Elucidating the structure-function attributes of a trypanosomal arginyl-tRNA synthetase.

Aminoacyl-tRNA synthetases (aaRSs) are fundamental components of the protein translation machinery. In light of their pivotal role in protein synthesis and structural divergence among species, they have always been considered potential targets for the development of antimicrobial compounds. Arginyl-tRNA synthetase from Trypanosoma cruzi (TcArgRS), the parasite responsible for causing Chagas Disease, contains a 100-amino acid insertion that was found to be completely absent in the human counterpart of similar length, as ascertained from multiple sequence alignment results. Thus, we were prompted to perform a preliminary characterization of TcArgRS using biophysical, biochemical, and bioinformatics tools. We expressed the protein in E. coli and validated its in-vitro enzymatic activity. Additionally, analysis of DTNB kinetics, Circular dichroism (CD) spectra, and ligand-binding studies using intrinsic tryptophan fluorescence measurements aided us to understand some structural features in the absence of available crystal structures. Our study indicates that TcArgRS can discriminate between L-arginine and its analogues. Among the many tested substrates, only L-canavanine and L-thioarginine, a synthetic arginine analogue exhibited notable activation. The binding of various substrates was also determined using in silico methods. This study may provide a viable foundation for studying small compounds that can be targeted against TcArgRS.

Unique Contributions of an Arginine Side Chain to Ligand Recognition in a Glutamate-gated Chloride Channel.

Glutamate recognition by neurotransmitter receptors often relies on Arg residues in the binding site, leading to the assumption that charge-charge interactions underlie ligand recognition. However, assessing the precise chemical contribution of Arg side chains to protein function and pharmacology has proven to be exceedingly difficult in such large and complex proteins. Using the in vivo nonsense suppression approach, we report the first successful incorporation of the isosteric, titratable Arg analog, canavanine, into a neurotransmitter receptor in a living cell, utilizing a glutamate-gated chloride channel from the nematode Haemonchus contortus Our data unveil a surprisingly small contribution of charge at a conserved arginine side chain previously suggested to form a salt bridge with the ligand, glutamate. Instead, our data show that Arg contributes crucially to ligand sensitivity via a hydrogen bond network, where Arg interacts both with agonist and with a conserved Thr side chain within the receptor. Together, the data provide a new explanation for the reliance of neurotransmitter receptors on Arg side chains and highlight the exceptional capacity of unnatural amino acid incorporation for increasing our understanding of ligand recognition.

Cell-free expression with the toxic amino acid canavanine.

Canavanine is a naturally occurring noncanonical amino acid, which is analogous to arginine. It is a potent antimetabolite and natural allelochemic agent, capable of affecting or blocking regulatory and catalytic reactions that involve arginine. Incorporated into proteins at arginine positions, canavanine can be detrimental to protein stability and functional integrity. Although incorporation of canavanine into proteins has long been documented, due to its toxicity, expression in Escherichia coli and other common hosts remains a considerable challenge. Here, we present a simple, cell-free expression system with markedly improved performance compared to heterologous expression. The cell-free expression system does not require any tuning besides substitution of arginine by canavanine. We show that our technique enables highly efficient protein expression in small volumes with arginine being fully replaced by canavanine for functional and structural studies.

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.

Replacement of all arginine residues with canavanine in MazF-bs mRNA interferase changes its specificity.

Replacement of a specific amino acid residue in a protein with nonnatural analogues is highly challenging because of their cellular toxicity. We demonstrate for the first time the replacement of all arginine (Arg) residues in a protein with canavanine (Can), a toxic Arg analogue. All Arg residues in the 5-base specific (UACAU) mRNA interferase from Bacillus subtilis (MazF-bs(arg)) were replaced with Can by using the single-protein production system in Escherichia coli. The resulting MazF-bs(can) gained a 6-base recognition sequence, UACAUA, for RNA cleavage instead of the 5-base sequence, UACAU, for MazF-bs(arg). Mass spectrometry analysis confirmed that all Arg residues were replaced with Can. The present system offers a novel approach to create new functional proteins by replacing a specific amino acid in a protein with its analogues.

