Human soluble CD39 displays substrate inhibition in a substrate-specific manner.

CD39 (ectonucleoside triphosphate diphosphohydrolase-1; ENTPD1) metabolizes extracellular ATP and ADP to AMP. AMP is subsequently metabolized by CD79 to adenosine. CD39 activity is therefore a key regulator of purinergic signalling in cancer, thrombosis, and autoimmune diseases. In this study we demonstrate that soluble, recombinant CD39 shows substrate inhibition with ADP or ATP as the substrate. Although CD39 activity initially increased with increasing substrate concentration, at high concentrations of ATP or ADP, CD39 activity was markedly reduced. Although the reaction product, AMP, inhibits CD39 activity, insufficient AMP was generated under our conditions to account for the substrate inhibition seen. In contrast, inhibition was not seen with UDP or UTP as substrates. 2-methylthio-ADP also showed no substrate inhibition, indicating the nucleotide base is an important determinant of substrate inhibition. Molecular dynamics simulations revealed that ADP can undergo conformational rearrangements within the CD39 active site that were not seen with UDP or 2-methylthio-ADP. Appreciating the existence of substrate inhibition of CD39 will help the interpretation of studies of CD39 activity, including investigations into drugs that modulate CD39 activity.

Time- and cost-efficient bacterial expression and purification of potato apyrase.

Apyrase from potato (Solanum tuberosum) is a divalent metal ion-dependent enzyme that catalyzes the hydrolysis of nucleoside di- and tri-phosphates with broad substrate specificity. The enzyme is widely used to manipulate nucleotide levels such as in the G protein-coupled receptor (GPCR) field where it is used to deplete guanine nucleotides to stabilize nucleotide-free ternary agonist-GPCR-G protein complexes. Potato apyrase is available commercially as the native enzyme purified from potatoes or as a recombinant protein, but these are prohibitively expensive for some research applications. Here, we report a relatively simple method for the bacterial production of soluble, active potato apyrase. Apyrase has several disulfide bonds, so we co-expressed the enzyme bearing a C-terminal (His)6 tag with the E. coli disulfide isomerase DsbC at low temperature (18 °C) in the oxidizing cytoplasm of E. coli Origami B (DE3). This allowed low level production of soluble apyrase. A two-step purification procedure involving Ni-affinity followed by Cibacron Blue-affinity chromatography yielded highly purified apyrase at a level of ∼0.5 mg per L of bacterial culture. The purified enzyme was functional for ATP hydrolysis in an ATPase assay and for GTP/GDP hydrolysis in a GPCR-G protein coupling assay. This methodology enables the time- and cost-efficient production of recombinant apyrase for various research applications.

Protein Corona Hinders N-CQDs Oxidative Potential and Favors Their Application as Nanobiocatalytic System.

The oxidative properties of nanomaterials arouse legitimate concerns about oxidative damage in biological systems. On the other hand, the undisputable benefits of nanomaterials promote them for biomedical applications; thus, the strategies to reduce oxidative potential are urgently needed. We aimed at analysis of nitrogen-containing carbon quantum dots (N-CQDs) in terms of their biocompatibility and internalization by different cells. Surprisingly, N-CQD uptake does not contribute to the increased oxidative stress inside cells and lacks cytotoxic influence even at high concentrations, primarily through protein corona formation. We proved experimentally that the protein coating effectively limits the oxidative capacity of N-CQDs. Thus, N-CQDs served as an immobilization support for three different enzymes with the potential to be used as therapeutics. Various kinetic parameters of immobilized enzymes were analyzed. Regardless of the enzyme structure and type of reaction catalyzed, adsorption on the nanocarrier resulted in increased catalytic efficiency. The enzymatic-protein-to-nanomaterial ratio is the pivotal factor determining the course of kinetic parameter changes that can be tailored for enzyme application. We conclude that the above properties of N-CQDs make them an ideal support for enzymatic drugs required for multiple biomedical applications, including personalized medical therapies.

