Cloning and sequencing of arg3 and arg11 genes of Schizosaccharomyces pombe on a 10-kb DNA fragment. Heterologous expression and mitochondrial targeting of their translation products.

The Schizosaccharomyces pombe arginine anabolic genes encoding ornithine carbamoyltransferase (arg3) and acetylglutamate kinase/acetylglutamyl-phosphate reductase (arg11) were cloned by functional complementation of S. pombe arg3 and arg11 mutant strains from S. pombe DNA genomic libraries. Restriction analysis and sequencing of the two clones showed that both genes are located on a common DNA fragment. The arg3 gene encodes a 327-amino-acid polypeptide presenting a strong identity to Saccharomyces cerevisiae and human ornithine carbamoyltransferases. The arg11 gene encodes a 884-amino-acid polypeptide. The acetylglutamate kinase and acetylglutamate-phosphate reductase domains have been defined by their identity with the S. cerevisiae ARG5,6 protein. The cloned arg11 gene from S. pombe does not complement an arg5,6 mutation in S. cerevisiae, nor does the ARG5,6 gene complement the S. pombe arg11- mutation. In contrast, both ornithine-carbamoyltransferase-encoding genes function in S. pombe. However, the S. pombe arg3 gene complements only weakly an arg3 S. cerevisiae strain, which is in agreement with the low level of expression of the S. pombe gene in S. cerevisiae. The subcellular localization of both ornithine carbamoyltransferases in the two yeasts indicates that, in contrast to the S. pombe enzyme, more than 95% of the S. cerevisiae enzyme remains in the S. pombe cytoplasm. The low expression of S. pombe ornithine carbamoyltransferases in S. cerevisiae did not allow its localization. The promoters of S. pombe arg3 and arg11 genes do not present striking similarities among themselves nor with the promoters of the equivalent genes of S. cerevisiae.     

A specific requirement for biotin in the synthesis of ornithine carbamoyltransferase by yeast

1. Growth of a biotin-requiring strain of Saccharomyces cerevisiae in a medium containing a suboptimum concentration of biotin for growth caused a decreased synthesis of ornithine carbamoyltransferase as compared with yeast grown in a medium containing an optimum concentration of biotin. Inclusion of the biotin homologues norbiotin or homobiotin, but not bishomobiotin, in the biotin-deficient medium caused an appreciable increase in ornithine carbamoyltransferase synthesis without affecting growth or synthesis of total RNA and protein. The addition of norbiotin to biotin-deficient medium had no effect on the respiratory activity of the yeast or on the synthesis of aspartate carbamoyltransferase, acid phosphatase, beta-fructofuranosidase or malate dehydrogenase. 2. Synthesis of acetylornithine deacetylase and acetylornithine acetyltransferase was slightly diminished by the imposition of biotin deficiency, but the effect was not as great as on ornithine carbamoyltransferase synthesis. Incorporation of norbiotin in the biotin-deficient medium had no marked effect on the synthesis of any other arginine-pathway enzyme except ornithine carbamoyltransferase. 3. l-Ornithine induced synthesis of ornithine carbamoyltransferase in yeast grown in biotin-deficient medium, but in yeast grown in this medium supplemented with norbiotin it repressed synthesis of the enzyme. l-Arginine had no detectable effect on ornithine carbamoyltransferase synthesis by the yeast grown in biotin-deficient medium with or without norbiotin. l-Aspartate repressed synthesis of ornithine carbamoyltransferase in biotin-deficient yeast and completely nullified the stimulatory effect of norbiotin on synthesis of the enzyme in this yeast. 4. There was no increase in ornithine carbamoyltransferase synthesis in biotin-deficient yeast incubated in phosphate buffer, pH4.5, containing glucose and biotin or norbiotin. In biotin-deficient yeast suspended in complete medium containing an optimum concentration of biotin, there was an increase in ornithine carbamoyltransferase synthesis only after the onset of growth.     

Genetic and physiological characterization of Pseudomonas aeruginosa mutants affected in the catabolic ornithine carbamoyltransferase.