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 Lcanavanine-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, 256e258). 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.

Inactivation of microbial arginine deiminases by L-canavanine.

Arginine deiminase (ADI) catalyzes the hydrolytic conversion of L-arginine to ammonia and L-citrulline as part of the energy-producing L-arginine degradation pathway. The chemical mechanism for ADI catalysis involves initial formation and subsequent hydrolysis of a Cys-alkylthiouronium ion intermediate. The structure of the Pseudomonas aeruginosa ADI-(L-arginine) complex guided the design of arginine analogs that might react with the ADIs to form inactive covalent adducts during catalytic turnover. One such candidate is L-canavanine, in which an N-methylene of L-arginine is replaced by an N-O. This substance was shown to be a slow substrate-producing O-ureido-L-homoserine. An in depth kinetic and mass spectrometric analysis of P. aeruginosa ADI inhibition by L-canavanine showed that two competing pathways are followed that branch at the Cys-alkylthiouronium ion intermediate. One pathway leads to direct formation of O-ureido-L-homoserine via a reactive thiouronium intermediate. The other pathway leads to an inactive form of the enzyme, which was shown by chemical model and mass spectrometric studies to be a Cys-alkylisothiourea adduct. This adduct undergoes slow hydrolysis to form O-ureido-L-homoserine and regenerated enzyme. In contrast, kinetic and mass spectrometric investigations demonstrate that the Cys-alkylthiouronium ion intermediate formed in the reaction of L-canavanine with Bacillus cereus ADI partitions between the product forming pathway (O-ureido-L-homoserine and free enzyme) and an inactivation pathway that leads to a stable Cys-alkylthiocarbamate adduct. The ADIs from Escherichia coli, Burkholderia mallei, and Giardia intestinalis were examined in order to demonstrate the generality of the L-canavanine slow substrate inhibition and to distinguish the kinetic behavior that defines the irreversible inhibition observed with the B. cereus ADI from the time controlled inhibition observed with the P. aeruginosa, E. coli, B. mallei, and G. intestinalis ADIs.

Proton affinity of canavanine and canaline, oxyanalogues of arginine and ornithine, from the extended kinetic method.

The absolute proton affinities of the nonprotein amino acids canavanine and canaline have been determined using the extended kinetic method in an electrospray ionization quadrupole ion trap instrument. Canavanine results from the substitution of an oxygen atom for the delta-CH2 group in the side chain of the protein amino acid arginine, whereas canaline results from a similar substitution at the delta-CH2 group in the side chain of ornithine. Absolute proton affinities of 1001+/-9 and 950+/-7 kJ/mol are obtained for canavanine and canaline, respectively. For canaline, this proton affinity is in excellent agreement with theoretical predictions obtained using the hybrid density functional theory method B3LYP/6-311++G**//B3LYP/6-31+G*. For canavanine, theory predicts a somewhat larger proton affinity of 1015 kJ/mol. Oxygen atom substitution in these nonprotein amino acids results in a decrease in their proton affinities of 40-50 kJ/mol compared to arginine and ornithine.

L-canavanine is a time-controlled mechanism-based inhibitor of Pseudomonas aeruginosa arginine deiminase.

The mechanism for inhibition of the Pseudomonas aeruginosa arginine deiminase (PaADI) by the arginine analogue l-canavanine was investigated. Inhibition by this substance (kinact = 0.31 +/- 0.03 min-1 and Ki = 1.7 +/- 0.5 mM) is associated with the formation of a modestly stable S-alkylthiouronium intermediate, detected by using kinetic techniques and identified by using electrospray ionization mass spectrometry. The electronic and/or orientation effects, caused by oxygen-for-methylene substitution in l-canavanine, on the rate of enzyme regeneration from the S-alkylthiouronium intermediate could serve as the basis for a strategy for the rational design of new slow substrate inhibitors of ADI.

