Nucleoside Triphosphate Pyrophosphohydrolase Research Articles

Structural analysis of the housecleaning nucleoside triphosphate pyrophosphohydrolase MazG from Mycobacterium tuberculosis.

The housecleaning enzyme of Mycobacterium tuberculosis (Mtb), MazG, is a nucleoside triphosphate pyrophosphohydrolase (NTP-PPase) and can hydrolyze all canonical or non-canonical NTPs into NMPs and pyrophosphate. The Mycobacterium tuberculosis MazG (Mtb-MazG) contributes to antibiotic resistance in response to oxidative or nitrosative stress under dormancy, making it a promising target for treating TB in latent infection patients. However, the structural basis of Mtb-MazG is not clear. Here we describe the crystal structure of Mtb-MazG (1-185) at 2.7 Å resolution, composed of two similar folded spherical domains in tandem. Unlike other all-α NTP pyrophosphatases, Mtb-MazG has an N-terminal extra region composed of three α-helices and five β-strands. The second domain is global, with five α-helices located in the N-terminal domain. Gel-filtration assay and SAXS analysis show that Mtb-MazG forms an enzyme-active dimer in solution. In addition, the metal ion Mg2+ is bound with four negative-charged residues Glu119, Glu122, Glu138, and Asp141. Different truncations and site-directed mutagenesis revealed that the full-length dimeric form and the metal ion Mg2+ are indispensable for the catalytic activity of Mtb-MazG. Thus, our work provides new insights into understanding the molecular basis of Mtb-MazG.

A Novel Broad Host Range Phage Infecting Alteromonas.

Bacteriophages substantially contribute to bacterial mortality in the ocean and play critical roles in global biogeochemical processes. Alteromonas is a ubiquitous bacterial genus in global tropical and temperate waters, which can cross-protect marine cyanobacteria and thus has important ecological benefits. However, little is known about the biological and ecological features of Alteromonas phages (alterophages). Here, we describe a novel alterophage vB_AmeP-R8W (R8W), which belongs to the Autographiviridae family and infects the deep-clade Alteromonas mediterranea. R8W has an equidistant and icosahedral head (65 ± 1 nm in diameter) and a short tail (12 ± 2 nm in length). The genome size of R8W is 48,825 bp, with a G + C content of 40.55%. R8W possesses three putative auxiliary metabolic genes encoding proteins involved in nucleotide metabolism and DNA binding: thymidylate synthase, nucleoside triphosphate pyrophosphohydrolase, and PhoB. R8W has a rapid lytic cycle with a burst size of 88 plaque-forming units/cell. Notably, R8W has a wide host range, such that it can infect 35 Alteromonas strains; it exhibits a strong specificity for strains isolated from deep waters. R8W has two specific receptor binding proteins and a compatible holin–endolysin system, which contribute to its wide host range. The isolation of R8W will contribute to the understanding of alterophage evolution, as well as the phage–host interactions and ecological importance of alterophages.

Comparative Analysis of the Global Transcriptomic Response to Oxidative Stress of Bacillus anthracis htrA-Disrupted and Parental Wild Type Strains.

We previously demonstrated that the HtrA (High Temperature Requirement A) protease/chaperone active in the quality control of protein synthesis, represents an important virulence determinant of Bacillus anthracis. Virulence attenuation of htrA-disrupted Bacillus anthracis strains was attributed to susceptibility of ΔhtrA strains to stress insults, as evidenced by affected growth under various stress conditions. Here, we report a comparative RNA-seq transcriptomic study generating a database of differentially expressed genes in the B. anthracis htrA-disrupted and wild type parental strains under oxidative stress. The study demonstrates that, apart from protease and chaperone activities, HtrA exerts a regulatory role influencing expression of more than 1000 genes under stress. Functional analysis of groups or individual genes exhibiting strain-specific modulation, evidenced (i) massive downregulation in the ΔhtrA and upregulation in the WT strains of various transcriptional regulators, (ii) downregulation of translation processes in the WT strain, and (iii) downregulation of metal ion binding functions and upregulation of sporulation-associated functions in the ΔhtrA strain. These modulated functions are extensively discussed. Fifteen genes uniquely upregulated in the wild type strain were further interrogated for their modulation in response to other stress regimens. Overexpression of one of these genes, encoding for MazG (a nucleoside triphosphate pyrophosphohydrolase involved in various stress responses in other bacteria), in the ΔhtrA strain resulted in partial alleviation of the H2O2-sensitive phenotype.