In Pseudomonas aeruginosa arginine can be degraded by the arginine "dihydrolase" system, consisting of arginine deiminase, catabolic ornithine carbamoyltransferase, and carbamate kinase. Mutants of P. aeruginosa strain PAO affected in the structural gene (arcB) of the catabolic ornithine carbamoyltransferase were isolated. Firt, and argF mutation (i.e., a block in the anabolic ornithine carbamoyltransferase) was suppressed specifically by a mutationally altered catabolic ornithine carbamoyltransferase capable of functioning in the anabolic direction. The suppressor locus arcB (Su) was mapped by transduction between hisII and argA. Second, mutants having lost suppressor activity were obtained. The Su- mutations were very closely linked to arcB (Su) and caused strongly reduced ornithine carbamoyltransferase activities in vitro. Under aerobic conditions, a mutant (PA0630) which had less than 1% of the wild-type catabolic ornithine carbamoyltransferase activity grew on arginine as the only carbon and nitrogen source, at the wild-type growth rate. When oxygen was limiting, strain PA0630 grown on arginine excreted citrulline in the stationary growth phase. These observations suggest that during aerobic growth arginine is not degraded exclusively via the dihydrolase pathway.     

New arginine-requiring mutants in Chlamydomonas reinhardtii

Twelve arginine-requiring mutants of the unicellular green alga Chlamydomonas reinhardtii previously isolated in our laboratory were investigated to find new blocks in the biosynthetic pathway of arginine. In addition to the already described mutants lacking acetylglutamyl phosphate reductase (arg 1), ornithine carbamoyltransferase (arg4) and argininosuccinate lyase (arg7), three new types of mutants were found lacking acetylornithine aminotransferase (arg9-1, arg9-2), acetylornithine glutamate transacetylase (arg10) and argininosuccinate synthetase (arg8-1, arg8-2, arg8-3) respectively. The genetic analysis of these new mutants showed that arg9 and arg8 are unlinked to the other arginine markers and that arg10 probably carries a chromosomal mutation inducing a very high lethality of meiotic products.Abbreviations WT wild-type - mt mating-type - SP spore plating - ZP zygote plating     

Complementation of the Aspergillus nidulans arg B1 mutation by ornithine transcarbamylase cDNA from rat liver

An Aspergillus nidulans strain which is deficient in ornithine transcarbamylase due to the arg B1 mutation was transformed with a plasmid containing the ornithine transcarbamylase cDNA from rat liver under the control of the amd S promoter. Stable transformants were obtained by selection on arginine free medium indicating complementation of the arg B mutation. Proof of expression of the rat enzyme in transformants was obtained by immunoprecipitation of all ornithine transcarbamylase activity from cell extracts with antibodies specific for the rat enzyme. The presence of catalytically active rat ornithine transcarbamylase in the transformants indicated that it is capable of being imported into mitochondria in A. nidulans, proteolytically processed and assembled into its homotrimeric form. In vitro uptake experiments using isolated A. nidulans mitochondria demonstrate that processing of the precursor of rat ornithine transcarbamylase occurs in two temporally separated steps as it does in rat liver mitochondria suggesting evolutionary conservation of the processing machinery. Up to 560 ng of active rat enzyme was produced per gm wet weight mycelia. Use of beta-D-alanine, an inducer of amd S, as sole N-source resulted in increased levels of active rat ornithine transcarbamylase relative to uninduced cultures.     

Isolation and heteroduplex mapping of a lambda transducing bacteriophage carrying the structural genes for carbamoylphosphate synthase: regulation of enzyme synthesis in Escherichia coli K-12 lysogens.

A N-lambda bacteriophage transducing the structural genes for Escherichia coli K-12 carbamoylphosphate synthase (glutamine) (CPSase; EC 2.7.2.9) has been isolated and analyzed both genetically and physically. The whole int-N region is substituted for a short chromosomal segment corresponding almost exactly to the car locus. The study of CPSase, ornithine carbamoyltransferase, and aspartate carbamoyltransferase regulation in carriers of lambdadcar confirms the previously reported participation of the argR gene product in the control of CPSase synthesis and points to the existence of a regulatory molecule involved in the control of both CPSase and aspartate carbamoyltransferase synthesis. The general usefulness of using N- lambda transducing bacteriophages for the recovery of large amounts of gene products is discussed.     

Phaseolotoxin-insensitive ornithine carbamoyltransferase of Pseudomonas syringae pv. phaseolicola: basis for immunity to phaseolotoxin.

Cell-free extracts from phaseolotoxin-producing strains of Pseudomonas syringae pv. phaseolicola grown at 18 degrees C, the optimum temperature for phaseolotoxin production, contain an ornithine carbamoyltransferase activity that is insensitive to phaseolotoxin. Extracts from the same strains grown at 30 degrees C, a temperature at which little or no detectable phaseolotoxin is produced, and from phaseolotoxin-nonproducing strains contain a phaseolotoxin-sensitive ornithine carbamoyltransferase activity. The phaseolotoxin-insensitive ornithine carbamoyltransferase activity is also less senstive to N delta-(phosphonacetyl)-L-ornithine than the phaseolotoxin-sensitive ornithine carbamoyltransferase activity of the corresponding strain.     

