Synthesis and inactivation of carbamyl phosphate synthetase isozymes of Bacillus subtilis during growth and sporulation.

Pyrimidine-repressible carbamyl phosphate synthetase P was synthesized in parallel with aspartate transcarbamylase during growth of Bacillus subtilis on glucose-nutrient broth. Both enzymes were inactivated at the end of exponential growth, but at different rates and by different mechanisms. Unlike the inactivation of aspartate transcarbamylase, the inactivation of carbamyl phosphate synthetase P was not interrupted by deprivation for oxygen or in a tricarboxylic acid cycle mutant. The arginine-repressible isozyme carbamyl phosphate synthetase A was synthesized in parallel with ornithine transcarbamylase during the stationary phase under these growth conditions. Again, both enzymes were subsequently inactivated, but at different rates and by apparently different mechanisms. The inactivation of carbamyl phosphate synthetase A was not affected in a protease-deficient mutatn the inactivation of ornithine transcarbamylase was greatly slowed.     

Biochemical and genetic characterization of a carbamyl phosphate synthetase mutant of Escherichia coli K12.

An unusual Escherichia coli K12 mutant for carbamyl phosphate synthetase is described. The mutation was generated by bacteriophage MUI insertion and left a 5% residual activity of the enzyme using either ammonia or glutamine as donors. The mutation is recessive to the wild-type allele and maps at or near the pyrA gene, but the mutant requires only arginine and not uracil for growth. By a second block in the pyrB gene it was possible to shift the accumulated carbamyl phosphate to arginine biosynthesis. The Km values and the levels of ornithine activation and inhibition by UMP were normal in the mutant enzyme.     

Carbamyl phosphate synthetase A of Neurospora crassa.

Carbamyl phosphate synthetase A of Neurospora crassa was partially purified from mitochondrial extracts. It is an extremely unstable enzyme (t 1/2 = 45 min at 25 detrees C) made up of two unequal subunits. The native enzyme has a molecular weight of approximately 175,000, and the large subunit has a molecular weight of about 125,000. Both the native enzyme and its large subunit are quite asymmetric, as revealed by slow sedimentation in sucrose gradents (7.3S and 6.6S, respectively). The small subunit has not been identified physically as a separate entity. The denaturation of the native, glutamine-dependent activity is correlated with dissociation of subunits, the larger of which retains a more stable, ammonia-dependent activity. Neither substrates nor any other agents except glycerol or polyethylene glycol appreciably stabilized the glutamine-dependent activity. Kinetic studies showed the native enzyme to have a Km for glutamine of about 0.16 mM, and a Km for NH4Cl of about 16 mM, at the optimal pH, 8.0. The enzyme, using either N donor, has a K+ requirement for activity, for which NH4+ can substitute. The glutamine leads to glutamate reaction, which requires the small subunit, also requires the large subunit and all reaction substrates for optimal activity. Other evidences of subunit interaction are the greater activity of the native enzyme, as opposed to the large subunit, with low concentrations of adenosine 5'-triphosphate-Mg2+, and in the stimulation of the ammonia-dependent activity of the native enzyme by glycine. Curiously, although the enzyme's role in biosynthesis is confined to the arginine pathway, it is completely indifferent to arginine or its precursors as feedback effectors or activators. The enzyme is compared with carbamyl phosphate synthetases of other organisms.     

Carbamyl phosphate and acetyl phosphate synthesis in Escherichia coli: analysis of associated enzyme activities by an antibody to acetokinase

