Amino acid discrimination by the nuclear encoded mitochondrial arginyl-tRNA synthetase of the larva of a bruchid beetle (Caryedes brasiliensis) from northwestern Costa Rica

l-canavanine, the toxic guanidinooxy analogue of l-arginine, is the product of plant secondary metabolism. The need for a detoxifying mechanism for the producer plant is self-evident but the larvae of the bruchid beetle Caryedes brasiliensis, that is itself a non-producer, have specialized in feeding on the l-canavanine-containing seeds of Dioclea megacarpa. The evolution of a seed predator that can imitate the enzymatic abilities of the host permits us to address the question of whether the same problem of amino acid recognition in two different kingdoms has been solved by the same mechanism. A discriminating arginyl-tRNA synthetase, detected in a crude C. brasiliensis larval extract, was proposed to be responsible for insect's ability to survive the diet of l-canavanine (Rosenthal, G. A., Dahlman, D. L., and Janzen, D. H. (1976) A novel means for dealing with L-canavanine, a toxic metabolite. Science 192, 256–258). Since the arginyl-tRNA synthetase of at least three genetic compartments (insect cytoplasmic, insect mitochondrial and insect gut microflora) may participate in conferring l-canavanine resistance, we investigated whether the nuclear-encoded C. brasiliensis mitochondrial arginyl-tRNA synthetase plays a role in this discrimination. Steady state kinetics of the cloned, recombinant enzyme have revealed and quantified an amino acid discriminating potential of the mitochondrial enzyme that is sufficient to account for the overall l-canavanine misincorporation rate observed in vivo. As in the cytoplasmic enzyme of the l-canavanine producer plant, the mitochondrial arginyl-tRNA synthetases from a specialist seed predator relies on a kinetic discrimination that prevents l-canavanine misincorporation into proteins.     

A specific arginase-based assay for l-canavanine in leguminous plants

A convenient method for the analysis of free l-canavanine in leguminous plants is described. Canavanine was specifically hydroloyzed to canaline and urea by the enzyme arginase (EC 3.5.3.1). The resulting amino-oxy functions of canaline were measured based on their ability to bleach the yellow colour of pyridoxal 5′-phosphate. Canavanine in the seeds of Canavalia ensiformis was determined with this method.     

The crystal structure of arginyl-tRNA synthetase from Homo sapiens

Arginyl-tRNA synthetase (ArgRS) is a tRNA-binding protein that catalyzes the esterification of l-arginine to its cognate tRNA. l-Canavanine, a structural analog of l-arginine, has recently been studied as an anticancer agent. Here, we determined the crystal structures of the apo, l-arginine-complexed, and l-canavanine-complexed forms of the cytoplasmic free isoform of human ArgRS (hArgRS). Similar interactions were formed upon binding to l-canavanine or l-arginine, but the interaction between Tyr312 and the oxygen of the oxyguanidino group was a little bit different. Detailed conformational changes that occur upon substrate binding were explained. The hArgRS structure was also compared with previously reported homologue structures. The results presented here may provide a basis for the design of new anticancer drugs, such as l-canavanine analogs.     

Further studies of the effect of L-canavanine on the tobacco hornworm, Manduca sexta.

Fifth instar Manduca sexta growth response to injected doses of canavanine was concentration-dependent over a range of 0·5 to 2·0 mg/g body weight. Twenty-four hr after injection of ¹⁴C-guanidinooxy-d,l-canavanine, M. sexta larvae incorporated approximately 3·6% of the labelled l-canavanine into protein of non-gut tissue. Adult M. sexta mortality was related to the level of injected canavanine over a range of 2 to 8 mg/g body weight. Injection of as little as 2 mg canavanine/g body weight caused hyperactivity in adult M. sexta. Arginine, able to negate the toxic effects of canavanine during larval growth, was only marginally capable of overcoming canavanine effects on larval-pupal ecdysis.     