Evidence that specific and "general" control of ornithine carbamoyltransferase production occurs at the level of transcription in Saccharomyces cerevisiae.

Ornithine carbamoyltransferase synthesis is subject to two major regulatory systems in Saccharomyces cerevisiae. One system is specific for the arginine biosynthetic enzymes, whereas the other appears to be general, acting on a variety of other amino acid pathways as well. We observed that the synthetic capacity for continued ornithine carbamoyltransferase synthesis had the same short half-life (ca. 5 to 7 min) whether repression of enzyme production was brought about by action of the specific or general control system. We present evidence suggesting that both control systems regulate accumulation or ornithine carbamoyltransferase-specific synthetic capacity, rather than modulating its expression.     

Methionine-321 in the C-terminal α-helix of catabolic ornithine carbamoyltransferase from Pseudomonas aeruginosa is important for positive homotropic cooperativity

Abstract Pseudomonas aeruginosa has a pair of distinct ornithine carbamoyltransferases. The anabolic ornithine carbamoyltransferase encoded by the argF gene catalyzes the formation of citrulline from ornithine and carbamoylphosphate. The catabolic ornithine carbamoyltransferase encoded by the arcB gene promotes the reverse reaction in vivo; although this enzyme can be assayed in vitro for citrulline synthesis, its unidirectionality in vivo is determined by its high concentration at half maximum velocity for carbamoylphosphate ([S]_0.5) and high cooperativity toward this substrate. We have mutant forms of catabolic ornithine carbamoyltransferase catalyzing the anabolic reaction in vivo. The corresponding arcB mutant alleles on a multicopy plasmid specifically suppressed an argF mutation of P. aeruginosa . Two new mutant enzymes were obtained. When methionine 321 was replaced by isoleucine, the mutant enzyme showed loss of homotropic cooperativity at physiological carbamoylphosphate concentrations. Substitution of glutamate 105 by lysine resulted in a partial loss of the sigmoidal response to increasing carbamoylphosphate concentrations. However, both mutant enzymes were still sensitive to the allosteric activator AMP and to the inhibitor spermidine. These results indicate that at least two residues of catabolic ornithine carbamoyltransferase are critically involved in positive carbamoylphisphate cooperativity: glutamate 105 (previously known to be important) and methionine 321. Mutational changes in either amino acid will affect the geometry of helix H2, which contains several residues required for carbamoylphosphate binding.     

Altered enzyme activities and citrulline synthesis in liver mitochondria from ornithine carbamoyltransferase-deficient sparse-furash mice.

Male mice carrying the spfash mutation have 5-10% of the normal activity of ornithine carbamoyltransferase, yet are only slightly hyperammonaemic and develop quite well. A study of liver mitochondria from normal and spfash males showed that they differ in important ways. (1) The spfash liver contains about 33% more mitochondrial protein per g than does normal liver. (2) The specific activities of carbamoyl-phosphate synthetase (ammonia) and glutamate dehydrogenase are about 15% lower than normal in mitochondria from spfash mice, whereas those of beta-hydroxybutyrate dehydrogenase and cytochrome oxidase are 22% higher and 30% lower respectively. (3) In the presence of 10 mM-ornithine and the substrates for carbamoyl phosphate synthesis, coupled and uncoupled mitochondria from spfash mice synthesize citrulline at unexpectedly high rates, about 25 and 44 nmol/min per mg respectively. Though these are somewhat lower than the corresponding rates obtained with normal mitochondria, the difference does not arise from the deficiency in ornithine carbamoyltransferase, but from the lower carbamoyl-phosphate synthetase activity of the mutant mitochondria. (4) At lower external [ornithine] (less than 2 mM), a smaller fraction of the carbamoyl phosphate synthesized is converted into citrulline in spfash than in normal mitochondria. These studies show that what appears to be a single mutation brings about major adaptations in the mitochondrial component of liver. In addition, they clarify the role of ornithine transport and of protein-protein interactions in citrulline synthesis in normal mitochondria.     