Recent Advances Clarifying the Structure and Function of Plant Apyrases (Nucleoside Triphosphate Diphosphohydrolases).

Studies implicating an important role for apyrase (NTPDase) enzymes in plant growth and development began appearing in the literature more than three decades ago. After early studies primarily in potato, Arabidopsis and legumes, especially important discoveries that advanced an understanding of the biochemistry, structure and function of these enzymes have been published in the last half-dozen years, revealing that they carry out key functions in diverse other plants. These recent discoveries about plant apyrases include, among others, novel findings on its crystal structures, its biochemistry, its roles in plant stress responses and its induction of major changes in gene expression when its expression is suppressed or enhanced. This review will describe and discuss these recent advances and the major questions about plant apyrases that remain unanswered.

Potato apyrase reduces granulomatous area and increases presence of multinucleated giant cells in murine schistosomiasis.

Granulomas are inflammatory tissue responses directed to a set of antigens. Trapped Schistosoma mansoni eggs promote productive granulomas in the tissues, and they are the main damage caused by schistosomiasis. Some S. mansoni antigenic proteins may have a direct involvement in the resolution of the granulomatous response. The ATP diphosphohydrolases isoforms of this parasite are immunogenic, expressed in all phases of the parasite life cycle and secreted by eggs and adult worms. Potato apyrase is a vegetable protein that cross-reactive with parasite ATP diphosphohydrolases isoforms. In this study, the vegetable protein was purified, before being inoculated in C57BL/6 mice that were later infected with cercariae. Sixty days after infection, adult worms were recovered, antibodies and cytokines were measured, and morphological granuloma alterations evaluated. Immunization of the animals induced significant levels of IgG and IgG1 antibodies and IFN-γ, IL-10 and IL-5 cytokines, but not IL-13, suggesting that potato apyrase is an immunoregulatory protein. Supporting this hypothesis, it was found that liver damage associated with schistosomiasis was mitigated, reducing the size of the areas affected by granuloma to 35% and increasing the presence of multinucleated giant cells in this environment. In conclusion, potato apyrase was found to be effective immunomodulatory antigen for murine schistosomiasis.

Structural and functional characterization of engineered bifunctional fusion proteins of CD39 and CD73 ectonucleotidases.

Extracellular diphosphate and triphosphate nucleotides are released from activated or injured cells to trigger vascular and immune P2 purinergic receptors, provoking inflammation and vascular thrombosis. These metabokines are scavenged by ectonucleoside triphosphate diphosphohydrolase-1 (E-NTPDase1 or CD39). Further degradation of the monophosphate nucleoside end products occurs by surface ecto-5'-nucleotidase (NMPase) or CD73. These ectoenzymatic processes work in tandem to promote adenosinergic responses, which are immunosuppressive and antithrombotic. These homeostatic ectoenzymatic mechanisms are lost in the setting of oxidative stress, which exacerbates inflammatory processes. We have engineered bifunctional enzymes made up from ectodomains (ECDs) of CD39 and CD73 within a single polypeptide. Human alkaline phosphatase-ectodomain (ALP-ECD) and human acid phosphatase-ectodomain (HAP-ECD) fusion proteins were also generated, characterized, and compared with these CD39-ECD, CD73-ECD, and bifunctional fusion proteins. Through the application of colorimetrical functional assays and high-performance liquid chromatography kinetic assays, we demonstrate that the bifunctional ectoenzymes express high levels of CD39-like NTPDase activity and CD73-like NMPase activity. Chimeric CD39-CD73-ECD proteins were superior in converting triphosphate and diphosphate nucleotides into nucleosides when compared with ALP-ECD and HAP-ECD. We also note a pH sensitivity difference between the bifunctional fusion proteins and parental fusions, as well as ectoenzymatic property distinctions. Intriguingly, these innovative reagents decreased platelet activation to exogenous agonists in vitro. We propose that these chimeric fusion proteins could serve as therapeutic agents in inflammatory diseases, acting to scavenge proinflammatory ATP and also generate anti-inflammatory adenosine.