Antagonistic interactions between osterix and pyrophosphate during cementum formation.

During cementum formation, the key roles of osterix (Osx) and inorganic pyrophosphate (PPi), mainly controlled by nucleotide pyrophosphatase 1 (Npp1; encoded by the Enpp1 gene) and progressive ankylosis protein (Ank), have been demonstrated by animal models displaying altered cementum formation. In this study, we analyzed the relationship of Osx and local PPi during cementum formation using compound mutant mice with their wildtype and corresponding single gene mutants. Importantly, functional defects in PPi regulation led to the induction of Osx expression at the cervical cementum as demonstrated by Enpp1 mutant mice and cementoblasts with the retroviral transduction of small hairpin RNA for Enpp1 or Ank. Conversely, cementoblasts exposed to inorganic PPi or with the enforced expression of Enpp1 or Ank reduced Osx expression in a concentration-dependent manner. Furthermore, the loss of Osx induced the higher expression of Npp1 and Ank at the apical region of the developing tooth root as observed in Osx-deficient mice. The activity of PPi-generating ectoenzymes (nucleoside triphosphate pyrophosphohydrolase, NTPPPHase) and the level of extracellular PPi were significantly increased in Osx-knockdown cementoblasts. However, the formation of ectopic cervical cementum was not completely diminished by inactivation of Osx in Enpp1 mutant mice. In addition, fibroblast growth factor (FGF) receptor 1 (Fgfr1) was strongly localized in cementoblasts lining the acellular cementum and involved in the inhibitory regulation of matrix accumulation and further mineralization by supporting PPi production. Taken together, these results suggest that local PPi suppresses matrix accumulation and further mineralization through an antagonistic interaction with Osx under the synergistic influence of FGF signaling during cementum formation.

Effect of Substitution Pro32Thr on the Interaction between Dimer Subunits of Human Phosphatase ITPA

Background: Cells have specific enzymes (nucleoside triphosphate pyrophosphohydrolase) that hydrolyze non-canonic nucleoside triphosphates into nucleoside monophosphophates and pyrophosphate, thus removing them from the metabolic processes. This class of enzymes includes inosine triphosphate pyrophosphatase (ITPA) which has specificity to ITP, dITP, XTP and dXTP. Objective: The mutation (94C→A) rather often occurs in humans and can affect the sensitivity of patients to medicines. This mutation leads to a Pro32Thr substitution in the human ITPA protein. The mechanism for the inactivating effect of the mutation is unknown yet. Methods: Molecular modeling of the polymorphic form of inosine triphosphate pyrophosphohydrolase Р32Т-hITPA showing the greatest decrease in the enzyme activity is performed. The analysis is given for four dimer variants: wild-type (P32/P32) and mutant (T32/T32) homodimers and two mutant heterodimers (Р32/Т32 and Т32/Р32). Results: The analysis does not show the motion of the loop between α2 and β2 where mutation localized. Thus, the hypothesis of the flipped-out hydrophobic residue and subsequent of protein degradation have not been confirmed. Dimer displacements were much higher than subunit displacements. The analysis of hydrogen bonds between subunits shows that there are the more stable hydrogen bonds in the wild-type homodimer and fewer in the mutant homodimer, while heterodimers have intermediate stability. Conclusion: The results confirm the assumption of possible weakening of bonds between the mutant subunits

Crystal structure of the Bacillus-conserved MazG protein, a nucleotide pyrophosphohydrolase.