Studies of X-chromosome inactivation with an improved histochemical technique for ornithine carbamoyltransferase

Studies of X-linked enzymes provide an approach to the study of tumour and normal cellular development. We have assessed the technique for the histochemical demonstration of one such enzyme, ornithine carbamoyltransferase (EC 2.1.3.3). Various stages in the Mizutani technique for ornithine carbamoyltransferase were re-examined, and the resulting improved technique applied to normal mice and to mice of the sparse fur strain (Spf) known to have an abnormal form of ornithine carbamoyltransferase inherited as an X-linked characteristic. Positive enzyme activity was present in all hepatocytes from normal mice, the strongest reaction being present in the periportal area with a gradual reduction of activity towards the centrilobular region. No activity was demonstrable in hepatocytes from hemizygous male Spf mice. In heterozygous female Spf mice, there was a clear-cut separation of ornithine carbamoyl-transferase-positive and -negative cells. These were present in very variable proportions in different liver lobes and different animals. Preliminary studies were also carried out using a high pH reaction mixture to detect the abnormal enzyme. These studies demonstrate conclusively the X-linkage of ornithine carbamoyltransferase in mice, showing the mosaic pattern of distribution predicted by the Lyon hypothesis. They show that the Spf strain of mice can be used for studies of both development and tumorigenesis in the liver, and that histochemical study of an animal strain with an X-linked enzyme abnormality provides a powerful investigative tool.     

Regulation of enzyme synthesis in the arginine deiminase pathway of Pseudomonas aeruginosa.

The three enzymes of the arginine deiminase pathway in Pseudomonas aeruginosa strain PAO were induced strongly (50- to 100-fold) by a shift from aerobic growth conditions to very low oxygen tension. Arginine in the culture medium was not essential for induction, but increased the maximum enzyme levels twofold. The induction of the three enzymes arginine deiminase (EC 3.5.3.6), catabolic ornithine carbamoyltransferase (EC 2.1.3.3), and carbamate kinase (EC 2.7.2.3) appeared to be coordinate. Catabolic ornithine carbamoyltransferase was studied in most detail. Nitrate and nitrite, which can replace oxygen as terminal electron acceptors in P. aeruginosa, partially prevented enzyme induction by low oxygen tension in the wild-type strain, but not in nar (nitrate reductase-negative) mutants. Glucose was found to exert catabolite repression of the deiminase pathway. Generally, conditions of stress, such as depletion of the carbon and energy source or the phosphate source, resulted in induced synthesis of catabolic ornithine carbamoyltransferase. The induction of the deiminase pathway is thought to mobilize intra- and extracellular reserves of arginine, which is used as a source of adenosine 5'-triphosphate in the absence of respiration.     

Anabolic ornithine carbamoyltransferase of Escherichia coli and catabolic ornithine carbamoyltransferase of Pseudomonas putida. Steady-state kinetic analysis.

The anabolic and catabolic ornithine carbamoyltransferases of Pseudomonas putida display an undirectional catalytic specialization: in citrulline synthesis for the anabolic enzyme, in citrulline phosphorolysis for the catabolic one. The irreversibility of the anabolic enzyme in vitro has been previously explained by its kinetic properties, whereas the irreversibility of the catabolic transferase in vivo was shown to be due to its allosteric behaviour. In this work a steady-state kinetic analysis has been carried out on the catabolic ornithine carbamoyltransferase at pH 6.8 in the presence of the allosteric activator, phosphate. The kinetic mechanism of Escherichia coli ornithine carbamoyltransferase serving as a reference was also determined. For the E. coli enzyme in the reverse direction, the initial velocity patterns converging on the abscissa were obtained with either citrulline or arsenate as variable substrate. The inhibition by the product ornithine was linear competitive with respect to citrulline and linear non-competitive with respect to arsenate. In the forward direction phosphate and its analogs induce an inhibition by ornithine which is partial and competitive with respect to carbamoylphosphate. Together with the results of thermo-inactivation studies in the presence of each reactant, this observation suggests a random kinetic mechanism, but with most of the reaction flux following the path where carbamoylphosphate adds before ornithine, when substrates are present at Km levels. The allosteric catabolic ornithine carbamoyltransferase of Pseudomonas displays qualitatively the same pattern as the E. coli enzyme.     

argJ mutations are highly inducible by ethidium bromide in proB strains of Streptomyces lividans: implication of pathway interactions.

Spontaneous Arg- mutants arose at high frequencies in Streptomyces lividans. Exposure to ethidium bromide increased the frequency of arg instability. In Pro+ strains the induced arg mutants were mainly argG, but in the proB mutants, a new mutation, argJ, prevailed which lacked ornithine acetyltransferase activity and required ornithine for growth. Introduction of the cloned proB gene of Streptomyces coelicolor A3(2) into the proB argJ mutants not only complemented the proB mutation but also suppressed the argJ mutation. The proB mutation was also suppressed by adding ornithine to the medium. These results indicated crossfeeding(s) between the arginine and proline pathways in S. lividans, which presumably circumvented the detection of argJ mutations in Pro+ strains.     