Brzozowski, Thomas H. (Stanford University School of Medicine, Palo Alto, Calif.), and Sumner M. Kalman. Carbamyl phosphate and acetyl phosphate synthesis in Escherichia coli: analysis of associated enzyme activities by an antibody to acetokinase. J. Bacteriol. 91:2286-2290. 1966.-Earlier studies have shown that the carbamyl phosphate synthesis from ammonia in cell extracts of wild-type Escherichia coli is due to at least two enzymes, acetokinase and the glutamine-dependent carbamyl phosphate synthetase. Partial purification of the glutamine-dependent carbamyl phosphate synthetase and acetokinase fails to separate from these enzymes this ammonia-dependent activity. An antibody to the partially purified acetokinase was prepared and used to determine the distribution of the ammonia-dependent activity in wild-type organisms and single-step arginine-uracil-requiring mutants with respect to the two enzymes. Such a study was possible because the antibody inhibits acetokinase but not the glutamine-utilizing carbamyl phosphate synthetase. Enzyme inhibition obtained by the stepwise addition of the antibody to cell extracts indicates that all of the ammonia-dependent carbamyl phosphate synthesis observed in the arginine-uracil-requiring mutants is due to a protein in the acetokinase fraction, presumably acetokinase itself, since acetyl phosphate and carbamyl phosphate synthesis were inhibited in a parallel fashion. In wild-type organisms, there is only partial inhibition of the ammonia-dependent activity, even when enough antibody is added to produce maximal inhibition of acetokinase. It is suggested that this residue is due to the glutamine-dependent carbamyl phosphate synthetase, for the ratio of the antibody insensitive to antibody sensitive ammonia-dependent activity present in cell extracts of the two wild-type organisms reported is qualitatively proportional to the level of carbamyl phosphate synthetase present relative to acetokinase.     

Enhanced production of arginine and urea by genetically engineered Escherichia coli K-12 strains.

Escherichia coli strains capable of enhanced synthesis of arginine and urea were produced by derepression of the arginine regulon and simultaneous overexpression of the E. coli carAB and argI genes and the Bacillus subtilis rocF gene. Plasmids expressing carAB driven by their natural promoters were unstable. Therefore, E. coli carAB and argI genes with and without the B. subtilis rocF gene were constructed as a single operon under the regulation of the inducible promoter ptrc. Arginine operator sequences (Arg boxes) from argI were also cloned into the same plasmids for titration of the arginine repressor. Upon overexpression of these genes in E. coli strains, very high carbamyl phosphate synthetase, ornithine transcarbamylase, and arginase catalytic activities were achieved. The biosynthetic capacity of these engineered bacteria when overexpressing the arginine biosynthetic enzymes was 6- to 16-fold higher than that of controls but only if exogenous ornithine was present (ornithine was rate limiting). Overexpression of arginase in bacteria with a derepressed arginine biosynthetic pathway resulted in a 13- to 20-fold increase in urea production over that of controls with the parent vector alone; in this situation, the availability of carbamyl phosphate was rate limiting.     

Identification of the junction between the glutamine amidotransferase and carbamyl phosphate synthetase domains of the mammalian CAD protein

The hamster CAD gene encodes a protein that catalyzes the first three steps of pyrimidine biosynthesis. We have sequenced a portion of a CAD cDNA and determined the location of the carbamyl phosphate synthetase II coding region. Subdomains coding for the glutamine hydrolyzing and carbamyl phosphate synthesizing functions have been identified through their high degree of similarity to carbamyl phosphate synthetase genes from a variety of organisms. The proline-rich junction between the glutaminase and synthetase domains, however, does not appear to be conserved among carbamyl phosphate synthetases.     

Carbamyl phosphate-dependent ATP synthesis catalyzed by formyltetrahydrofolate synthetase.

Formyltetrahydrofolate synthetase (formate:tetrahydrofolate ligase (ADP-forming), EC 6.3.4.3) from Clostridium cylindrosporum catalyzes phosphate transfer from carbamyl phosphate to ADP. This activity is lost when monovalent cations are removed and is recovered when K+ is added back. Carbamyl phosphate is an inhibitor of the formyltetrahydrolfolate synthetase forward reaction, and formate as well as phosphate inhibit the ATP synthesis reaction. Acetyl phosphate and phosphonoacetate are inhibitors of both reactions. The results of kinetic studies support the concept that carbamyl phosphate is an analog of the putative intermediate of the formyltetrahydrofolate synthetase reaction, formyl phosphate.     

Cloning and characterization of the metE gene encoding S-adenosylmethionine synthetase from Bacillus subtilis.

The metE gene, encoding S-adenosylmethionine synthetase (EC 2.5.1.6) from Bacillus subtilis, was cloned in two steps by normal and inverse PCR. The DNA sequence of the metE gene contains an open reading frame which encodes a 400-amino-acid sequence that is homologous to other known S-adenosylmethionine synthetases. The cloned gene complements the metE1 mutation and integrates at or near the chromosomal site of metE1. Expression of S-adenosylmethionine synthetase is reduced by only a factor of about 2 by exogenous methioinine. Overproduction of S-adenosylmethionine synthetase from a strong constitutive promoter leads to methionine auxotrophy in B. subtilis, suggesting that S-adenosylmethionine is a corepressor of methionine biosynthesis in B. subtilis, as others have already shown for Escherichia coli.     

