The potential use of arginine kinase from the brown tick Rhipicephalus sanguineus as a biomarker for vector exposure in the surveillance of Rocky Mountain spotted fever.

The brown dog tick (Rhipicephalus sanguineus) is the vector of Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever (RMSF) in Northern Mexico and Southwestern United States. The immune response to a tick protein in the sera of humans or animals may reveal the zones with a high propensity to acquire RMSF, and vector control strategies may be focused on these zones. Arginine kinase (AK) is a highly antigenic invertebrate protein that may serve as a marker for tick exposure. We used R. sanguineus recombinant AK in an indirect ELISA assay with RMSF-positive patient sera. The response to AK was significantly higher against the sera of RMSF patients than the control sera from healthy participants without contact with dogs. To validate the antigenicity of tick AK, we mutated one predicted conformational epitope to alanine residues, which reduced the recognition by RMSF patients' immunoglobulins. This preliminary result opens a perspective towards the development of a complimentary technique based on RsAK as an antigen biomarker for vector serological surveillance for Rickettsia RMSF prevention.

Arginine kinase shows nucleoside diphosphate kinase-like activity toward deoxythymidine diphosphate.

Arginine kinase (AK) (ATP: L-arginine phosphotransferase, E.C. 2.7.3.3) catalyzes the reversible transfer of ATP γ-phosphate group to L-arginine to synthetize phospho-arginine as a high-energy storage. Previous studies suggest additional roles for AK in cellular processes. Since AK is found only in invertebrates and it is homologous to creatine kinase from vertebrates, the objective of this work was to demonstrate nucleoside diphosphate kinase-like activity for shrimp AK. For this, AK from marine shrimp Litopenaeus vannamei (LvAK) was purified and its activity was assayed for phosphorylation of TDP using ATP as phosphate donor. Moreover, by using high-pressure liquid chromatography (HPLC) the phosphate transfer reaction was followed. Also, LvAK tryptophan fluorescence emission changes were detected by dTDP titration, suggesting that the hydrophobic environment of Trp 221, which is located in the top of the active site, is perturbed upon dTDP binding. The kinetic constants for both substrates Arg and dTDP were calculated by isothermal titration calorimetry (ITC). Besides, docking calculations suggested that dTDP could bind LvAK in the same cavity where ATP bind, and LvAK basic residues (Arg124, 126 and 309) stabilize the dTDP phosphate groups and the pyrimidine base interact with His284 and Ser122. These results suggest that LvAK bind and phosphorylate dTDP being ATP the phosphate donor, thus describing a novel alternate nucleoside diphosphate kinase-like activity for this enzyme.

Binding of green tea epigallocatechin gallate to the arginine kinase active site from the brown recluse spider (Loxosceles laeta): A potential synergist to chemical pesticides.

Loxosceles spp. spiders can cause serious public health issues. Chemical control is commonly used, leading to health and environmental problems. Identifying molecular targets and using them with natural compounds can help develop safer and eco-friendlier biopesticides. We studied the kinetics and predicted structural characteristics of arginine kinase (EC 2.7.3.3) from Loxosceles laeta (LlAK), a key enzyme in the energy metabolism of these organisms. Additionally, we explored (-)-epigallocatechin gallate (EGCG), a green tea flavonoid, as a potential lead compound for the LlAK active site through fluorescence and in silico analysis, such as molecular docking and molecular dynamics (MD) simulation and MM/PBSA analyses. The results indicate that LlAK is a highly efficient enzyme (K m Arg 0.14 mM, K m ATP 0.98 mM, k cat 93 s-1, k cat/K m Arg 630 s-1 mM-1, k cat/K m ATP 94 s-1 mM-1), which correlates with its structure similarity to others AKs (such as Litopenaeus vannamei, Polybetes pythagoricus, and Rhipicephalus sanguineus) and might be related to its important function in the spider's energetic metabolism. Furthermore, the MD and MM/PBSA analysis suggests that EGCG interacted with LlAK, specifically at ATP/ADP binding site (RMSD <1 nm) and its interaction is energetically favored for its binding stability (-40 to -15 kcal/mol). Moreover, these results are supported by fluorescence quenching analysis (K d 58.3 µM and K a 1.71 × 104 M-1). In this context, LlAK is a promising target for the chemical control of L. laeta, and EGCG could be used in combination with conventional pesticides to manage the population of Loxosceles species in urban areas.

