Mammalian Nudix proteins cleave nucleotide metabolite caps on RNAs.

We recently reported the presence of nicotinamide adenine dinucleotide (NAD)-capped RNAs in mammalian cells and a role for DXO and the Nudix hydrolase Nudt12 in decapping NAD-capped RNAs (deNADding) in cells. Analysis of 5'caps has revealed that in addition to NAD, mammalian RNAs also contain other metabolite caps including flavin adenine dinucleotide (FAD) and dephosphoCoA (dpCoA). In the present study we systematically screened all mammalian Nudix proteins for their potential deNADing, FAD cap decapping (deFADding) and dpCoA cap decapping (deCoAping) activity. We demonstrate that Nudt16 is a novel deNADding enzyme in mammalian cells. Additionally, we identified seven Nudix proteins-Nudt2, Nudt7, Nudt8, Nudt12, Nudt15, Nudt16 and Nudt19, to possess deCoAping activity in vitro. Moreover, our screening revealed that both mammalian Nudt2 and Nudt16 hydrolyze FAD-capped RNAs in vitro with Nudt16 regulating levels of FAD-capped RNAs in cells. All decapping activities identified hydrolyze the metabolite cap substrate within the diphosphate linkage. Crystal structure of human Nudt16 in complex with FAD at 2.7 Å resolution provide molecular insights into the binding and metal-coordinated hydrolysis of FAD by Nudt16. In summary, our study identifies novel cellular deNADding and deFADding enzymes and establishes a foundation for the selective functionality of the Nudix decapping enzymes on non-canonical metabolite caps.

Crystal Structure of African Swine Fever Virus dUTPase Reveals a Potential Drug Target.

E165R, a highly specific dUTP nucleotidohydrolase (dUTPase) encoded by the African swine fever virus (ASFV) genome, is required for productive replication of ASFV in swine macrophages. Here, we solved the high-resolution crystal structures of E165R in its apo state and in complex with its product dUMP. Structural analysis explicitly defined the architecture of the active site of the enzyme as well as the interaction between the active site and the dUMP ligand. By comparing the ASFV E165R structure with dUTPase structures from other species, we found that the active site of E165R is highly similar to those of dUTPases from Mycobacterium tuberculosis and Plasmodium falciparum, against which small-molecule chemicals have been developed, which could be the potential drug or lead compound candidates for ASFV. Our results provide important basis for anti-ASFV drug design by targeting E165R.IMPORTANCE African swine fever virus (ASFV), an Asfivirus affecting pigs and wild boars with up to 100% case fatality rate, is currently rampaging throughout China and some other countries in Asia. There is an urgent need to develop therapeutic and preventive reagents against the virus. Our crystallographic and biochemical studies reveal that ASFV E165R is a member of trimeric dUTP nucleotidohydrolase (dUTPase) family that catalyzes the hydrolysis of dUTP into dUMP. Our apo-E165R and E165R-dUMP structures reveal the constitutive residues and the configuration of the active center of this enzyme in rich detail and give evidence that the active center of E165R is very similar to that of dUTPases from Plasmodium falciparum and Mycobacterium tuberculosis, which have already been used as targets for designing drugs. Therefore, our high-resolution structures of E165R provide useful structural information for chemotherapeutic drug design.

A cryptic activity in the Nudix hydrolase superfamily.

The Nudix hydrolase superfamily is identified by a conserved cassette of 23 amino acids, and it is characterized by its pyrophosphorylytic activity on a wide variety of nucleoside diphosphate derivatives. Of the 13 members of the family in Escherichia coli, only one, Orf180, has not been identified with a substrate, although a host of nucleoside diphosphate compounds has been tested. Several reports have noted a strong similarity in the three-dimensional structure of the unrelated enzyme, isopentenyl diphosphate isomerase (IDI) to the Nudix structure, and the report that a Nudix enzyme was involved in the synthesis of geraniol, a product of the two substrates of IDI, prompted an investigation of whether the IDI substrates, isopentenyl diphosphate (IPP), and dimethylallyl diphosphate (DAPP) could be substrates of Orf180. This article demonstrates that Orf180 does have a very low activity on IPP, DAPP, and geranyl pyrophosphate (GPP). However, several of the other Nudix enzymes with established nucleoside diphosphate substrates hydrolyze these compounds at substantial rates. In fact, some Nudix hydrolases have higher activities on IPP, DAPP, and GPP than on their signature nucleoside diphosphate derivatives.

A comprehensive structural, biochemical and biological profiling of the human NUDIX hydrolase family.

The NUDIX enzymes are involved in cellular metabolism and homeostasis, as well as mRNA processing. Although highly conserved throughout all organisms, their biological roles and biochemical redundancies remain largely unclear. To address this, we globally resolve their individual properties and inter-relationships. We purify 18 of the human NUDIX proteins and screen 52 substrates, providing a substrate redundancy map. Using crystal structures, we generate sequence alignment analyses revealing four major structural classes. To a certain extent, their substrate preference redundancies correlate with structural classes, thus linking structure and activity relationships. To elucidate interdependence among the NUDIX hydrolases, we pairwise deplete them generating an epistatic interaction map, evaluate cell cycle perturbations upon knockdown in normal and cancer cells, and analyse their protein and mRNA expression in normal and cancer tissues. Using a novel FUSION algorithm, we integrate all data creating a comprehensive NUDIX enzyme profile map, which will prove fundamental to understanding their biological functionality.