The Mechanism of l-Canavanine Cytotoxicity: Arginyl tRNA Synthetase as a Novel Target for Anticancer Drug Discovery

There is a clear need for agents with novel mechanisms of action to provide new therapeutic approaches for the treatment of pancreatic cancer. Owing to its structural similarity to l-arginine, l-canavanine, the δ-oxa-analog of l-arginine, is a substrate for arginyl tRNA synthetase and is incorporated into nascent proteins in place of l-arginine. Although l-arginine and l-canavanine are structurally similar, the oxyguanidino group of l-canavanine is significantly less basic than the guanidino group of l-arginine. Consequently, l-canavanyl proteins lack the capacity to form crucial ionic interactions, resulting in altered protein structure and function, which leads to cellular death. Since l-canavanine is selectively sequestered by the pancreas, it may be especially useful as an adjuvant therapy in the treatment of pancreatic cancer. This novel mechanism of cytotoxicity forms the basis for the anticancer activity of l-canavanine and thus, arginyl tRNA synthetase may represent a novel target for the development of such therapeutic agents.     

Structural Aberrations in T-Even Bacteriophage IV. Parameters of Induction and Formation of Lollipops

Previous results from our laboratory have shown that when a T-even bacteriophage-infected bacterial cell was exposed to l-canavanine followed by an l-arginine chase, a monster phage particle, termed a lollipop, was formed. We now describe certain parameters concerning (i) the induction and (ii) the formation of T4 lollipops. The induction step involves a T4 late function, and can require only a 3-min exposure to l-canavanine. Short pulses of l-canavanine result in the formation of shorter lollipops indicating the presence of a possible "precursor substance" which is influenced by l-canavanine. DNA synthesis is inhibited by l-canavanine but is stimulated 20 to 40 min after the addition of l-arginine. Chloramphenicol prevents both responses indicating a possible protein involvement. The appearance of lollipops and phage was noted only after 25 min after the addition of l-arginine.     

Structural Aberrations in T-Even Bacteriophage V. Effects of Canavanine on the Maturation and Utilization of Specific Gene Products

Previous results have shown that when a T-even bacteriophage-infected cell was exposed to l-canavanine followed by an exposure to l-arginine, a monster phage particle, termed a lollipop, was formed. l-Canavanine was necessary for the induction event but l-arginine was required for the maturation of the particle. We now describe the effects of canavanine on the maturation of certain T4 proteins and their role in the induction of lollipops. The cleavage reactions of the head proteins P22, P23, P24, and IPIII are prevented by l-canavanine as shown by the accumulation of the precursor proteins and the failure of the cleaved products to appear. l-Canavanine also prevents the appearance of P12 (tailplate protein) and P20 (head protein) indicating that these proteins may undergo a proteolytic cleavage during normal assembly. The formation of P10 (tailplate protein) and P18 (tail sheath protein) is also affected by l-canavanine. The data suggest that P23 in conjunction with P20 plays a major role in the determination of the length of the phage head.     

Is canavanine a natural component of mammalian metabolism?

A group of non-protein amino acids of higher plants, namely l-canavanine, l-canaline, 0-ureido-l-homoserine, and l-canavaninosuccinic acid, have been implicated in mammalian intermediary metabolism. The clinical observations and biochemical basis for this hypothesis as well as conflicting experimental evidence are presented. A possible explanation for the apparent role of these non-protein amino acids in mammalian metabolism is offered.     

Preparation and colorimetric analysis of l-Canavanine

Procedures for the preparation and colorimetric assay of l-canavanine, a structural analog of l-arginine, are presented.     

Allelochemical induced stress: Effects of l-canavanine on the pathogenicity of Bacillus thuringiensis in Manduca sexta

Increased susceptibility of Manduca sexta to commercial formulations of the microbial insecticide, Bacillus thuringiensis, as evidenced by lower LD₅₀ and LT₅₀ values, was observed when M. sexta were reared on an artificial diet supplemented with a sublethal concentration (2.5 mm) of l-canavanine. At several dosages of B. thuringiensis, which were administered either by diet contamination or by per os forced feeding, a greater than 70% reduction (P < 0.05) occurred in the LT₅₀ response with canavanine-treated larvae. The LD₅₀ values also were lowered by canavanine treatment. This constitutes the first report of a plant allelochemical enhancing the effect of B. thuringiensis in vivo. It is suggested that canavanine enhances the effect of B. thuringiensis on gut permeability and active transport.     