Crystal structure of shrimp arginine kinase in binary complex with arginine-a molecular view of the phosphagen precursor binding to the enzyme.

Arginine kinase (AK) is a key enzyme for energetic balance in invertebrates. Although AK is a well-studied system that provides fast energy to invertebrates using the phosphagen phospho-arginine, the structural details on the AK-arginine binary complex interaction remain unclear. Herein, we determined two crystal structures of the Pacific whiteleg shrimp (Litopenaeus vannamei) arginine kinase, one in binary complex with arginine (LvAK-Arg) and a ternary transition state analog complex (TSAC). We found that the arginine guanidinium group makes ionic contacts with Glu225, Cys271 and a network of ordered water molecules. On the zwitterionic side of the amino acid, the backbone amide nitrogens of Gly64 and Val65 coordinate the arginine carboxylate. Glu314, one of proposed acid-base catalytic residues, did not interact with arginine in the binary complex. This residue is located in the flexible loop 310-320 that covers the active site and only stabilizes in the LvAK-TSAC. This is the first binary complex crystal structure of a guanidine kinase in complex with the guanidine substrate and could give insights into the nature of the early steps of phosphagen biosynthesis.

Expression, purification, crystallization and X-ray crystallographic studies of different redox states of the active site of thioredoxin 1 from the whiteleg shrimp Litopenaeus vannamei.

Thioredoxin (Trx) is a 12 kDa cellular redox protein that belongs to a family of small redox proteins which undergo reversible oxidation to produce a cystine disulfide bond through the transfer of reducing equivalents from the catalytic site cysteine residues (Cys32 and Cys35) to a disulfide substrate. In this study, crystals of thioredoxin 1 from the Pacific whiteleg shrimp Litopenaeus vannamei (LvTrx) were successfully obtained. One data set was collected from each of four crystals at 100 K and the three-dimensional structures of the catalytic cysteines in different redox states were determined: reduced and oxidized forms at 2.00 Šresolution using data collected at a synchrotron-radiation source and two partially reduced structures at 1.54 and 1.88 Šresolution using data collected using an in-house source. All of the crystals belonged to space group P3212, with unit-cell parameters a = 57.5 (4), b = 57.5 (4), c = 118.1 (8) Å. The asymmetric unit contains two subunits of LvTrx, with a Matthews coefficient (VM) of 2.31 Å(3) Da(-1) and a solvent content of 46%. Initial phases were determined by molecular replacement using the crystallographic model of Trx from Drosophila melanogaster as a template. In the present work, LvTrx was overexpressed in Escherichia coli, purified and crystallized. Structural analysis of the different redox states at the Trx active site highlights its reactivity and corroborates the existence of a dimer in the crystal. In the crystallographic structures the dimer is stabilized by several interactions, including a disulfide bridge between Cys73 of each LvTrx monomer, a hydrogen bond between the side chain of Asp60 of each monomer and several hydrophobic interactions, with a noncrystallographic twofold axis.

Shrimp oncoprotein nm23 is a functional nucleoside diphosphate kinase.

Biosynthesis of nucleoside triphosphates is critical for bioenergetics and nucleic acid replication, and this is achieved by nucleoside diphosphate kinase (NDK). As an emerging biological model and the global importance of shrimp culture, we have addressed the study of the Pacific whiteleg shrimp (Litopenaeus vannamei) NDK. We demonstrated its activity and affinity towards deoxynucleoside diphosphates. Also, the quaternary structure obtained by gel filtration chromatography showed that shrimp NDK is a trimer. Affinity was in the micro-molar range for dADP, dGDP, dTDP and except for dCDP, which presented no detectable interaction by isothermal titration calorimetry, as described previously for Plasmodium falciparum NDK. This information is particularly important, as this enzyme could be used to test nucleotide analogs that can block white spot syndrome virus (WSSV) viral replication and to study its bioenergetics role during hypoxia and fasting.

Gene expression and protein levels of thioredoxin in the gills from the whiteleg shrimp (Litopenaeus vannamei) infected with two different viruses: the WSSV or IHHNV.