Carbamyl phosphate synthetase of Escherichia coli uses the same diastereomer of adenosine-5'-[2-thiotriphosphate] at both ATP sites.

Carbamyl phosphate synthetase from Escherichia coli has been shown to use only the A isomer of adenosine-5'-[2-thiotriphosphate] in both the ATPase reaction (MgATP HCO3- leads to MgADP + Pi) and the carbamyl phosphate synthesis reaction (2MgATP + HCO3- + L-glutamine leads to 2MgADP + Pi + carbamyl-P + L-glutamate). The B isomer was less than 5% as reactive. In the reverse reaction, only the A isomer of adenosine-5'-[2-thiotriphosphate] is synthesized from adenosine-5'-[2-thiodiphosphate] and carbamyl-P as determined by 31P NMR and a coupled enzymatic assay with Cd2+- hexokinase. It is therefore proposed that carbamyl phosphate synthetase uses the same diastereomer of MgATP at both ATP sites.     

Inhibition of carbamyl phosphate synthetase by P1, P5-di(adenosine 5')-pentaphosphate: evidence for two ATP binding sites.

Studies on the effect of a series of alpha, omega-diadenosine 5'-polyphosphate (ApnA; n = 2 to 6) on carbamyl phosphate synthetase showed that only Ap5A is an effective inhibitor. Ap5A also inhibits two partial reactions catalyzed by the enzyme: bicarbonate-dependent ATPase and ATP synthesis from carbamyl phosphate and ADP. The data indicate that Ap5A binds to the enzyme sites that interact with ATP. Of a variety of ATP-utilizing enzymes (kinases, hydrolases, synthetases), only adenylate kinase (Leinhard, G. E., and Secemski, I. I. (1973) J. Biol. Chem. 248, 1121--1123) and carbamyl phosphate synthetase are inhibited by Ap5A. The present findings provide strong evidence that carbamyl phosphate synthetase has two separate binding sites for ATP in which the gamma-phosphate moeities of ATP are bound in close proximity to the bicarbonate binding site of the enzyme.     

Regulation and expression of carbamyl phosphate synthetase I mRNA in developing rat liver and Morris hepatoma 5123D

A cDNA clone complementary to mRNA encoding the precursor (Mr = 165,000) to the rat liver mitochondrial matrix enzyme carbamyl phosphate synthetase I (Mr = 160,000) was employed to compare relative amounts of the messenger in adult and fetal liver and in Morris hepatoma 5123D and 3924A cells. Northern blot analysis gave a size estimate for the messenger of 6,500-6,700 nucleotides. Carbamyl phosphate synthetase mRNA levels in 15-day-old fetal liver were less than 10% of adult levels; 5123D cells expressed the messenger at levels about 2-fold higher than normal adult liver, but the messenger was undetectable in 3924A cells. Albumin mRNA was also expressed in the former but not in the latter. Maintaining rats for 5 days on a diet containing 60% casein augmented the relative amount of carbamyl phosphate synthetase mRNA by about 2-fold, while a protein-free diet resulted in reduced levels of the mRNA (about 50% compared to animals on a normal diet). Finally, the pattern of hybridization of carbamyl phosphate synthetase cDNA to HindIII-digested genomic DNA showed no differences between normal liver and its corresponding hepatoma; however, a HindIII site polymorphism was observed between Buffalo and ACI rats.     

Carbamyl phosphate synthesis in Bacillus subtilis

In vitro and "in situ" assays have been developed to test the carbamyl phosphate synthetase (CPSase) activity of a series of pyrimidine-requiring mutants of Bacillus subtilis. The enzyme has been shown to be highly unstable, and was successfully extracted only in the presence of 10% glycerol and 1 mM dithiothreitol (Cleland's reagent). It loses activity rapidly when sonicated or when treated with lysozyme. Genetic studies, using mutants, indicate that B. subtilis may possess two CPSases. This possibility and its physiological consequences were probed enzymatically. CPSase activity has been shown to undergo inhibition by both uridine triphosphate and dihydroorotate; activation has been demonstrated in response to phosphoribosyl pyrophosphate (PRPP) and (to a lesser extent) ornithine.     

Coordinate synthesis of the enzymes of pyrimidine biosynthesis in Bacillus subtilis.