BA1544 from Bacillus anthracis was previously annotated as a transcription factor for the gene cluster ba1554 - ba1558, but has not been experimentally characterized. B. anthracis is an obligate pathogen causing fatal inhalational anthrax, and BA1544 is absolutely conserved in Bacillus species, including Bacillus cereus, Bacillus thuringiensis and Bacillus mycoides, with 100% sequence identity. To address the function of BA1544, we performed structural and biochemical studies, which revealed that BA1544 is a MazG protein. Thus, herein, the protein is defined as Bacillus-conserved MazG (BcMazG). Like other MazG structures, BcMazG assembles into a tetrameric architecture. Each monomer adopts a four-α-helix bundle that accommodates a metal ion using four acidic residues, and presents one putative substrate-binding site. Enzymatic characterization demonstrated that BcMazG is a nucleoside triphosphate (NTP) pyrophosphohydrolase and prefers adenosine triphosphate as a substrate among canonical NTPs. Moreover, structural comparison of BcMazG with its homologues revealed a potential regulation mechanism whereby the enzymatic activity of BcMazG is regulated by its C-terminal region.

Crystal structure of the MazG-related nucleoside triphosphate pyrophosphohydrolase from Thermotoga maritima MSB8.

The MazG family proteins, which are highly conserved in bacteria, are nucleoside triphosphate pyrophosphohydrolases that hydrolyze all canonical nucleoside triphosphates, and are also involved in removing noncanonical nucleoside triphosphates to prevent their incorporation into DNA or RNA. The primary structure of TM0360 from Thermotoga maritima MSB8 suggested that TM0360 is a MazG-related nucleoside triphosphate pyrophosphohydrolase. The crystal structure of the TM0360 protein was determined by the MAD technique at 2.0Å resolution. The asymmetric unit contains an intact dimer molecule. The overall structure of TM0360 is similar to the known structures of the dimeric MazG protein and dUTPases. The putative NTP binding pocket in TM0360, identified by considering the probable NTP-interacting residues and structural features, suggested that TM0360 resembles the C-terminal domain of Escherichia coli MazG, although TM0360 may be a truncated paralog of the N-terminal domain of T. maritima MazG (TM0913), according to its primary structure. The putative function of TM0360 is discussed, based on structural homology.

Genomic Analysis of Xanthomonas translucens Pathogenic on Wheat and Barley Reveals Cross-Kingdom Gene Transfer Events and Diverse Protein Delivery Systems

In comparison to dicot-infecting bacteria, only limited numbers of genome sequences are available for monocot-infecting and in particular cereal-infecting bacteria. Herein we report the characterisation and genome sequence of Xanthomonas translucens isolate DAR61454 pathogenic on wheat and barley. Based on phylogenetic analysis of the ATP synthase beta subunit (atpD) gene, DAR61454 is most closely related to other X. translucens strains and the sugarcane- and banana- infecting Xanthomonas strains, but shares a type III secretion system (T3SS) with X. translucens pv. graminis and more distantly related xanthomonads. Assays with an adenylate cyclase reporter protein demonstrate that DAR61454's T3SS is functional in delivering proteins to wheat cells. X. translucens DAR61454 also encodes two type VI secretion systems with one most closely related to those found in some strains of the rice infecting strain X. oryzae pv. oryzae but not other xanthomonads. Comparative analysis of 18 different Xanthomonas isolates revealed 84 proteins unique to cereal (i.e. rice) infecting isolates and the wheat/barley infecting DAR61454. Genes encoding 60 of these proteins are found in gene clusters in the X. translucens DAR61454 genome, suggesting cereal-specific pathogenicity islands. However, none of the cereal pathogen specific proteins were homologous to known Xanthomonas spp. effectors. Comparative analysis outside of the bacterial kingdom revealed a nucleoside triphosphate pyrophosphohydrolase encoding gene in DAR61454 also present in other bacteria as well as a number of pathogenic Fusarium species, suggesting that this gene may have been transmitted horizontally from bacteria to the Fusarium lineage of pathogenic fungi. This example further highlights the importance of horizontal gene acquisition from bacteria in the evolution of fungi.