Cleavage of head and tail proteins during bacteriophage T5 assembly: selective host involvement in the cleavage of a tail protein

Electrophoresis studies showed that at least three phage-specified proteins undergo proteolytic cleavage during the development of bacteriophage T5. One of these proteins has a molecular weight of about 135,000 and the product of this cleavage reaction is a minor component of the T5 tail, having a molecular weight of about 128,000. All of the tail-defective T5 mutants studied in this report failed to induce this cleavage reaction under restrictive conditions. This reaction also failed to occur in Escherichia coli groEA639 and groEA36 infected with wild type T5. Examination of lysates of infected groE cells in the electron microscope revealed the presence of filled and empty heads as well as tubular head structures, but no tails were detected. The filled heads were able to combine with separately prepared T5 tails in vitro to form infectious phage particles. Therefore, propagation of T5 in these groE mutants is prevented primarily by a specific block in tail assembly. A T5 mutant, T5?6, was isolated, which has the capacity to propagate in these groE hosts. The gene locus in T5?6 was mapped.The second T5 protein which is cleaved has a molecular weight of 50,000 and is related to head morphogenesis. Treatment of infected cells with l-canavanine (50 μg/ml) inhibited cleavage of this polypeptide. Only small quantities of the major head protein (32,000 mol. wt) were produced in these treated cells. Treatment with canavanine lead to production of tubular heads. The major protein component of partially purified tubular heads has a molecular weight of 50,000. Cells infected with T5 amber H30b, a mutant defective in head gene D20, does not produce the 50,000 and 32,000 molecular weight proteins. These findings suggest that the 50,000 molecular weight protein undergoes cleavage to form the major head polypeptide. A third T5 protein is cleaved to form a minor head component with a molecular weight of 43,000 and its cleavage is linked to that involving the major head protein.     

Studies on the metabolism of guanidine compounds in mammals

[¹⁴C]Guanidine was observed in the urine after subcutaneous administration to rats of l-[guanidino-¹⁴C]arginine or l-[guanidino-¹⁴C]canavanine. [${}^{14}\text{C}$]Hydroxyguanidine was additionally detected in the urine after injection of dl-[guanidino-¹⁴C]canavanine. These ${}^{14}\text{C}$ metabolites were characterized by high-voltage electrophoresis and paper chromatography, by enzymatic conversion of [¹⁴C]hydroxyguanidine to [¹⁴C]guanidine, and by repeated recrystallization of isolated urinary [${}^{14}\text{C}$]guanidine as the picrate salt with no significant loss of specific activity. These experiments demonstrate that both l-arginine and l-canavanine can serve as precursors of guanidine in the rat.     

Induced Structural Defects in T-Even Bacteriophage

Multiple aberrant substructures of T-even bacteriophage particles occurred when amino acid analogues or antimetabolites were present during phage growth. Certain aberrant substructures were induced by specific analogues or antimetabolites. In particular, it was observed by electron microscopy that l-canavanine, an arginine analogue, gave rise to polyheads; l-azetidine-2-carboxylic acid, a proline analogue, gave rise to polytail tubes; and 1,2,4-trizaole-3-alanine, a histidine analogue, proflavine, and actinomycin D all gave rise to small heads. These aberrant substructures were similar to those reported earlier with conditional lethal mutants (amber) of T4D in a restrictive host.     