Strains of Bacillus subtilis that were resistant to repression of pyrimidine nucleotide biosynthetic enzymes were selected by isolating spontaneous uracil-tolerant derivatives of a uracil-sensitive strain, which lacks arginine-repressible carbamyl phosphate synthetase. The relative content of all six enzymes of uridylic acid biosynthesis de novo in these strains was in a constant ratio over a 10-fold range of derepression, which indicates that synthesis of these enzymes is coordinately regulated.     

Chloroplastic and cytoplasmic valyl- and leucyl-tRNA synthetases from Euglena gracilis. Comparative study of their structural properties

Chloroplastic and cytoplasmic valyl- and leucyl-tRNA synthetases purified from Euglena gracilis show a monomeric structure. The molecular weights of the two valyl-tRNA synthetases are identical (126,000) while those of the leucyl-tRNA synthetases are different (100 000 for the chloroplastic and 116 000 for the cytoplasmic enzyme). The tryptic maps and the amino acid compositions reveal differences between the chloroplastic valyl- and leucyl-tRNA synthetases and their cytoplasmic homologues. These results suggest that a chloroplastic aminoacyl-tRNA synthetase and its cytoplasmic counterpart are coded for by distinct genes.     

A gene encoding a tyrosine tRNA synthetase is located near sacS in Bacillus subtilis.

Within the frame of an attempt to sequence the whole Bacillus subtilis genome, a region of 5.5 kbp of the B. subtilis chromosome near the sacS locus has been sequenced. It contains five complete coding sequences, including the sequence of sacY, three unknown CDS and a sequence coding for a tyrosine tRNA synthetase. That the corresponding CDS encodes a functional synthetase has been demonstrated by complementation of an Escherichia coli mutant possessing a thermosensitive tRNA synthetase. Insertion of a kanamycin resistance cassette in the B. subtilis chromosome at the corresponding locus resulted, however, in no apparent phenotype, demonstrating that this synthetase is dispensable. Finally phylogenetic relationships between known tyrosine and tryptophan tRNA synthetases are discussed.     

Chloroplastic and cytoplasmic valyl- and leucyl- tRNA synthetases from Euglena gracilis: Comparative study of their structural properties

Chloroplastic and cytoplasmic valyl- and leucyl-tRNA synthetases purified from Euglena gracilis show a monomeric structure. The molecular weights of the two valyl-tRNA synthetases are identical (126 000) while those of the leucyl-tRNA synthetases are different (100 000 for the chloroplastic and 116 000 for the cytoplasmic enzyme). The tryptic maps and the amino acid compositions reveal differences between the chloroplastic valyl- and leucyl-tRNA synthetases and their cytoplasmic homologues. These results suggest that a chloroplastic aminoacyl-tRNA synthetase and its cytoplasmic counterpart are coded for by distinct genes.     

An essential residue at the active site of aspartate transcarbamylase.

Reaction of phenylglyoxal with aspartate transcarbamylase and its isolated catalytic subunit results in complete loss of enzymatic activity. This modification reaction is markedly influenced by pH and is partially reversible upon dialysis. Carbamyl phosphate or carbamyl phosphate with succinate partially protect the catalytic subunit and the native enzyme from inactivation by phenylglyoxal. In the native enzyme complete protection from inactivation is afforded by N-(phosphonacetyl)-L-aspartate. The decrease in enzymatic activity correlates with the modification of 6 arginine residues on each aspartate transcarbamylase molecule, i.e. 1 arginine per catalytic site. The data suggest that the essential arginine is involved in the binding of carbamyl phosphate to the enzyme. Reaction of the single thiol on the catalytic chain with 2-chloromercuri-4-nitrophenol does not prevent subsequent reaction with phenylglyoxal. If N-(phosphonacetyl)-L-aspartate is used to protect the active site we find that phenylglyoxal also causes the loss of activation of ATP and inhibition by CTP. The rate of loss of heterotropic effects is exactly the same for both nucleotides indicating that the two opposite regulatory effects originate at the same location on the enzyme, or are transmitted by the same mechanism between the subunits, or both.     