Intracellular localization of Aspergillus nidulans ornithine carbamoyltransferase in native host cells and in Saccharomyces cerevisiae cells harbouring its cloned structural gene

Differential centrifugation of the Aspergillus nidulans cell lysate shows that ornithine carbamoyltransferase (EC 2.1.3.3) appears mainly in the particulate (organellar) fraction. The enzyme was located to the mitochondria by co-sedimentation with cytochrome oxidase in isopycnic density gradient and by cytochemical-electron microscopic means. Arginase (EC 3.5.3.1) and ornithine delta-aminotransferase (E.C. 2.6.1.13) were found to reside in cytosol. The release of ornithine carbamoyltransferase from the organellar fraction by various agents indicates that the enzyme resides in the mitochondrial matrix. In Saccharomyces cerevisiae the plasmid pSAL43, carrying cloned Aspergillus nidulans ornithine carbamoyltransferase gene, directs the synthesis of the enzyme partially associated with yeast mitochondria even though the homologous yeast enzyme is exclusively cytosolic. The implications of these findings are discussed.     

Oxygen and nitrate in utilization by Bacillus licheniformis of the arginase and arginine deiminase routes of arginine catabolism and other factors affecting their syntheses.

Bacillus licheniformis has two pathways of arginine catabolism. In well-aerated cultures, the arginase route is present, and levels of catabolic ornithine carbamoyltransferase were low. An arginase pathway-deficient mutant, BL196, failed to grow on arginine as a nitrogen source under these conditions. In anaerobiosis, the wild type contained very low levels of arginase and ornithine transaminase. BL196 grew normally on glucose plus arginine in anaerobiosis and, like the wild type, had appreciable levels of catabolic transferase. Nitrate, like oxygen, repressed ornithine carbamoyltransferase and stimulated arginase synthesis. In aerobic cultures, arginase was repressed by glutamine in the presence of glucose, but not when the carbon-energy source was poor. In anaerobic cultures, ammonia repressed catabolic ornithine carbamoyltransferase, but glutamate and glutamine stimulated its synthesis. A second mutant, derived from BL196, retained the low arginase and ornithine transaminase levels of BL196 but produced high levels of deiminase pathway enzymes in the presence of oxygen.     

Escherichia coli ornithine carbamolytransferase isoenzymes: evolutionary significance and the isolation of lambdaargF and lambdaargI transducing bacteriophages.

Among the Enterobacteriaceae, Escherichia coli K-12 is the only strain known to have two structural genes (argF and argI) for ornithine carbamoyltransferase. The two gene products interact to form a family of four functional isoenzymes, respectively designated FFF, FFI, FII, and III. The FFF and III isoenzymes exhibit nearly identical kinetic parameters in the conditions applied. FFF is more thermolabile than III; this allows the straightforward characterization of new transducing phages carrying either argF or argI. The bearing of the available information regarding ornithine carbamoyltransferase isoenzymes on the evolution of the ancestral E. coli chromosome is reconsidered.     

Urea cycle enzymes in human liver: ontogenesis and interaction with the synthesis of pyrimidines and polyamines

Summary All the five enzymes of urea synthesis and the formation of urea in vitro can already be demonstrated in human liver as early as the 9th week of fetal development. At this stage the activity of carbamoyl phosphate synthetase is the highest, whereas that of ornithine carbamoyltransferase is the lowest as compared to those in the adult. The kinetic parameters of the urea cycle enzymes are the same in fetal liver as in adult liver, except that the Km values of ornithine carbamoyltransferase for L-ornithine are 3.5 mM and 0.42 mM in the fetus and in adult liver, respectively.Urea formation in vivo seems to begin in the second half of fetal life, and a gradual increase can be detected in the activity of the enzymes of urea synthesis. The activity of ortnithine decarboxylase, the glutamine-dependent carbamoyl phosphate synthetase and aspartate carbamoyltransferase, however, changes in the opposite direction.The concentration of carbamoyl phosphate and aspartate remains constant, but that of ornithine gradually decreases during ontogenesis. The ornithine, carbamoyl phosphate and aspartate pools are probably utilized in the polyamine, pyrimidine and urea syntheses at varying rates.