DCTP pyrophosphohydrase exhibits nucleic accumulation in multiple carcinomas

Nucleoside triphosphate pyrophosphohydrolase (NTP-PPase) functions as one of the mechanisms to guarantee the fidelity of DNA replication through the cleavage of non-canonical nucleotides into di- or monophosphates. Human NTP-PPase is poorly understood and investigated. In the present study, by using tissue microarrays with the paired cancer and adjacent regions, we found that with the prevalent expression of dCTP pyrophosphohydrase (DCTPP1) in the cytosol and nucleus in tumors investigated, DCTPP1 was inclined to accumulate in the nucleus of cancer cells compared to the paired adjacent tissue cells in multiple carcinomas including lung, breast, liver, cervical, gastric and esophagus cancer. More significantly, the higher DCTPP1 expression in the nucleus of lung, gastric and esophagus cancer cells was associated with histological subtypes. The nucleic accumulation of DCTPP1 was apparently observed as well when tumor cell line MCF-7 was treated with H2O2 in vitro. Considering the roles of DCTPP1 on restricting the concentration of non-canonical nucleotides in the nucleotide pool, accumulation of DCTPP1 in the nucleus of tumor cells might suffice for maintaining the proper DNA replication in order to fulfill the requirement for the survival and proliferation of tumor cells.

Thrombin and plasmin induced proteoglycan release in human cartilage is par-dependent

Thrombin and plasmin induced proteoglycan release in human cartilage is par-dependent

Ectonucleotide Pyrophosphatase/Phosphodiesterase-1 (ENPP1) Protein Regulates Osteoblast Differentiation

ENPP1 (ectonucleotide pyrophosphatase/phosphodiesterase-1) is an established regulator of tissue mineralization. Previous studies demonstrated that ENPP1 is expressed in differentiated osteoblasts and that ENPP1 influences matrix mineralization by increasing extracellular levels of inorganic pyrophosphate. ENPP1 is also expressed in osteoblastic precursor cells when stimulated with FGF2, but the role of ENPP1 in preosteoblastic and other precursor cells is unknown. Here we investigate the function of ENPP1 in preosteoblasts. We find that ENPP1 expression is critical for osteoblastic differentiation and that this effect is not mediated by changes in extracellular concentration levels of phosphate or pyrophosphate or ENPP1 catalytic activity. MC3T3E1(C4) preosteoblastic cells, in which ENPP1 expression was suppressed by ENPP1-specific shRNA, and calvarial cells isolated from Enpp1 knock-out mice show defective osteoblastic differentiation upon stimulation with ascorbate, as indicated by a lack of cellular morphological change, a lack of osteoblast marker gene expression, and an inability to mineralize matrix. Additionally, MC3T3E1(C4) cells, in which wild type or catalytic inactive ENPP1 expression was increased, exhibited an increased tendency to differentiate, as evidenced by increased osteoblast marker gene expression and increased mineralization. Notably, treatment of cells with inorganic phosphate or pyrophosphate inhibited, as opposed to enhanced, expression of multiple genes that are expressed in association with osteoblast differentiation, matrix deposition, and mineralization. Our results indicate that ENPP1 plays multiple and distinct roles in the development of mineralized tissues and that the influence of ENPP1 on osteoblast differentiation and gene expression may include a mechanism that is independent of its catalytic activity.

Cell response of Escherichia coli to cisplatin‐induced stress

Cisplatin is undoubtedly one of the most common and successful anticancer drugs worldwide. Though its DNA-based mechanism of action is well established, the contribution of the proteome to this process remains unclear. The possible impact of particular Escherichia coli proteins on the cytostatic activity of cisplatin was the subject of this study. Our main focus was not only the "bottom-up" identification of novel cisplatin protein targets through LC/LC-MS/MS analysis, but also a label-free quantification of their regulation profile by spectral-counting. The regulation of two proteins, aconitate hydratase 2 and 60 kDa chaperonin 1, could be linked to a platinated amino acid in the protein sequence, whereas in the cases of 30S ribosomal protein S1 and enolase, it could be shown that cisplatin fragments are coordinated to an essential site for the functionality of the protein. Nucleoside triphosphate pyrophosphohydrolase (MazG) regulates the programmed cell death and was found to be platinated on the protein surface, which probably correlates with the established mode of action. A possible new chapter in the understanding of cisplatin's mechanism of action and its severe side effects is opened, since evidence is provided that platinated proteins are not only involved in cellular stress response but also in energy metabolism through glycolysis and catabolic processes, in gene regulatory mechanisms and protein synthesis.

