eukaryote.Operator regions, one for arginase and another for ornithine transaminase, are identified. The operator mutations are fully constitutive. A mutation compatible with the occurrence of a catabolic represser, CARGR, leads to partial pleiotropic constitutivity.The connection between the induction process and the repression of biosynthetic enzymes is due to a common receptor of metabolic signals, an ambivalent repressor ARGR endowed with the property of a usual repressor for anabolic enzymes and playing the role of inducer at the level of CARGR; this cascade process simulates a positive control. argR^? mutations, by producing defective ARGR, “turn on” anabolic enzyme synthesis and “turn off” the synthesis of catabolic enzymes (Fig. 2). The dual role of ARGR is confirmed by the isolation of a mutation argRII^d which, in contrast to the defective properties caused by usual argR^? mutations, causes a dominant hyperactivity toward induction of a catabolic enzyme, but retains recessive hypoactivity toward repression of an anabolic enzyme. Such an ambivalent repressor is a function necessary for mutual, balanced exclusion between opposite metabolisms.Many operator constitutive mutations for arginase, cargA⁺O^?, change the level of enzyme to a similar value, thus defining a genetic function. One of these mutations, cargA⁺O^h, in addition to having unusual genetic behaviour, leads to production of twice as much arginase as cargA⁺O^?. This suggests the existence of another genetic region near the structural gene for this enzyme and an additional regulatory function to be analyzed in a separate paper (Dubois &; Wiame, 1978).     

Organization and expression of a two-gene cluster in the arginine biosynthesis of Saccharomyces cerevisiae.

Summary Using the pMB9 recombinant plasmid pMY3, which contains a functional gene for the tRNA^Try mutant Su⁺7, the EcoRI fragment containing the tRNA^Try gene is mapped and oriented with respect to the HindIII site in the tetracycline region of pMB9. Complete HpaII and HaeIII maps of the EcoRI fragment are derived. The Su⁺7 tRNA gene is placed by hybridization to these fragments, and the tRNA gene is oriented by using the restriction sites for HinfI, TaqI, and HpaII in the tRNA gene itself. A tRNA^Asp gene is shown to lie adjacent to tRNA^Try, and is also placed and oriented in the map. The RI fragment itself originates in a locus adjacent to, and transcribed in the same direction as, the ribosomal RNA genes of 80d₃.The implications of the structure of the cloned DNA for its previously measured regulatory and tRNA gene activities are discussed. In particular, the effect on the regulation of RNA synthesis is attributable to an E. coli DNA sequence, but cannot be due to the presence of a normal tRNA promoter on the plasmid.Abbreviations MD megadaltons; expressions of the form HpaII:0.075 refer to a fragment generated by the indicated restriction nuclease, having the indicated molecular weight, in MD     

Regulation of isoleucine and valine biosynthesis in Salmonella typhimurium: the effect of hisU on repression control.

The control of isoleucine and valine biosynthesis was examined in a hisU mutant of Salmonella typhimurium. It was found that the levels of expression of the ilvEDA operon and the ilvC gene were significantly reduced relative to an isogenic normal strain when grown in unsupplemented medium. In contrast, this hisU mutant exhibited only a slight reduction in total acetohydroxy acid synthase activity relative to that of the wild type. The hisU and hisU⁺ strains were examined to determine their derepressibility upon either leucine, valine or isoleucine limitation. Only during leucine limitation did the hisU strain exhibit impaired derepressibility relative to the hisU⁺ strain. In addition, repression control of threonine deaminase (the ilvA product of the ilvEDA operon) in this hisU mutant was refractory to exogenous supplementation with either leucine or valine. This response is in distinct contrast to that of the normal strain, in which the single addition of leucine or valine results in a significant reduction in the level of threonine deaminase.     

Enzyme repression in the arginine pathway ofSaccharomyces cerevisiae

Enzyme repression in the arginine pathway ofSaccharomyces cerevisiae was demonstrated by comparison of specific enzyme activities in yeast grown with and without arginine in various mineral salts media. Of the enzymes tested only ornithine transcarbamoylase was found to be repressed by exogenous arginine. Acetylornithine-glutamate transacetylase and argininosuccinate lyase were not affected. No relationship between specific enzyme activities and intracellular arginine concentration was observed.During the adaptation of yeast grown in a medium supplemented with amino acids to a mineral salts medium, the enzymes ornithine transcarbamoylase and argininosuccinate lyase were not derepressed beyond their specific activities normally present in yeast grown in mineral salts media. Neither were the arginine-degrading enzymes arginase and ornithine transaminase broken down during this adaptation.Thanks are due to Professor E. G. Mulder and to Professor H. Veldkamp for stimulatory discussions; to the Heineken's Brouwerij, Rotterdam, and to the Landbouwhogeschoolfonds for research grants.     