l-Canavanine Metabolism in Jack Bean, Canavalia ensiformis (L.) DC. (Leguminosae)

l-Canavanine, a highly toxic arginine antimetabolite, is the principal nonprotein amino acid of many leguminous plants. Labeled-precursor feeding studies, conducted primarily with [(14)C]carbamoyl phosphate, and utilization of the seedlings of jack bean, Canavalia ensiformis (L.) DC. (Leguminosae), have provided evidence for l-canavanine biosynthesis from l-canaline via O-ureido-l-homoserine. This reaction pathway appears to constitute an important in vivo route of canavanine production. Canavanine cleavage to canaline may represent a degradative phase of canavanine metabolism distinct from the anabolic reactions described above. Thus, while these reactions of canavanine metabolism bear analogy to the mammalian Krebs-Henseleit ornithine-urea cycle, no evidence has been obtained at present for the reutilization of canaline in ureidohomoserine formation.     

Properties and structure of a gene 24-controlled T4 giant phage

Giant T4 bacteriophage were found by Doermann et al. (1973a) with point mutants in gene 23 and by Cummings et al. (1973) after l-canavanine induction followed by an arginine chase. We now find T4 giant phage with 14 out of 15 tested temperature-sensitive mutants in gene 24 grown at intermediate temperatures between 33 °C and 37 °C.For one of these mutants, T4,24(tsB86), we found that (a) the optimum temperature for giant phage production is 34.8 °C, (b) the head-length distribution peaks sharply between 10 and 12 normal T4 phage head lengths, (c) about 75% of our giant phage have two tails, (d) the buoyant density in CsCl is greater than that of normal phage, (e) they are infectious and show an increased u.v. resistance, (f) their sodium dodecyl sulphate gel electrophoresis pattern is qualitatively similar to that of normal T4 phage, although the relative intensities of some of the bands are different, showing for example, a decreased $\text{P24}{}^1\text{P23}{}^1$² ratio, (g) optical diffraction and filtering of the flattened cylindrical part of the giant heads show a p6 surface net with a lattice constant of approximately 130 Å, a unique $\text{u}\text{v}$ ratio of $\text{15}\text{5}$ and a capsomer morphology of the type 1 + 6 + 6.Mixed infections with T4 wild type and T4.24(amN65) also yield giant phage. These are produced in highest amounts with a multiplicity of infection ratio of 5:5; no giants are observed at ratios of 1:9 or 9:1, suggesting that their formation may be caused by a dosage effect of P24.     

Urea cycle of Fasciola gigantica: purification and characterization of arginase

The ornithine-urea cycle has been investigated in Fasciola gigantica. Agrinase had very high activity compared to the other enzymes. Carbamoyl phosphate synthetase and ornithine carbamoyltransferase had very low activity. A moderate enzymatic activity was recorded for argininosuccinate synthetase and argininosuccinate lyase. The low levels of F. gigantica urea cycle enzymes except to the arginase suggest the urea cycle is operative but its role is of a minor important. The high level of arginase activity may benefit for the hydrolysis of the exogenous arginine to ornithine and urea. Two arginases Arg I and Arg II were separated by DEAE-Sepharose column. Further purification was restricted to Arg II with highest activity. The molecular weight of Arg II, as determined by gel filtration and SDS-PAGE, was 92,000. The enzyme was capable to hydrolyze l-arginine and to less extent l-canavanine at arginase:canavanase ratio (>10). The enzyme exhibited a maximal activity at pH 9.5 and Km of 6 mM. The optimum temperature of F. gigantica Arg II was 40 degrees C and the enzyme was stable up to 30 degrees C and retained 80% of its activity after incubation at 40 degrees C for 15 min and lost all of its activity at 50 degrees C. The order of effectiveness of amino acids as inhibitors of enzyme was found to be lysine>isoleucine>ornithine>valine>leucine>proline with 67%, 43%, 31%, 25%, 23% and 15% inhibition, respectively. The enzyme was activated with Mn2+, where the other metals Fe2+, Ca2+, Hg2+, Ni2+, Co2+ and Mg2+ had inhibitory effects.     