Regulation of Glutamine Synthetase II. Patterns of Feedback Inhibition in Microorganisms

The feedback inhibition of glutamine synthetase was investigated by use of partially purified enzyme preparations from Salmonella typhimurium, Micrococcus sodonensis, Pseudomonas fluorescens, Bacillus cereus, Bacillus licheniformis, Clostridium pasteurianum, Rhodospirillum rubrum, Neurospora crassa, Candida utilis, and Chlorella pyrenoidosa. Inhibition analyses indicated that the enzyme of each organism can be effectively regulated with mixtures of end products from the diverse pathways of glutamine metabolism. When tested individually, tryptophan, histidine, alanine, glycine, glutamine, 5'-adenylate (AMP), cytidine-5'-triphosphate, carbamyl phosphate, and glucosamine-6-phosphate gave limited inhibition. In most cases, the inhibitors were independent in their action, and cumulative degrees of inhibition were obtained with mixtures of these end products. In contrast, with the glutamine synthetases of the two Bacillus species, the simultaneous presence of AMP and histidine (or AMP and glutamine) gave inhibition greater than the sum of the amounts of inhibition caused by either inhibitor alone. Also, alanine and carbamyl phosphate acted synergistically to inhibit the enzyme from N. crassa. The remarkable similarity in the overall patterns of end-product inhibition observed with the enzymes from different sources indicates that these diverse organisms have evolved comparable mechanisms for the regulation of glutamine metabolism. Nevertheless, the enzymes from different sources do differ significantly in their physical and catalytic properties, as was demonstrated by dissimilarities in their purification behaviors, specificity for nucleotide substrate, ability to catalyze the glutamyl transfer reaction, and ability to utilize Mn(++) and Mg(++) as activators for the biosynthetic reaction.     

Biochemical genetic analysis of pyrimidine biosynthesis in mammalian cells: III. Association of carbamyl phosphate synthetase, aspartate transcarbamylase, and dihydroorotase in mutants of cultured Chinese hamster cells.

Carbamyl phosphate synthetase (EC 2.7.2.9), aspartate transcarbamylase (EC 2.1.3.2), and dihydroorotase (EC 3.5.2.3), the first three enzymes in de novo pyrimidine synthesis in Chinese hamster ovary cell strain Kl (CHO-Kl), cose diment through a glycerol gradient. When an extract from Urd- A, a pyrimidine-requiring auxotroph reduced in all three activities, is run on a glycerol gradient, the enzyme activities appear in two peaks higher in the gradient, a peak of aspartate transcarbamylase separated from a peak of carbamyl phosphate synthetase and dihydroorotase. Revertants of Urd- A have increased activity of all three enzymes and give glycerol gradient patterns similar to either CHO-Kl or Urd- A. The gradient pattern for Urd- A and some of its revertants can be mimicked by treating the CHO-Kl cell extract with trypsin. Hybrids made between a CHO-Kl purine-requiring auxotroph (Ade- C) and a Urd- A revertant gave a glycerol gradient pattern which is a composite of the CHO-Kl and revertant patterns. A model is presented for the structure of this multifunctional protein.     

Regulation of phosphorylation of aminoacyl-tRNA synthetases in the high molecular weight core complex in reticulocytes

Five aminoacyl-tRNA synthetases found in the high molecular weight core complex were phosphorylated in rabbit reticulocytes following labeling with 32P. The synthetases were isolated by affinity chromatography on tRNA-Sepharose followed by immunoprecipitation. The five synthetases phosphorylated were the glutamyl-, glutaminyl-, lysyl-, and aspartyl-tRNA synthetases and, to a lesser extent, the methionyl-tRNA synthetase. In addition, a 37,000-dalton protein, associated with the synthetase complex and tentatively identified as casein kinase I, was also phosphorylated in intact cells. Phosphoamino acid analysis of the proteins indicated all of the phosphate was on seryl residues. Incubation of reticulocytes with 32P in the presence of 8-bromo-cAMP and 3-isobutyl-1-methylxanthine resulted in a 6-fold increase in phosphorylation of the glutaminyl-tRNA synthetase and a 2-fold increase in phosphorylation of the aspartyl-tRNA synthetase. When the high molecular weight core complex was isolated by gel filtration/affinity chromatography, the profile of phosphorylation was similar to that observed by immunoprecipitation with a 9- and 3-fold stimulation of the glutaminyl- and aspartyl tRNA-synthetase, respectively. From this data it was concluded that the increased phosphorylation of the glutaminyl- and aspartyl-tRNA synthetases obtained with 8-bromo-cAMP did not appear to be involved in dissociation of the high molecular weight core complex.