Structural and Functional Insights into DR2231 Protein, the MazG-like Nucleoside Triphosphate Pyrophosphohydrolase from Deinococcus radiodurans

Deinococcus radiodurans is among the very few bacterial species extremely resistant to ionizing radiation, UV light, oxidizing agents, and cycles of prolonged desiccation. The proteome of D. radiodurans reflects the evolutionary pressure exerted by chronic exposure to (nonradioactive) forms of DNA and protein damage. A clear example of this adaptation is the overrepresentation of protein families involved in the removal of non-canonical nucleoside triphosphates (NTPs) whose incorporation into nascent DNA would promote mutagenesis and DNA damage. The three-dimensional structure of the DR2231 protein has been solved at 1.80 Å resolution. This protein had been classified as an all-α-helical MazG-like protein. The present study confirms that it holds the basic structural module characteristic of the MazG superfamily; two helices form a rigid domain, and two helices form a mobile domain and connecting loops. Contrary to what is known of MazG proteins, DR2231 protein shows a functional affinity with dUTPases. Enzymatic and isothermal calorimetry assays have demonstrated high specificity toward dUTP but an inability to hydrolyze dTTP, a typical feature of dUTPases. Co-crystallization with the product of hydrolysis, dUMP, in the presence of magnesium or manganese cations, suggests similarities with the dUTP/dUDP hydrolysis mechanism reported for dimeric dUTPases. The genome of D. radiodurans encodes for all enzymes required for dTTP synthesis from dCMP, thus bypassing the need of a dUTPase. We postulate that DR2231 protein is not essential to D. radiodurans and rather performs "house-cleaning" functions within the framework of oxidative stress response. We further propose DR2231 protein as an evolutionary precursor of dimeric dUTPases.

Mycobacterial MazG Is a Novel NTP Pyrophosphohydrolase Involved in Oxidative Stress Response

MazG nucleoside triphosphate pyrophosphohydrolase (NTP-PPase, EC 3.6.1.8) from the avirulent Mycobacterium tuberculosis H37Ra contains a spontaneous mutation on a highly conserved residue, resulting in an A219E substitution (MtMazG[A219E]). In this work, we show that mycobacterial MazG from either the virulent M. tuberculosis H37Rv (MtMazG) or the fast-growing Mycobacterium smegmatis (MsMazG) is a potent NTP-PPase capable of hydrolyzing all canonical (d)NTPs, as well as the mutagenic dUTP and 8-oxo-7,8-dihydro-2'-dGTP. However, this hydrolysis activity is diminished by the MtMazG[A219E] mutation. Moreover, deletion of mazG in M. smegmatis rendered the mycobacteria defective in response to oxidative stress. Importantly, expression of the wild-type MtMazG, but not the A219E mutant, restored cell viability under oxidative stress. Intriguingly, under oxidative stress, both the mazG-null and MtMazG[A219E]-expressing M. smegmatis strains failed to elevate relA, while retaining their ability to up-regulate sigE, suggesting a specific role for the MazG NTP-PPase activity in oxidative stress-triggered, transcriptional activation of relA. The MtMazG is a homotetramer with each subunit containing a single MazG core domain flanked by two regions, both of which are essential for NTP-PPase activity. Taken together, these results demonstrate that the mycobacterial MazG is a potent NTP-PPase and that this activity is required to maintain the full capacity of the mycobacteria to respond to oxidative stress. Our work implicates a role for the MazG activity in the virulence of M. tuberculosis.