Targeting Metabolism of Extracellular Nucleotides via Inhibition of Ectonucleotidases CD73 and CD39.

In the tumor microenvironment, unusually high concentrations of extracellular adenosine promote tumor proliferation through various immunosuppressive mechanisms. Blocking adenosine production by inhibiting nucleotide-metabolizing enzymes, such as ectonucleotidases CD73 and CD39, represents a promising therapeutic strategy that may synergize with other immuno-oncology mechanisms and chemotherapies. Emerging small-molecule ectonucleotidase inhibitors have recently entered clinical trials. This Perspective will outline challenges, strategies, and recent advancements in targeting this class with small-molecule inhibitors, including AB680, the first small-molecule CD73 inhibitor to enter clinical development. Specific case studies, including structure-based drug design and lead optimization, will be outlined. Preclinical data on these molecules and their ability to enhance antitumor immunity will be discussed.

Screening of plant derived chalcones on the inhibition of potato apyrase: Potential protein biotechnological applications in health.

NTPDases (EC 3.6.1.5) are enzymes belonging to a protein family which have as a common feature the ability to hydrolyze di- and triphosphate nucleotides (ADP and ATP) to monophosphate nucleosides (AMP) in the presence of Ca+2 and Mg+. The potato apyrase has been the first protein of the NTPDase family to be purified. In mammals, these enzymes are involved in physiologic and sick processes as thromboregulation, inflammatory and immunologic responses. In this study, we investigated the in vitro potential of synthetic chalcones on the inhibition of potato apyrase purified from Solanum tuberosum. The protein was purified with high grade purity and its identity was confirmed by electrophoresis, western blot, and LC-MS/MS. Five out of the eight chemically synthetized chalcones analyzed in this study showed significant inhibition of the apyrase activity. The compound with the best rate of inhibition of ATP hydrolytic activity was able to promote 54% inhibition with a concentration of 3.125 µM. Ticlopidine, used as an inhibition drug control, was able to promote inhibitions around 50% of the activity (IC50 = 2.167 µM). Our results with the potato apyrase inhibition with the synthetic chalcones suggest that these compounds may use as potential lead candidates for the treatment of some diseases associated with nucleotides.

Ectonucleotidases in Blood Malignancies: A Tale of Surface Markers and Therapeutic Targets.

Leukemia develops as the result of intrinsic features of the transformed cell, such as gene mutations and derived oncogenic signaling, and extrinsic factors, such as a tumor-friendly, immunosuppressed microenvironment, predominantly in the lymph nodes and the bone marrow. There, high extracellular levels of nucleotides, mainly NAD+ and ATP, are catabolized by different ectonucleotidases, which can be divided in two families according to substrate specificity: on one side those that metabolize NAD+, including CD38, CD157, and CD203a; on the other, those that convert ATP, namely CD39 (and other ENTPDases) and CD73. They generate products that modulate intracellular calcium levels and that activate purinergic receptors. They can also converge on adenosine generation with profound effects, both on leukemic cells, enhancing chemoresistance and homing, and on non-malignant immune cells, polarizing them toward tolerance. This review will first provide an overview of ectonucleotidases expression within the immune system, in physiological and pathological conditions. We will then focus on different hematological malignancies, discussing their role as disease markers and possibly pathogenic agents. Lastly, we will describe current efforts aimed at therapeutic targeting of this family of enzymes.

Characterization of soluble CD39 (SolCD39/NTPDase1) from PiggyBac nonviral system as a tool to control the nucleotides level.