The thioredoxin (TRX) system in crustaceans has demonstrated to act as a cell antioxidant being part of the immune response by dealing with the increased production of reactive oxygen species during bacterial or viral infection. Since the number of marine viruses has increased in the last years significantly affecting aquaculture practices of penaeids, and due to the adverse impact on wild and cultured shrimp populations, it is important to elucidate the dynamics of the shrimp response to viral infections. The role of Litopenaeus vannamei thioredoxin (LvTRX) was compared at both, mRNA and protein levels, in response to two viruses, the white spot syndrome virus (WSSV) and the infectious hypodermal and hematopoietic necrosis virus (IHHNV). The results confirmed changes in the TRX gene expression levels of WSSV-infected shrimp, but also demonstrated a more conspicuous response of TRX to WSSV than to IHHNV. While both the dimeric and monomeric forms of LvTRX were detected by Western blot analysis during the WSSV infection, the dimer on its reduced form was only detected through the IHHNV infectious process. These findings indicate that WSSV or IHHNV infected shrimp may induce a differential response of the LvTRX protein.

Crystallization and X-ray diffraction studies of arginine kinase from the white Pacific shrimp Litopenaeus vannamei.

Crystals of an unligated monomeric arginine kinase from the Pacific whiteleg shrimp Litopenaeus vannamei (LvAK) were successfully obtained using the microbatch method. Crystallization conditions and preliminary X-ray diffraction analysis to 1.25 Šresolution are reported. Data were collected at 100 K on NSLS beamline X6A. The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 56.5, b = 70.2, c = 81.7 Å. One monomer per asymmetric unit was found, with a Matthews coefficient (V(M)) of 2.05 Å(3) Da(-1) and 40% solvent content. Initial phases were determined by molecular replacement using a homology model of LvAK as the search model. Refinement was performed with PHENIX, with final R(work) and R(free) values of 0.15 and 0.19, respectively. Biological analysis of the structure is currently in progress.

The greening reaction of skipjack tuna (Katsuwonus pelamis) metmyoglobin promoted by free cysteine during thermal treatment.

Background: Tuna muscle greening is a problem that occurs after heating. A hypothesis has been postulated to address this problem, involving a conserved Cys residue at position 10 (Cys-10) present on tuna myoglobin (Mb) that is exposed during the thermic treatment, forming a disulfide bond with free cysteine (Cys) in the presence of trimethylamine oxide (TMAO), resulting in the greening of the tuna Mb. Methods: We present a study using skipjack tuna (Katsuwonus pelamis) metmyoglobin (MbFe(III)-H2O) where the effect of free Cys (1-6 mM), TMAO (1.33 mM), and catalase on the greening reaction (GR) was monitored by UV-vis spectrometry during thermal treatment at 60 °C for 30 min. Moreover, the participation of Cys-10 on the GR was evaluated after its blocking with N-ethymaleimide. Results: The GR occurred in tuna MbFe(III)-H2O after heat treatment with free Cys, forming sulfmyoglobin (MbFe(II)-S) as the responsible pigment for the tuna greening. However, the rate constants of MbFe(II)-S production depended on Cys concentration (up to 4 mM) and occurred regardless of the TMAO presence. We postulate that two consecutive reactions involve an intermediate ferrylmyoglobin (promoted by H2O2) species with a subsequent MbFe(II)-S formation since the presence of catalase fosters the reduction of the rate reaction. Moreover, GR occurred even with blocked Cys-10 residues in tuna Mb and horse Mb (without Cys in its sequence). Discussion: We found that GR is not exclusive to tuna Mb´s, and it can be promoted in other muscle systems. Moreover, Cys and thermal treatment are indispensable for promoting this pigmentation anomaly.

White spot syndrome virus Orf514 encodes a bona fide DNA polymerase.

White spot syndrome virus (WSSV) is the causative agent of white spot syndrome, one of the most devastating diseases in shrimp aquaculture. The genome of WSSV includes a gene that encodes a putative family B DNA polymerase (ORF514), which is 16% identical in amino acid sequence to the Herpes virus 1 DNA polymerase. The aim of this work was to demonstrate the activity of the WSSV ORF514-encoded protein as a DNA polymerase and hence a putative antiviral target. A 3.5 kbp fragment encoding the conserved polymerase and exonuclease domains of ORF514 was overexpressed in bacteria. The recombinant protein showed polymerase activity but with very low level of processivity. Molecular modeling of the catalytic protein core encoded in ORF514 revealed a canonical polymerase fold. Amino acid sequence alignments of ORF514 indicate the presence of a putative PIP box, suggesting that the encoded putative DNA polymerase may use a host processivity factor for optimal activity. We postulate that WSSV ORF514 encodes a bona fide DNA polymerase that requires accessory proteins for activity and maybe target for drugs or compounds that inhibit viral DNA replication.