Dexamethasone Promotes Calcium Pyrophosphate Dihydrate Crystal Formation by Articular Chondrocytes

Calcium pyrophosphate dihydrate (CPPD) crystals are commonly found in osteoarthritic joints and correlate with a poor prognosis. Intraarticular corticosteroids, such as dexamethasone (Dxm), are commonly used therapies for osteoarthritis with or without CPPD deposition. Dxm has variable effects in mineralization models. We investigated the effects of Dxm on CPPD crystal formation in a well established tissue culture model. Porcine articular chondrocytes were incubated with ATP to generate CPPD crystals. Chondrocytes incubated with or without ATP were exposed to 1-100 nM Dxm in the presence of 45Ca. Mineralization was measured by 45Ca uptake in the cell layer. We also investigated the effect of Dxm on mineralization-regulating enzymes such as alkaline phosphatase, nucleoside triphosphate pyrophosphohydrolase (NTPPPH), and transglutaminase. Dxm significantly increased ATP-induced mineralization by articular chondrocytes. While alkaline phosphatase and NTPPPH activities were unchanged by Dxm, transglutaminase activity increased in a dose-responsive manner. Levels of Factor XIIIA mRNA and protein were increased by Dxm, while type II Tgase protein was unchanged. Transglutaminase inhibitors suppressed Dxminduced increases in CPPD crystal formation. These findings suggest a potential for Dxm to contribute to pathologic mineralization in cartilage and reinforce a central role for the transglutaminase enzymes in CPPD crystal formation.

Crystal Structure of Escherichia coli MazG, the Regulator of Nutritional Stress Response

MazG is a nucleoside triphosphate pyrophosphohydrolase that hydrolyzes all canonical nucleoside triphosphates. The mazG gene located downstream from the chromosomal mazEF "addiction module," that mediated programmed cell death in Escherichia coli. MazG activity is inhibited by the MazEF complex both in vivo and in vitro. Enzymatic activity of MazG in vivo affects the cellular level of guanosine 3',5'-bispyrophosphate (ppGpp), synthesized by RelA under amino acid starvation. The reduction of ppGpp, caused by MazG, may extend the period of cell survival under nutritional stress. Here we describe the first crystal structure of active MazG from E. coli, which is composed of two similarly folded globular domains in tandem. Among the two putative catalytic domains, only the C-terminal domain has well ordered active sites and exhibits an NTPase activity. The MazG-ATP complex structure and subsequent mutagenesis studies explain the peculiar active site environment accommodating all eight canonical NTPs as substrates. In vivo nutrient starvation experiments show that the C terminus NTPase activity is responsible for the regulation of bacterial cell survival under nutritional stress.

Crystal Structure of RS21-C6, Involved in Nucleoside Triphosphate Pyrophosphohydrolysis

RS21-C6, which is highly expressed in all vertebrate genomes and green plants, is proposed to have nucleoside triphosphate pyrophosphohydrolase activity. Here, we report the crystal structures of the core fragment of RS21-C6, named RSCUT, and the complex with the substrate 5-methyl dCTP. The refined structure of RSCUT consists mainly of α-helices and shows formation of a tightly associated tetramer. On the basis of the structure of the RSCUT–m5dCTP complex and the results of pyrophosphatase activity assays, several key residues involved in the substrate binding of RS21-C6 have been identified. Tetramer formation is shown to be required for substrate binding.