Extracellular ATP (eATP) and its metabolites have emerged as key modulators of different diseases and comprise a complex pathway called purinergic signaling. An increased number of tools have been developed to study the role of nucleotides and nucleosides in cell proliferation and migration, influence on the immune system and tumor progression. These tools include receptor agonists/antagonists, engineered ectonucleotidases, interference RNAs and ectonucleotidase inhibitors that allow the control and quantification of nucleotide levels. NTPDase1 (also called apyrase, ecto-ATPase and CD39) is one of the main enzymes responsible for the hydrolysis of eATP, and purified enzymes, such as apyrase purified from potato, or engineered as soluble CD39 (SolCD39), have been widely used in in vitro and in vivo experiments. However, the commercial apyrase had its effects recently questioned and SolCD39 exhibits limitations, such as short half-life and need of high doses to reach the expected enzymatic activity. Therefore, this study investigated a non-viral method to improve the overexpression of SolCD39 and evaluated its impact on other enzymes of the purinergic system. Our data demonstrated that PiggyBac transposon system proved to be a fast and efficient method to generate cells stably expressing SolCD39, producing high amounts of the enzyme from a limited number of cells and with high hydrolytic activity. In addition, the soluble form of NTPDase1/CD39 did not alter the expression or catalytic activity of other enzymes from the purinergic system. Altogether, these findings set the groundwork for prospective studies on the function and therapeutic role of eATP and its metabolites in physiological and pathological conditions.

Kinetic study of NTPDase immobilization and its effect of haemocompatibility on polyethylene terephthalate.

Poor haemocompatibility of material surfaces is a serious limitation that can lead to failure of blood-contacting devices and implants. In this work, we have improved the haemocompatibility of polyethylene terephthalate (PET) surfaces by immobilizing apyrase/ecto-nucleoside triphosphate diphosphohydrolase (NTPDase) on to the carboxylated PET. NTPDase immobilized PET surfaces scavenge the ADP released by activated platelets, which prevents further platelet activation and aggregation. The surface properties of the modified PET were characterized by scanning electron microscope (SEM), energy dispersive X-ray spectroscopy (EDAX), and contact angle measurement. The enzyme attachment and stability on the modified PET surfaces were evaluated. The kinetics of free enzyme and immobilized enzyme were studied and fitted using the Michaelis-Menten kinetic model. Both free and immobilized NTPDase followed Michaelis-Menten kinetics with similar Michaelis-Menten constants (Km). This suggests that the activity of NTPDase was unchanged upon immobilization. Protein adsorption and %hemolysis was significantly reduced for carboxylated PET and NTPDase immobilized PET surfaces compared to unmodified PET. Lactate dehydrogenase assay showed that the number of adhered platelets reduced by more than an order of magnitude for the NTPDase immobilized PET surface compared to unmodified PET. These results clearly indicate that NTPDase immobilization significantly enhances the haemocompatibility of PET surfaces.

Schiff bases of tryptamine as potent inhibitors of nucleoside triphosphate diphosphohydrolases (NTPDases): Structure-activity relationship.

Overexpression of NTPDases leads to a number of pathological situations such as thrombosis, and cancer. Thus, effective inhibitors are required to combat these pathological situations. Different classes of NTPDase inhibitors are reported so far including nucleotides and their derivatives, sulfonated dyes such as reactive blue 2, suramin and its derivatives, and polyoxomatalates (POMs). Suramin is a well-known and potent NTPDase inhibitor, nonetheless, a range of side effects are also associated with it. Reactive blue 2 also had non-specific side effects that become apparent at high concentrations. In addition, most of the NTPDase inhibitors are high molecular weight compounds, always required tedious chemical steps to synthesize. Hence, there is still need to explore novel, low molecular weight, easy to synthesize, and potent NTPDase inhibitors. Keeping in mind the known NTPDase inhibitors with imine functionality and nitrogen heterocycles, Schiff bases of tryptamine, 1-26, were synthesized and characterized by spectroscopic techniques such as EI-MS, HREI-MS, 1H-, and 13C NMR. All the synthetic compounds were evaluated for the inhibitory avidity against activities of three major isoforms of NTPDases: NTPDase-1, NTPDase-3, and NTPDase-8. Cumulatively, eighteen compounds were found to show potent inhibition (Ki = 0.0200-0.350 µM) of NTPDase-1, twelve (Ki = 0.071-1.060 µM) of NTPDase-3, and fifteen compounds inhibited (Ki = 0.0700-4.03 µM) NTPDase-8 activity. As a comparison, the Kis of the standard inhibitor suramin were 1.260 ±â€¯0.007, 6.39 ±â€¯0.89 and 1.180 ±â€¯0.002 µM, respectively. Kinetic studies were performed on lead compounds (6, 5, and 21) with human (h-) NTPDase-1, -3, and -8, and Lineweaver-Burk plot analysis showed that they were all competitive inhibitors. In silico study was conducted on compound 6 that showed the highest level of inhibition of NTPDase-1 to understand the binding mode in the active site of the enzyme.