A Novel Glutathione S-Transferase Gtt2 Class (VpGSTT2) Is Found in the Genome of the AHPND/EMS Vibrio parahaemolyticus Shrimp Pathogen.

Glutathione S-transferases are a family of detoxifying enzymes that catalyze the conjugation of reduced glutathione (GSH) with different xenobiotic compounds using either Ser, Tyr, or Cys as a primary catalytic residue. We identified a novel GST in the genome of the shrimp pathogen V. parahaemolyticus FIM- S1708+, a bacterial strain associated with Acute Hepatopancreatic Necrosis Disease (AHPND)/Early Mortality Syndrome (EMS) in cultured shrimp. This new GST class was named Gtt2. It has an atypical catalytic mechanism in which a water molecule instead of Ser, Tyr, or Cys activates the sulfhydryl group of GSH. The biochemical properties of Gtt2 from Vibrio parahaemolyticus (VpGSTT2) were characterized using kinetic and crystallographic methods. Recombinant VpGSTT2 was enzymatically active using GSH and CDNB as substrates, with a specific activity of 5.7 units/mg. Low affinity for substrates was demonstrated using both Michaelis-Menten kinetics and isothermal titration calorimetry. The crystal structure showed a canonical two-domain structure comprising a glutathione binding G-domain and a hydrophobic ligand H domain. A water molecule was hydrogen-bonded to residues Thr9 and Ser 11, as reported for the yeast Gtt2, suggesting a primary role in the reaction. Molecular docking showed that GSH could bind at the G-site in the vicinity of Ser11. G-site mutationsT9A and S11A were analyzed. S11A retained 30% activity, while T9A/S11A showed no detectable activity. VpGSTT2 was the first bacterial Gtt2 characterized, in which residues Ser11 and Thr9 coordinated a water molecule as part of a catalytic mechanism that was characteristic of yeast GTT2. The GTT2 family has been shown to provide protection against metal toxicity; in some cases, excess heavy metals appear in shrimp ponds presenting AHPND/EMS. Further studies may address whether GTT2 in V. parahaemolyticus pathogenic strains may provide a competitive advantage as a novel detoxification mechanism.

Inhibition by Cu2+ and Cd2+ of a mu-class glutathione S-transferase from shrimp Litopenaeus vannamei.

Glutathione S-transferases (GSTs) are a family of detoxifying enzymes that catalyze the conjugation of glutathione (GSH) to electrophiles, thereby increasing the solubility of xenobiotics and aiding its excretion from the cell. The present work presents the inhibition of a mu-class GST of the marine shrimp Litopenaeus vannamei by copper (Cu2+) and cadmium (Cd2+). The protein was overexpressed in bacteria and its enzymatic activity measured using 1-chloro-2,4-dinitrobenzene. The mean inhibitory concentration (IC(50)) for shrimp GST against Cu2+) was 4.77 microM and for Cd2+ was 0.39 microM. A molecular model of the protein based on the crystal structure of a maize GST bound to cadmium showed that the metal binds in the GSH-binding site by coordination with Asp and Gln residues. These results are consistent with the experimental data and suggest that sublethal concentration of metals may affect the capacity of the organism to detoxify pesticides or xenobiotics.

Gallotannins are uncompetitive inhibitors of pancreatic lipase activity.

Inhibition of pancreatic lipase (PL) is used to treat dyslipidemias and obesity. Phenolic compounds are highly bioactive molecules that can inhibit various enzymes. Our aim was to evaluate the inhibitory activity of selected phenolic compounds of increasing molecular complexity, namely, phenolic acids, mangiferin, penta-O-galloyl-ß-d-glucose (PGG) and tannic acid (TA) against porcine PL, according to in vitro and in silico methodologies. TA and PGG were effective inhibitors (IC50 22.4 and 64.6 µM, respectively), with strong affinity towards the enzyme-substrate complex (uncompetitive inhibition). Fluorescence quenching suggested phenolic-enzyme interactions, which may occur at the PL-colipase complex interface, according to molecular docking. Interactions are likely between hydroxyl groups and polar amino acid residues. We conclude that TA and PGG, but not simple phenolic acids, are effective PL inhibitors, likely due to their numerous hydroxyl groups, which promote phenolic-enzyme interactions. Thus, their consumption may exert health benefits derived from their effects on this digestive enzyme.

Biochemical Characterization of a Novel α/ß-Hydrolase/FSH from the White Shrimp Litopenaeus vannamei.