S100A8 and S100A9 in Human Arterial Wall: IMPLICATIONS FOR ATHEROGENESIS

Atherogenesis is a complex process involving inflammation. S100A8 and S100A9, the Ca2+-binding neutrophil cytosolic proteins, are associated with innate immunity and regulate processes leading to leukocyte adhesion and transmigration. In neutrophils and monocytes the S100A8-S100A9 complex regulates phosphorylation, NADPH-oxidase activity, and fatty acid transport. The proteins have anti-microbial properties, and S100A8 may play a role in oxidant defense in inflammation. Murine S100A8 is regulated by inflammatory mediators and recruits macrophages with a proatherogenic phenotype. S100A9 but not S100A8 was found in macrophages in ApoE-/- murine atherosclerotic lesions, whereas both proteins are expressed in human giant cell arteritis. Here we demonstrate S100A8 and S100A9 protein and mRNA in macrophages, foam cells, and neovessels in human atheroma. Monomeric and complexed forms were detected in plaque extracts. S100A9 was strongly expressed in calcifying areas and the surrounding extracellular matrix. Vascular matrix vesicles contain high levels of Ca2+-binding proteins and phospholipids that regulate calcification. Matrix vesicles characterized by electron microscopy, x-ray microanalysis, nucleoside triphosphate pyrophosphohydrolase assay and cholesterol/phospholipid analysis contained predominantly S100A9. We propose that S100A9 associated with lipid structures in matrix vesicles may influence phospholipid-Ca2+ binding properties to promote dystrophic calcification. S100A8 and S100A9 were more sensitive to hypochlorite oxidation than albumin or low density lipoprotein and immunoaffinity confirmed S100A8-S100A9 complexes; some were resistant to reduction, suggesting that hypochlorite may contribute to protein cross-linking. S100A8 and S100A9 in atherosclerotic plaque and calcifying matrix vesicles may significantly influence redox- and Ca2+-dependent processes during atherogenesis and its chronic complications, particularly dystrophic calcification.

Identification of an ITPase/XTPase in Escherichia coli by Structural and Biochemical Analysis

Inosine triphosphate (ITP) and xanthosine triphosphate (XTP) are formed upon deamination of ATP and GTP as a result of exposure to chemical mutagens and oxidative damage. Nucleic acid synthesis requires safeguard mechanisms to minimize undesired lethal incorporation of ITP and XTP. Here, we present the crystal structure of YjjX, a protein of hitherto unknown function. The three-dimensional fold of YjjX is similar to those of Mj0226 from Methanococcus janschii, which possesses nucleotidase activity, and of Maf from Bacillus subtilis, which can bind nucleotides. Biochemical analyses of YjjX revealed it to exhibit specific phosphatase activity for inosine and xanthosine triphosphates and have a possible interaction with elongation factor Tu. The enzymatic activity of YjjX as an inosine/xanthosine triphosphatase provides evidence for a plausible protection mechanism by clearing the noncanonical nucleotides from the cell during oxidative stress in E. coli.

Thermotoga maritima MazG Protein Has Both Nucleoside Triphosphate Pyrophosphohydrolase and Pyrophosphatase Activities

MazG proteins form a widely conserved family among bacteria, but their cellular function is still unknown. Here we report that Thermotoga maritima MazG protein (Tm-MazG), the product of the TM0913 gene, has both nucleoside triphosphate pyrophosphohydrolase (NTPase) and pyrophosphatase activities. Tm-MazG catalyzes the hydrolysis of all eight canonical ribo- and deoxyribonucleoside triphosphates to their corresponding nucleoside monophosphates and PPi and subsequently hydrolyzes the resultant PPi to Pi. The NTPase activity with deoxyribonucleoside triphosphates as substrate is higher than corresponding ribonucleoside triphosphates. dGTP is the best substrate among the deoxyribonucleoside triphosphates, and GTP is the best among the ribonucleoside triphosphates. Both NTPase and pyrophosphatase activities were enhanced at higher temperatures and blocked by the alpha,beta-methyleneadenosine triphosphate, which cannot be hydrolyzed by Tm-MazG. Furthermore, PPi is an inhibitor for the Tm-MazG NTPase activity. Significant decreases in the NTPase activity and concomitant increases in the pyrophosphatase activity were observed when mutations were introduced at the highly conserved amino acid residues in Tm-MazG N-terminal region (E41Q/E42Q, E45Q, E61Q, R97A/R98A, and K118A). These results demonstrated that Tm-MazG has dual enzymatic functions, NTPase and pyrophosphatase, and that these two enzymatic activities are coordinated.