Development of a selective and highly sensitive fluorescence assay for nucleoside triphosphate diphosphohydrolase1 (NTPDase1, CD39).

Ecto-nucleoside triphosphate diphosphohydrolase1 (NTPDase1, CD39) is a major ectonucleotidase that hydrolyzes proinflammatory ATP via ADP to AMP, which is subsequently converted by ecto-5'-nucleotidase (CD73) to immunosuppressive adenosine. Activation of CD39 has potential for treating inflammatory diseases, while inhibition was suggested as a novel strategy for the immunotherapy of cancer. In the present study, we developed a selective and highly sensitive capillary electrophoresis (CE) assay using a novel fluorescent CD39 substrate, a fluorescein-labelled ATP (PSB-170621A) that is converted to its AMP derivative. To accelerate the assays, a two-directional (forward and reverse) CE system was implemented using 96-well plates, which is suitable for the screening of compound libraries (Z'-factor: 0.7). The detection limits for the forward and reverse operation were 11.7 and 2.00 pM, respectively, indicating a large enhancement in sensitivity as compared to previous methods (e.g. malachite-green assay: 1 000 000-fold, CE-UV assay: 500 000-fold, fluorescence polarization immunoassay: 12 500-fold). Enzyme kinetic studies at human CD39 revealed a Km value of 19.6 µM, and a kcat value of 119 × 10-3 s-1 for PSB-170621A, which shows similar substrate properties as ATP (11.4 µM and 82.5 × 10-3 s-1). The compound displayed similar properties at rat and mouse CD39. Subsequent docking studies into a homology model of human CD39 revealed a hydrophobic pocket that accommodates the fluorescein tag. PSB-170621A was found to be preferably hydrolyzed by CD39 as compared to other ectonucleotidases. The new assay was validated by performing inhibition assays with several standard CD39 inhibitors yielding results that were consonant with data using the natural substrates.

Cationic Europium Complexes for Visualizing Fluctuations in Mitochondrial ATP Levels in Living Cells.

The ability to study cellular metabolism and enzymatic processes involving adenosine triphosphate (ATP) is impeded by the lack of imaging probes capable of signalling the concentration and distribution of intracellular ATP rapidly, with high sensitivity. We report here the first example of a luminescent lanthanide complex capable of visualizing changes in the concentration of ATP in the mitochondria of living cells. Four cationic europium(III) complexes [Eu.1-4]+ have been synthesized and their binding capabilities towards nucleoside polyphosphate anions examined in aqueous solution at physiological pH. Complexes [Eu.1]+ and [Eu.3]+ bearing hydrogen bond donor groups in the pendant arms showed excellent discrimination between ATP, ADP and monophosphate species. Complex [Eu.3]+ showed relatively strong binding to ATP (logKa =5.8), providing a rapid, long-lived luminescent signal that enabled its detection in a highly competitive aqueous medium containing biologically relevant concentrations of Mg2+ , ADP, GTP, UTP and human serum albumin. This EuIII complex responds linearly to ATP within the physiological concentration range (1-5 mm), and was used to continuously monitor the apyrase-catalyzed hydrolysis of ATP to ADP in vitro. We demonstrate that [Eu.3]+ can permeate mammalian (NIH-3T3) cells efficiently and localize to the mitochondria selectively, permitting real-time visualization of elevated mitochondrial ATP levels following treatment with a broad spectrum kinase inhibitor, staurosporine, as well as depleted ATP levels upon treatment with potassium cyanide under glucose starvation conditions.