L-Canavanine made by Medicago sativa interferes with quorum sensing in Sinorhizobium meliloti

Sinorhizobium meliloti is a gram-negative soil bacterium, capable of establishing a nitrogen-fixing symbiosis with its legume host, alfalfa (Medicago sativa). Quorum sensing plays a crucial role in this symbiosis, where it influences the nodulation process and the synthesis of the symbiotically important exopolysaccharide II (EPS II). S. meliloti has three quorum-sensing systems (Sin, Tra, and Mel) that use N-acyl homoserine lactones as their quorum-sensing signal molecule. Increasing evidence indicates that certain eukaryotic hosts involved in symbiotic or pathogenic relationships with gram-negative bacteria produce quorum-sensing-interfering (QSI) compounds that can cross-communicate with the bacterial quorum-sensing system. Our studies of alfalfa seed exudates suggested the presence of multiple signal molecules capable of interfering with quorum-sensing-regulated gene expression in different bacterial strains. In this work, we choose one of these QSI molecules (SWI) for further characterization. SWI inhibited violacein production, a phenotype that is regulated by quorum sensing in Chromobacterium violaceum. In addition, this signal molecule also inhibits the expression of the S. meliloti exp genes, responsible for the production of EPS II, a quorum-sensing-regulated phenotype. We identified this molecule as l-canavanine, an arginine analog, produced in large quantities by alfalfa and other legumes.     

Effects of l-canavanine on arginine catabolism in manduca sexta (sphingidae: lepidoptera)

• 1.1. Hemolymph ornithine concentrations in tobacco horn worm larvae fed a 2.5 mM l-canavanine plus 25 mM l-arginine-supplemented artificial diet (CAAM) were higher than those in larvae fed diets supplemented with 2.5 mM canavanine (CAV), 25 mM arginine (ARG), or controls (CON).
• 2.2. Ornithine concentrations in CAV-treated larvae were significantly greater than the control or ARG treatment, but less than the CAAM treatment during the latter part of the wandering larval stage and during the pharate pupal stage.
• 3.3. Urea concentrations were greater during the active feeding stage with the CAAM- and ARG-treated larvae having significantly higher levels than control or CAV-treated larvae.
• 4.4. Urea concentrations in all treatments never exceeded 36.5% of the ornithine concentration.
• 5.5. Canavanine concentrations were higher in CAV-treated larvae than in CAAM-treated larvae.
• 6.6. During active feeding, arginine concentrations for all treatments were similar, but were lower in CAV- and CAAM-treated larvae during the pharate pupal stage.

     

Proteasomal components required for cell growth and stress responses in the haloarchaeon Haloferax volcanii

Little is known regarding the biological roles of archaeal proteases. The haloarchaeon Haloferax volcanii is an ideal model for understanding these enzymes, as it is one of few archaea with an established genetic system. In this report, a series of H. volcanii mutant strains with markerless and/or conditional knockouts in each known proteasome gene was systematically generated and characterized. This included single and double knockouts of genes encoding the 20S core alpha1 (psmA), beta (psmB), and alpha2 (psmC) subunits as well as genes (panA and panB) encoding proteasome-activating nucleotidase (PAN) proteins closely related to the regulatory particle triple-A ATPases (Rpt) of eukaryotic 26S proteasomes. Our results demonstrate that 20S proteasomes are required for growth. Although synthesis of 20S proteasomes containing either alpha1 or alpha2 could be separately abolished via gene knockout with little to no impact on growth, conditional depletion of either beta alone or alpha1 and alpha2 together rendered the cells inviable. In contrast, the PAN proteins were not essential based on the robust growth of the panA panB double knockout strain. Deletion of genes encoding either alpha1 or PanA did, however, render cells more sensitive to growth on organic versus inorganic nitrogen sources and hypo-osmotic stress and limited growth in the presence of l-canavanine. Abolishment of alpha1 synthesis also had a severe impact on the ability of cells to withstand thermal stress. This contrasted with what was seen for panA knockouts, which displayed enhanced thermotolerance. Together, these results provide new and important insight into the biological role of proteasomes in archaea.