Regulation of pyrimidine and arginine biosynthesis investigated by the use of phaseolotoxin and 5-Fluorouracil

Purified phaseolotoxin inhibits the growth of carrot cells. Such inhibitions can be reversed completely by citrulline but not by arginine. This toxin inhibits ornithine transcarbamylase activity in vitro, which leads to an accumulation of ornithine and a decrease in arginine levels intracellularly. In carrot cells, 5-fluorouracil (5-FU) toxicity can be reduced by the addition of purified toxin and citrulline, or ornithine. The toxin also decreases the incorporation of [¹⁴C]uracil and [¹⁴C]5-FU into trichloroacetic acid precipitable material by 50%. Finally, a 5-FU-resistant line, F5 (Sung ZR, Jacques S 1980 Planta 148: 389-396), was found to be more sensitive to the toxin than were 5-FU-sensitive cells. One millimolar 5-FU roughly doubled the ability of F5 to tolerate phaseolotoxin. These results demonstrate a close regulation between the pyrimidine and arginine path-ways in carrots.     

Proline production via the arginine biosynthetic pathway: transfer of regulatory mutations of arginine biosynthesis into a proline-producing strain ofSerratia marcescens

Summary Proline production via a part of the arginine biosynthetic pathway was examined. About 20 mg/ml ofl-proline was produced by using arginine biosynthetic enzymes. Accordingly, three mutations of arginine biosynthesis, namely, derepression of arginine biosynthetic enzymes (assigned byargR2), feedback inhibition-resistant N-acetylglutamate synthase (assigned byargA2) and defectiveness in N-acetylornithine aminotransferase (assigned byargD ^–) were introduced by three transductional crosses into a proline-producing strain which produced about 55 mg/ml ofl-proline. The constructed strain produced 62 mg/ml ofl-proline, although about 10 mg/ml ofl-arginine and 1 mg/ml of N-acetylglutamate--semialdehyde were produced as by-products.     

De novo arginine biosynthesis in leaves of phosphorus-deficient citrus and poncirus species

Young, fully expanded leaves from 7-month-old P-deficient citrus rootstock seedlings had levels of nonprotein arginine that were 10- to 50-fold greater than those from P-sufficient control plants. Arginine content of the protein fraction increased 2- to 4-fold in P-deficient leaves. Total arginine content, which averaged 72 ± 6 micromoles per gram dry weight of P-sufficient leaf tissue (mean ± se, n = the four rootstocks) was 207, 308, 241, and 178 micromoles in P-deficient leaves from Citrus limon cv rough lemon, Poncirus trifoliata × C. sinensis cv Carrizo citrange and cv Troyer citrange, and P. trifoliata cv Australian trifoliate orange, respectively. For each rootstock, the accumulation of arginine paralleled an increase in the activity of the pathway for the de novo biosynthesis of arginine. The ratio of the nanomoles NaH¹⁴CO₃ incorporated into the combined pool of arginine plus urea per gram fresh weight intact leaf tissue during a 3-hour labeling period for P-deficient to P-sufficient plants was 91:34, 49:11, 35:11, and 52:41, respectively. When P-deficient plants were supplied with P, incorporation of NaH¹⁴CO₃ into arginine plus urea was reduced to the level observed for the P-sufficient control plants of the same age and arginine ceased to accumulate. Arginase and arginine decarboxylase activity were either unaffected or slightly increased during phosphorus deficiency. Taken together, these results provide strong evidence that arginine accumulation during phosphorus deficiency is due to increased activity of the de novo arginine biosynthetic pathway.     

Deregulation of arginine biosynthesis in Synechococcus sp. PCC7942

Summary In order to deregulate arginine biosynthesis in Synechococcus sp. PCC7942, d-arginine-resistant cell lines were selected following ethyl methanesulfonate mutagenesis of wild-type (WT) cells. Three of these arginine-producing mutant (APM) cell lines, APM1, APM31 and APM40, were putative regulatory mutants based upon secretion of l-arginine into their growth medium. HPLC of lyophilized post-harvest supernatants of APM 31 and 40 resolved two predominant amino acids, arginine and citrulline. In-vitro activity of N-acetylglutamate kinase (NAGK), the proposed regulatory enzyme of the arginine pathway, was about 100-fold less sensitive to l-arginine inhibition in extracts from APM 31 and 40 than the enzyme in WT extracts. The enzyme from APM 1 was 20-fold less sensitive to l-arginine inhibition than WT. The most likely site of mutation in each of the APM cell lines is in the gene for NAGK, rendering the enzymes insensitive to l-arginine feedback control. These strains can be utilized for the phototrophic production of arginine. Offprint requests to: S. E. Bingham     