NTPDase activities: possible roles on Leishmania spp infectivity and virulence.

Nucleoside triphosphate diphosphohydrolases (NTPDases) are enzymes that belong to the GDA1/CD39 protein superfamily. These enzymes catalyze the hydrolysis of ATP and ADP to the monophosphate form (AMP). Biochemical characterization of the nucleotidases/NTPDases from various types of cells, including those from plants, animals, and pathogenic organisms, has revealed the existence of several isoforms with different specificities with respect to divalent cations (magnesium, calcium, manganese, and zinc) and substrates. In mammals, the NTPDases play important roles in the regulation of thrombosis and inflammation. In parasites of the genus Leishmania, the causative agents of leishmaniasis, two NTPDase isoforms, termed NTPDase-1 and NTPDase-2 have been described. Independently of their cellular localization, whether cell-surface localized, secreted or targeted to other organelles, in some Leishmania species these NTPDases could be involved in parasite growth, infectivity, and virulence. Experimental evidence has suggested that the hydrolysis of ATP and ADP by parasite ecto-nucleotidases can down-modulate the host immune response. In this context, the present work provides an overview of recent works that show strong evidence not only of the involvement of the nucleotidases/NTPDases in Leishmania spp infectivity and virulence but also of the molecular mechanisms that lead to the success of the parasitic infection.

Molecular dynamic simulations reveal structural insights into substrate and inhibitor binding modes and functionality of Ecto-Nucleoside Triphosphate Diphosphohydrolases.

Ecto-nucleotidase enzymes catalyze the hydrolysis of extracellular nucleotides to their respective nucleosides. Herein, we place the focus on the elucidation of structural features of the cell surface located ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDase1-3 and 8). The physiological role of these isozymes is crucially important as they control purinergic signaling by modulating the extracellular availability of nucleotides. Since, crystal or NMR structure of the human isozymes are not available - structures have been obtained by homology modeling. Refinement of the homology models with poor stereo-chemical quality is of utmost importance in order to derive reliable structures for subsequent studies. Therefore, the resultant models obtained by homology modelling were refined by running molecular dynamic simulation. Binding mode analysis of standard substrates and of competitive inhibitor was conducted to highlight important regions of the active site involved in hydrolysis of the substrates and possible mechanism of inhibition.

A bio-image sensor for simultaneous detection of multi-neurotransmitters.

We report here a new bio-image sensor for simultaneous detection of spatial and temporal distribution of multi-neurotransmitters. It consists of multiple enzyme-immobilized membranes on a 128 × 128 pixel array with read-out circuit. Apyrase and acetylcholinesterase (AChE), as selective elements, are used to recognize adenosine 5'-triphosphate (ATP) and acetylcholine (ACh), respectively. To enhance the spatial resolution, hydrogen ion (H+) diffusion barrier layers are deposited on top of the bio-image sensor and demonstrated their prevention capability. The results are used to design the space among enzyme-immobilized pixels and the null H+ sensor to minimize the undesired signal overlap by H+ diffusion. Using this bio-image sensor, we can obtain H+ diffusion-independent imaging of concentration gradients of ATP and ACh in real-time. The sensing characteristics, such as sensitivity and detection of limit, are determined experimentally. With the proposed bio-image sensor the possibility exists for customizable monitoring of the activities of various neurochemicals by using different kinds of proton-consuming or generating enzymes.

First report of the characterization of a snake venom apyrase (Ruviapyrase) from Indian Russell's viper (Daboia russelii) venom.