(1) Background: Lipases and esterases are important enzymes that share the α/ß hydrolase fold. The activity and cellular localization are important characteristics to understand the role of such enzymes in an organism. (2) Methods: Bioinformatic and biochemical tools were used to describe a new α/ß hydrolase from a Litopenaeus vannamei transcriptome (LvFHS for Family Serine Hydrolase). (3) Results: The enzyme was obtained by heterologous overexpression in Escherichia coli and showed hydrolytic activity towards short-chain lipid substrates and high affinity to long-chain lipid substrates. Anti-LvFHS antibodies were produced in rabbit that immunodetected the LvFSH enzyme in several shrimp tissues. (4) Conclusions: The protein obtained and analyzed was an α/ß hydrolase with esterase and lipase-type activity towards long-chain substrates up to 12 carbons; its immunodetection in shrimp tissues suggests that it has an intracellular localization, and predicted roles in energy mobilization and signal transduction.

Role of an invariant lysine residue in folate binding on Escherichia coli thymidylate synthase: calorimetric and crystallographic analysis of the K48Q mutant.

Thymidylate synthase (TS) catalyzes the reductive methylation of deoxyuridine monophosphate (dUMP) using methylene tetrahydrofolate (CH(2)THF) as cofactor, the glutamate tail of which forms a water-mediated hydrogen bond with an invariant lysine residue of this enzyme. To understand the role of this interaction, we studied the K48Q mutant of Escherichia coli TS using structural and biophysical methods. The k(cat) of the K48Q mutant was 430-fold lower than wild-type TS in activity, while the K(m) for the (R)-stereoisomer of CH(2)THF was 300 microM, about 30-fold larger than K(m) from the wild-type TS. Affinity constants were determined using isothermal titration calorimetry, which showed that binding was reduced by one order of magnitude for folate-like TS inhibitors, such as propargyl-dideazafolate (PDDF) or compounds that distort the TS active site like BW1843U89 (U89). The crystal structure of the K48Q-dUMP complex revealed that dUMP binding is not impaired in the mutant, and that U89 in a ternary complex of K48Q-nucleotide-U89 was bound in the active site with subtle differences relative to comparable wild-type complexes. PDDF failed to form ternary complexes with K48Q and dUMP. Thermodynamic data correlated with the structural determinations, since PDDF binding was dominated by enthalpic effects while U89 had an important entropic component. In conclusion, K48 is critical for catalysis since it leads to a productive CH(2)THF binding, while mutation at this residue does not affect much the binding of inhibitors that do not make contact with this group.

A novel thymidylate synthase from the Vibrionales, Alteromonadales, Aeromonadales, and Pasteurellales (VAAP) clade with altered nucleotide and folate binding sites.

Thymidylate synthase (TS, E.C. 2.1.1.45) is a crucial enzyme for de novo deoxythymidine monophosphate (dTMP) biosynthesis. The gene for this enzyme is thyA, which encodes the folate-dependent TS that converts deoxyuridine monophosphate group (dUMP) into (dTMP) using the cofactor 5,10-methylenetetrahydrofolate (mTHF) as a carbon donor. We identified the thyA gene in the genome of the Vibrio parahaemolyticus strain FIM-S1708+ that is innocuous to humans but pathogenic to crustaceans. Surprisingly, we found changes in the residues that bind the substrate dUMP and mTHF, previously postulated as invariant among all TSs known (Finer-Moore, Santi & Stroud, 2003). Interestingly, those amino acid changes were also found in a clade of microorganisms that contains Vibrionales, Alteromonadales, Aeromonadales, and Pasteurellales (VAAP) from the Gammaproteobacteria class. In this work, we studied the biochemical properties of recombinant TS from V. parahemolyticus FIM-S1708+ (VpTS) to address the natural changes in the TS amino acid sequence of the VAAP clade. Interestingly, the Km for dUMP was 27.3 ± 4.3 µM, about one-fold larger compared to other TSs. The Km for mTHF was 96.3 ± 18 µM, about three- to five-fold larger compared to other species, suggesting also loss of affinity. Thus, the catalytic efficiency was between one or two orders of magnitude smaller for both substrates. We used trimethoprim, a common antibiotic that targets both TS and DHFR for inhibition studies. The IC50 values obtained were high compared to other results in the literature. Nonetheless, this molecule could be a lead for the design antibiotics towards pathogens from the VAAP clade. Overall, the experimental results also suggest that in the VAAP clade the nucleotide salvage pathway is important and should be investigated, since the de novo dTMP synthesis appears to be compromised by a less efficient thymidylate synthase.