Implication of a repression system,homologous to those of other bacteria,in the control of arginine biosynthesis genes inStreptomyces coelicolor

As with most amino acid biosynthetic pathways in streptomycetes, enzymes of arginine biosynthesis inStreptomyces coelicolor show only slight derepression in minimal medium without, as opposed to with, exogenous arginine. However, when an arginine auxotroph was cultured in limiting arginine, ornithine carbamoyltransferase (OCT) activities rose by as much as 100-fold. The response was not due to a general starvation effect. To elucidate the repression-derepression mechanism, a DNA fragment containing the upstream region of the previously isolatedS. coelicolor argCJB cluster was cloned into a multicopy vector and transformed into wild-typeS. coelicolor; a slight transient derepression of OCT was observed in minimal medium without, though not with, added arginine, consistent with titration by the insert of a negatively acting macromolecule such as a repressor. A sub-fragment carrying the 5′ end ofargC and the region immediately upstream showed specific binding, in mobility shift assays, to purified AhrC, the repressor/activator of genes of arginine metabolism inBacillus subtilis. It is therefore likely that inS. coelicolor, expression of arginine biosynthesis genes is controlled by a protein homologous to the well-characterisedB. subtilis andEscherichia coli repressors.     

Immobilization of cyclodextrin glucanotransferase on amberlite IRA-900 for biosynthesis of transglycosylated xylitol

Cyclodextrin glucanotransferase (CGTase) fromThermoanaerobacter sp. was adsorbed on the ion exchange resin Amberlite IRA-900. The optimum conditions for the immobilization of the CGTase were pH 6.0 and 600 U CGTase/g resin, and the maximum yield of immobilization was around 63% on the basis of the amount ratio of the adsorbed enzyme to the initial amount in the solution. Immobilization of CGTase shifted the optimum temperature for the enzyme to produce transglycosylated xylitol from 70°C to 90°C and improved the thermal stability of immobilized CGTase, especially after the addition of soluble starch and calcium ions. Transglycosylated xylitol was continuously produced using immobilized CGTase in the column type packed bed reactor, and the operating conditions for maximum yield were 10% (w/v) dextrin (13 of the dextrose equivalent) as the glycosyl donor, 10% (w/v) xylitol as the glycosyl acceptor, 20 mL/h of medium flow rate, and 60°C. The maximum yield of transglycosylated xylitol and productivity were 25% and 7.82 g·L⁻¹·h⁻¹, respectively. The half-life of the immobilized CGTase in a column type packed bed reactor was longer than 30 days.     

Studies in phytosterol biosynthesis. Mechanism of biosynthesis of cycloartenol

1. The mechanism of cycloartenol biosynthesis in leaves of Solanum tuberosum was investigated with the use of [2-¹⁴C,(4R)-4-³H₁]mevalonic acid. 2. The ³H/¹⁴C atomic ratio in cycloartenol was 6:6, the same as that in squalene; this eliminates lanosterol as a possible biosynthetic precursor of cycloartenol, and indicates that a hydrogen migration from C-9 to C-8 occurs. 3. Chemical isomerization of the cycloartenol to lanosterol (³H/¹⁴C ratio 5:6) and parkeol (³H/¹⁴C ratio 6:6) confirms the hydrogen migration from C-9 to C-8. 4. Possible mechanisms for the biosynthesis of cycloartenol and parkeol are discussed. 5. The ³H/¹⁴C ratio for 24-methylenecycloartanol was 6:6, demonstrating that the hydrogen atom at C-24 is retained during alkylation of the cycloartenol side chain.     

Coordinate repression of cholesterol biosynthesis and cytoplasmic 3-hydroxy-3-methylglutaryl coenzyme A synthase by glucocorticoids in HeLa cells.