A novel apyrase from Russell's viper venom (RVV) was purified and characterized, and it was named Ruviapyrase (Russell's viper apyrase). It is a high molecular weight (79.4 kDa) monomeric glycoprotein that contains 2.4% neutral sugars and 58.4% N-linked oligosaccharides and strongly binds to Concanavalin A. The LC-MS/MS analysis did not identify any protein in NCBI protein database, nevertheless some de novo sequences of Ruviapyrase showed putative conserved domain of apyrase superfamily. Ruviapyrase hydrolysed adenosine triphosphate (ATP) to a significantly greater extent (p < .05) as compared to adenosine diphosphate (ADP); however, it was devoid of 5'-nucleotidase and phosphodiesterase activities. The Km and Vmax values for Ruviapyrase towards ATP were 2.54 µM and 615 µM of Pi released min-1, respectively with a turnover number (Kcat) of 24,600 min-1. Spectrofluorometric analysis demonstrated interaction of Ruviapyrase with ATP and ADP at Kd values of 0.92 nM and 1.25 nM, respectively. Ruviapyrase did not show cytotoxicity against breast cancer (MCF-7) cells and haemolytic activity, it exhibited marginal anticoagulant and strong antiplatelet activity, and dose-dependently reversed the ADP-induced platelet aggregation. The catalytic activity and platelet deaggregation property of Ruviapyrase was significantly inhibited by EDTA, DTT and IAA, and neutralized by commercial monovalent and polyvalent antivenom.

Structures and kinetics for plant nucleoside triphosphate diphosphohydrolases support a domain motion catalytic mechanism.

Extracellular nucleoside triphosphate diphosphohydrolases (NTPDases) are enzymes that hydrolyze extracellular nucleotides to the respective monophosphate nucleotides. In the past 20 years, NTPDases belonging to mammalian, parasitic and prokaryotic domains of life have been discovered, cloned and characterized. We reveal the first structures of NTPDases from the legume plant species Trifolium repens (7WC) and Vigna unguiculata subsp. cylindrica (DbLNP). Four crystal structures of 7WC and DbLNP were determined at resolutions between 1.9 and 2.6 Å. For 7WC, structures were determined for an -apo form (1.89 Å) and with the product AMP (2.15 Å) and adenine and phosphate (1.76 Å) bound. For DbLNP, a structure was solved with phosphate and manganese bound (2.60 Å). Thorough kinetic data and analysis is presented. The structure of 7WC and DbLNP reveals that these NTPDases can adopt two conformations depending on the molecule and co-factor bound in the active site. A central hinge region creates a "butterfly-like" motion of the domains that reduces the width of the inter-domain active site cleft upon molecule binding. This phenomenon has been previously described in Rattus norvegicus and Legionella pneumophila NTPDaseI and Toxoplasma gondii NTPDaseIII suggesting a common catalytic mechanism across the domains of life.

Structural and functional characterization of a cold adapted TPM-domain with ATPase/ADPase activity.

The Pfam PF04536 TPM_phosphatase family is a broadly conserved family of domains found across prokaryotes, plants and invertebrates. Despite having a similar protein fold, members of this family have been implicated in diverse cellular processes and found in varied subcellular localizations. Very recently, the biochemical characterization of two evolutionary divergent TPM domains has shown that they are able to hydrolyze phosphate groups from different substrates. However, there are still incorrect functional annotations and uncertain relationships between the structure and function of this family of domains. BA41 is an uncharacterized single-pass transmembrane protein from the Antarctic psychrotolerant bacterium Bizionia argentinensis with a predicted compact extracytoplasmic TPM domain and a C-terminal cytoplasmic low complexity region. To shed light on the structural properties that enable TPM domains to adopt divergent roles, we here accomplish a comprehensive structural and functional characterization of the central TPM domain of BA41 (BA41-TPM). Contrary to its predicted function as a beta-propeller methanol dehydrogenase, light scattering and crystallographic studies showed that BA41-TPM behaves as a globular monomeric protein and adopts a conserved Rossmann fold, typically observed in other TPM domain structures. Although the crystal structure reveals the conservation of residues involved in substrate binding, no putative catalytic or intramolecular metal ions were detected. Most important, however, extensive biochemical studies demonstrated that BA41-TPM has hydrolase activity against ADP, ATP, and other di- and triphosphate nucleotides and shares properties of cold-adapted enzymes. The role of BA41 in extracellular ATP-mediated signaling pathways and its occurrence in environmental and pathogenic microorganisms is discussed.