Arabidopsis ubiquitin-specific protease 6 (AtUBP6) interacts with calmodulin.

Calmodulin (CaM), a key Ca(2+) sensor in eukaryotes, regulates diverse cellular processes by interacting with many proteins. To identify Ca(2+)/CaM-mediated signaling components, we screened an Arabidopsis expression library with horseradish peroxidase-conjugated Arabidopsis calmodulin2 (AtCaM2) and isolated a homolog of the UBP6 deubiquitinating enzyme family (AtUBP6) containing a Ca(2+)-dependent CaM-binding domain (CaMBD). The CaM-binding activity of the AtUBP6 CaMBD was confirmed by CaM mobility shift assay, phosphodiesterase competition assay and site-directed mutagenesis. Furthermore, expression of AtUBP6 restored canavanine resistance to the Deltaubp6 yeast mutant. This is the first demonstration that Ca(2+) signaling via CaM is involved in ubiquitin-mediated protein degradation and/or stabilization in plants.

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 beta-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.

Spectrophotometric and steady-state kinetic analysis of the biosynthetic arginine decarboxylase of Yersinia pestis utilizing arginine analogues as inhibitors and alternative substrates.

The PLP-dependent, biosynthetic arginine decarboxylase (ADC) of Yersinia pestis was investigated using steady-state kinetics employing structural analogues of arginine as both alternative substrates and competitive inhibitors. The inhibitor analysis indicates that binding of the carboxyl and guanidinium groups of the substrate, l-arginine, provides essentially all of the free energy change realized upon substrate binding in the ground state. Furthermore, recognition of the guanidinium group is primarily responsible for substrate specificity. Comparison of the steady-state parameters for a series of alternative substrates that contained chemically modified guanidinium moieties provides evidence of a role for induced fit in ADC catalysis. ADC was also characterized by UV/vis and fluorescence spectrophotometry in the presence or absence of a number of arginine analogues. The enzyme complexes formed served as models for the adsorption complex and the external aldimine complex of the enzyme with the substrate.

Computational docking of L-arginine and its structural analogues to C-terminal domain of Escherichia coli arginine repressor protein (ArgRc).

The arginine repressor (ArgR) of Escherichia coli binds to six L-arginine molecules that act as its co-repressor in order to bind to DNA. The binding of L-arginine molecules as well as its structural analogues is compared by means of computational docking. A grid-based energy evaluation method combined with a Monte Carlo simulated annealing process was used in the automated docking. For all ligands, the docking procedure proposed more than one binding site in the C-terminal domain of ArgR (ArgRc). Interaction patterns of ArgRc with L-arginine were also observed for L-canavanine and L-citrulline. L-lysine and L-homoarginine, on the other hand, were shown to bind poorly at the binding site. Figure A general overview of the sites found from docking the various ligands into ArgRc ( grey ribbons). Red coloured sticks: residues in binding site H that was selected for docking

L-Canavanine: a higher plant insecticidal allelochemical.

L-Canavanine, L-2-amino-4-(guanidinooxy)butyric acid, is a potentially toxic nonprotein amino acid of certain leguminous plants. Many species are prolific canavanine producers; they divert enormous nitrogen resource to the storage of this single natural product. Canavanine, a highly effective protective allelochemical, provides a formidable chemical barrier to predation and disease. The accumulated experimental evidence leaves little doubt that the key element in the ability of canavanine to function as an effective protective allelochemical is its subtle structural mimicry of arginine which makes it an effective substrate for amino acid activation and aminoacylation, and its marked diminution in basicity relative to arginine which mediates the production of structural aberrant, dysfunctional canavanyl proteins. The biological burdens of canavanyl protein formation by canavanine-treated Manduca sexta larvae were carried throughout their remaining life cycle. Protein-based sequestration of canavanine prevented turnover and clearance of the free amino acid, and undoubtedly contributed significantly to the antimetabolic character of this protective allelochemical.