The stability constants for the calcium and magnesium complexes of rhodanese are >10⁵m^?1 at both high and low substrate concentrations. The stoichiometry of alkaline earth metal ion binding totals close to 1 per 18,500 molecular weight. The usual assay reagents contain sufficient amounts of these metal ions to maintain added enzyme in its metal-complexed form. When reaction mixtures are treated with oxalate to remove calcium ions, inhibition of rhodanese activity is virtually complete under circumstances such that the contribution of magnesium ion is low.Zinc and a number of transition metal ions are inhibitors of rhodanese activity. Studies of the concentration dependence of these effects with zinc, copper, and nickel showed that: 1) Some cyanide complexes of these metals are competitive with the donor substrate, thiosulfate ion. The binding of the copper and zinc complexes is mutually competitive. 2) Another cyanide species of copper appears to combine with the free enzyme to form a functionally active complex. 3) The zinc cyanide species with a net positive charge is an inhibitor competitive with the acceptor substrate, cyanide ion.All of these observations are consistent with a model in which metal ions serve as the electrophilic site of rhodanese.     

Two arginine repressors regulate arginine biosynthesis in Lactobacillus plantarum

The repression of the carAB operon encoding carbamoyl phosphate synthase leads to Lactobacillus plantarum FB331 growth inhibition in the presence of arginine. This phenotype was used in a positive screening to select spontaneous mutants deregulated in the arginine biosynthesis pathway. Fourteen mutants were genetically characterized for constitutive arginine production. Mutations were located either in one of the arginine repressor genes (argR1 or argR2) present in L. plantarum or in a putative ARG operator in the intergenic region of the bipolar carAB-argCJBDF operons involved in arginine biosynthesis. Although the presence of two ArgR regulators is commonly found in gram-positive bacteria, only single arginine repressors have so far been well studied in Escherichia coli or Bacillus subtilis. In L. plantarum, arginine repression was abolished when ArgR1 or ArgR2 was mutated in the DNA binding domain, or in the oligomerization domain or when an A123D mutation occurred in ArgR1. A123, equivalent to the conserved residue A124 in E. coli ArgR involved in arginine binding, was different in the wild-type ArgR2. Thus, corepressor binding sites may be different in ArgR1 and ArgR2, which have only 35% identical residues. Other mutants harbored wild-type argR genes, and 20 mutants have lost their ability to grow in normal air without carbon dioxide enrichment; this revealed a link between arginine biosynthesis and a still-unknown CO2-dependent metabolic pathway. In many gram-positive bacteria, the expression and interaction of different ArgR-like proteins may imply a complex regulatory network in response to environmental stimuli.     

Role of oxygen fixation in hydroxyproline biosynthesis by etiolated seedlings

Etiolated maize and soybean seedlings were grown for several days in atmospheres enriched with O¹⁸. Hydroxyproline subsequently isolated from the seedlings by column and thin-layer chromatography was labeled with excess O¹⁸, but proline was not. Control experiments in which seedlings were grown in H₂O¹⁸ and unlabeled atmospheres demonstrated that neither proline nor hydroxyproline was labeled with excess O¹⁸. It was concluded that oxygen fixation is an essential feature of hydroxyproline biosynthesis in these seedlings, and that the hydroxyl oxygen atom in hydroxyproline is derived from molecular oxygen and not from water; similar results have been reported previously for sycamore cell suspensions.     

Genes, enzymes and regulation of arginine biosynthesis in plants.

Arabidopsis genes encoding enzymes for each of the eight steps in L-arginine (Arg) synthesis were identified, based upon sequence homologies with orthologs from other organisms. Except for N-acetylglutamate synthase (NAGS; EC 2.3.1.1), which is encoded by two genes, all remaining enzymes are encoded by single genes. Targeting predictions for these enzymes, based upon their deduced sequences, and subcellular fractionation studies, suggest that most enzymes of Arg synthesis reside within the plastid. Synthesis of the L-ornthine (Orn) intermediate in this pathway from L-glutamate occurs as a series of acetylated intermediates, as in most other organisms. An N-acetylornithine:glutamate acetyltransferase (NAOGAcT; EC 2.3.1.35) facilitates recycling of the acetyl moiety during Orn formation (cyclic pathway). A putative N-acetylornithine deacetylase (NAOD; EC 3.5.1.16), which participates in the "linear" pathway for Orn synthesis in some organisms, was also identified. Previous biochemical studies have indicated that allosteric regulation of the first and, especially, the second steps in Orn synthesis (NAGS; N-acetylglutamate kinase (NAGK), EC 2.7.2.8) by the Arg end-product are the major sites of metabolic control of the pathway in organisms using the cyclic pathway. Gene expression profiling for pathway enzymes further suggests that NAGS, NAGK, NAOGAcT and NAOD are coordinately regulated in response to changes in Arg demand during plant growth and development. Synthesis of Arg from Orn is further coordinated with pyrimidine nucleotide synthesis, at the level of allocation of the common carbamoyl-P intermediate.