De novo assembly and transcriptome characterization of the freshwater prawn Palaemonetes argentinus: Implications for a detoxification response.

Palaemonetes argentinus, an abundant freshwater prawn species in the northern and central region of Argentina, has been used as a bioindicator of environmental pollutants as it displays a very high sensitivity to pollutants exposure. Despite their extraordinary ecological relevance, a lack of genomic information has hindered a more thorough understanding of the molecular mechanisms potentially involved in detoxification processes of this species. Thus, transcriptomic profiling studies represent a promising approach to overcome the limitations imposed by the lack of extensive genomic resources for P. argentinus, and may improve the understanding of its physiological and molecular response triggered by pollutants. This work represents the first comprehensive transcriptome-based characterization of the non-model species P. argentinus to generate functional genomic annotations and provides valuable resources for future genetic studies. Trinity de novo assembly consisted of 24,738 transcripts with high representation of detoxification (phase I and II), anti-oxidation, osmoregulation pathways and DNA replication and bioenergetics. This crustacean transcriptome provides valuable molecular information about detoxification and biochemical processes that could be applied as biomarkers in further ecotoxicology studies.

Molecular characterization of arginine kinase, an allergen from the shrimp Litopenaeus vannamei.

BACKGROUND: Consumption of seafood can produce allergic symptoms in susceptible individuals and crustacean allergies are the most frequently reported causes of allergic reactions. METHODS: An allergen from the muscle of the white shrimp Litopenaeus vannamei was purified by ion exchange chromatography and identified by mass spectrometry of tryptic peptides and its specific enzymatic activity. Moreover, the corresponding full-length cDNA was obtained from an L. vannamei muscle cDNA library. RESULTS: A 40-kDa protein was purified and identified as arginine kinase and its cDNA of 1.4 kb encoded a 356 amino acid protein. The obtained arginine kinase was recognized by IgE in serum from shrimp-allergic individuals using ELISA and immunoblotting analysis. CONCLUSIONS: This is the first allergen reported for the Pacific white shrimp species; it was named Lit v 2 and has a 96% identity to Pen m 2 from Penaeus monodon.

The lysozyme from insect (Manduca sexta) is a cold-adapted enzyme.

Enzymatic activity is dependent on temperature, although some proteins have evolved to retain activity at low temperatures at the expense of stability. Cold adapted enzymes are present in a variety of organisms and there is ample interest in their structure-function relationships. Lysozyme (E.C. 3.2.1.17) is one of the most studied enzymes due to its antibacterial activity against Gram positive bacteria and is also a cold adapted protein. In this work the characterization of lysozyme from the insect Manduca sexta and its activity at low temperatures is presented. Both M. sexta lysozymes natural and recombinant showed a higher content of alpha-helix secondary structure compared to that of hen egg white lysozyme and a higher specific enzymatic activity in the range of 5-30 degrees C. These results together with measured thermodynamic activation parameters support the designation of M. sexta lysozyme as a cold adapted enzyme. Therefore, the insect recombinant lysozyme is feasible as a model for structure-function studies for cold-adapted proteins.

Biochemical and structural characterization of a novel arginine kinase from the spider Polybetes pythagoricus.

Energy buffering systems are key for homeostasis during variations in energy supply. Spiders are the most important predators for insects and therefore key in terrestrial ecosystems. From biomedical interest, spiders are important for their venoms and as a source of potent allergens, such as arginine kinase (AK, EC 2.7.3.3). AK is an enzyme crucial for energy metabolism, keeping the pool of phosphagens in invertebrates, and also an allergen for humans. In this work, we studied AK from the Argentininan spider Polybetes pythagoricus (PpAK), from its complementary DNA to the crystal structure. The PpAK cDNA from muscle was cloned, and it is comprised of 1068 nucleotides that encode a 384-amino acids protein, similar to other invertebrate AKs. The apparent Michaelis-Menten kinetic constant (Km ) was 1.7 mM with a kcat of 75 s-1. Two crystal structures are presented, the apoPvAK and PpAK bound to arginine, both in the open conformation with the active site lid (residues 310-320) completely disordered. The guanidino group binding site in the apo structure appears to be organized to accept the arginine substrate. Finally, these results contribute to knowledge of mechanistic details of the function of arginine kinase.