Effects of newly synthesized analogs of MIF-1 containing unnatural amino acids on electrically evoked smooth muscle contractions.

New MIF-1 (Pro-Leu-Gly-NH2) analogs containing unnatural amino acids such as L-canavanine (Cav) and L-cysteic acid S-(2-aminoethyl)amide (sLys) have been synthesized and in vitro experiments were performed to study their action on neurotransmission in target tissues with adrenergic and cholinergic neurotransmission. The experiments were carried out on electrically stimulated proximal guinea pig ileum (GPI) and the prostatic part of rat and rabbit vasa deferentia (VDR, VDRabb). The present results show that the newly synthesized Cav2-MIF and sLys2-MIF might affect electrically evoked smooth muscle contractions.

Effect of citrulline for arginine replacement on the structure and turnover of phosphopeptide substrates of protein phosphatase-1.

Phosphorylated and nonphosphorylated forms of a decapeptide corresponding to residues 9 to 18 of glycogen phosphorylase were compared using two-dimensional nuclear magnetic resonance with assignment of both peptides done by the sequential method. Both forms had little secondary structure, but there was evidence for an interaction between arginine-16 and phosphorylated serine at position 14. A change in the chemical shift for the epsilon-nitrogen hydrogen of arginine in position 16 was observed in the spectrum of the phosphorylated peptide and was not evident in a phosphopeptide having citrulline in place of arginine-16. Hydrolysis catalyzed by protein phosphatase-1 was decreased with the citrulline-containing phosphopeptide compared to the arginine-containing phosphopeptide with effects observed on both kcat and Km of the phosphatase reaction. Alkaline phosphatase hydrolyzed these peptides and a di-citrulline peptide equally well. These results are consistent with arginine being favorable in the recognition of substrates by phosphatase-1, possibly recognition as an arginine-phosphoserine complex. As a model study, arginine and two analogs, citrulline and canavanine, were examined for association with inorganic phosphate by nuclear magnetic resonance spectrometry. 31P-NMR measurements showed that arginine and canavanine caused a shift in the phosphate resonance at 20 degreesC. Citrulline caused no change. Changes in chemical shift were measured over the pH range 5-9 with arginine and canavanine both causing a slight decrease in the apparent pKa of inorganic phosphate (DeltapKa approximately 0.15). NaCl, NH4Cl, and guanidine hydrochloride showed little effect on the resonance signal position of inorganic phosphate at pH 6.5, consistent with selectivity for the guanidino group. Temperature (6 degrees, 20 degrees, and 37 degreesC) caused little change in the effect of arginine, but there was some dependency with canavanine, decreasing with temperature. Citrulline caused no change in the chemical shift of phosphate at any temperature. It was concluded that hydrogen bonded complexes were formed between the dianion of phosphate and the protonated form of arginine or canavanine with a bifurcated structure having preference for the omega-hydrogens.

L-homoarginine studies provide insight into the antimetabolic properties of L-canavanine.

A method for the chemical synthesis of L-homoarginine, based on the guanidination of L-lysine with O-methylisourea, has been developed; this procedure provides radiochemically pure L-[guanidino-14C]homoarginine in high yield. Radiolabeled homoarginine is incorporated readily into the newly synthesized hemolymphic proteins of larvae of the tobacco hornworm, Manduca sexta without adversely affecting larval growth and development. This finding stands in sharp contrast to the effect of L-canavanine, another L-arginine analog, which is markedly deleterious to these larvae. Homoarginine is incorporated into M. sexta lysozyme, and the antibacterial proteins of the fly, Phormia terranovae with impunity. In contrast, the comparable canavanine-containing enzymes are inhibited severely. Experimental evidence is presented that the innocuous nature of homoarginine results from the elevated pKa value of its guanidino group which arguably exceeds even that of arginine. As a result, homoarginine does not disrupt essential residue interactions. In contrast canavanine, which is much less basic than arginine, does adversely affect R group interactions forming the requisite three-dimensional conformation of the protein.