The evolution of function within the Nudix homology clan.

The Nudix homology clan encompasses over 80,000 protein domains from all three domains of life, defined by homology to each other. Proteins with a domain from this clan fall into four general functional classes: pyrophosphohydrolases, isopentenyl diphosphate isomerases (IDIs), adenine/guanine mismatch-specific adenine glycosylases (A/G-specific adenine glycosylases), and nonenzymatic activities such as protein/protein interaction and transcriptional regulation. The largest group, pyrophosphohydrolases, encompasses more than 100 distinct hydrolase specificities. To understand the evolution of this vast number of activities, we assembled and analyzed experimental and structural data for 205 Nudix proteins collected from the literature. We corrected erroneous functions or provided more appropriate descriptions for 53 annotations described in the Gene Ontology Annotation database in this family, and propose 275 new experimentally-based annotations. We manually constructed a structure-guided sequence alignment of 78 Nudix proteins. Using the structural alignment as a seed, we then made an alignment of 347 "select" Nudix homology domains, curated from structurally determined, functionally characterized, or phylogenetically important Nudix domains. Based on our review of Nudix pyrophosphohydrolase structures and specificities, we further analyzed a loop region downstream of the Nudix hydrolase motif previously shown to contact the substrate molecule and possess known functional motifs. This loop region provides a potential structural basis for the functional radiation and evolution of substrate specificity within the hydrolase family. Finally, phylogenetic analyses of the 347 select protein domains and of the complete Nudix homology clan revealed general monophyly with regard to function and a few instances of probable homoplasy. Proteins 2017; 85:775-811. © 2016 Wiley Periodicals, Inc.

In vitro characterization of a (E)-ß-farnesene synthase from Matricaria recutita L. and its up-regulation by methyl jasmonate.

(E)-ß-farnesene is a sesquiterpene semiochemical that is used extensively by both plants and animals for communication. This acyclic olefin is found in the essential oil of chamomile (Matricaria recutita) and was demonstrated that it could attract natural enemies to reduce cabbage aphids in the Chinese cabbage fields. However, little is known regarding the sequence and function of (E)-ß-farnesene synthase in M. recutita. In this study, we reported a new full-length cDNA encoding (E)-ß-farnesene synthase from M. recutita (Mr-ßFS). The cDNA of Mr-ßFS consisted of 2010bp including 1725bp of coding sequence encoding a protein of 574 amino acids with a molecular weight of 67kDa. The deduced amino acid sequence exhibits a considerably higher homology to ßFS from Artemisia annua (about 92% identity) than to ßFSs from other plants (about 20-40% identity). The recombinant enzyme, produced in Escherichia coli, catalyzed the formation of a single product, (E)-ß-farnesene, from farnesyl diphosphate. Real-time quantitative PCR (qRT-PCR) analysis showed that Mr-ßFS expression was highest in leaves and lowest in disk florets. The treatment of M. recutita with methyl jasmonate (MeJA) significantly enhanced the transcriptional level of ßFS gene and the content of (E)-ß-farnesene in M. recutita. The transcriptional level of ßFS gene was approximately 11.5-fold higher than the control sample and the (E)-ß-farnesene emission level ranged from approximately from 0.082 to 0.695µg/g after 24h induction. Our results laid a solid foundation for later improving crop aphid resistance by transgenic technology and provided an important basic data for the regulation of valuable products from M. recutita.

The role of the NTPDase enzyme family in parasites: what do we know, and where to from here?

Nucleoside triphosphate diphosphohydrolases (NTPDases, GDA1_CD39 protein superfamily) play a diverse range of roles in a number of eukaryotic organisms. In humans NTPDases function in regulating the inflammatory and immune responses, control of vascular haemostasis and purine salvage. In yeast NTPDases are thought to function primarily in the Golgi, crucially involved in nucleotide sugar transport into the Golgi apparatus and subsequent protein glycosylation. Although rare in bacteria, in Legionella pneumophila secreted NTPDases function as virulence factors. In the last 2 decades it has become clear that a large number of parasites encode putative NTPDases, and the functions of a number of these have been investigated. In this review, the available evidence for NTPDases in parasites and the role of these NTPDases is summarized and discussed. Furthermore, the processes by which NTPDases could function in pathogenesis, purine salvage, thromboregulation, inflammation and glycoconjugate formation are considered, and the data supporting such putative roles reviewed. Potential future research directions to further clarify the role and importance of NTPDases in parasites are proposed. An attempt is also made to clarify the nomenclature used in the parasite field for the GDA1_CD39 protein superfamily, and a uniform system suggested.

The RelA/SpoT homolog (RSH) superfamily: distribution and functional evolution of ppGpp synthetases and hydrolases across the tree of life.

RelA/SpoT Homologue (RSH) proteins, named for their sequence similarity to the RelA and SpoT enzymes of Escherichia coli, comprise a superfamily of enzymes that synthesize and/or hydrolyze the alarmone ppGpp, activator of the "stringent" response and regulator of cellular metabolism. The classical "long" RSHs Rel, RelA and SpoT with the ppGpp hydrolase, synthetase, TGS and ACT domain architecture have been found across diverse bacteria and plant chloroplasts, while dedicated single domain ppGpp-synthesizing and -hydrolyzing RSHs have also been discovered in disparate bacteria and animals respectively. However, there is considerable confusion in terms of nomenclature and no comprehensive phylogenetic and sequence analyses have previously been carried out to classify RSHs on a genomic scale. We have performed high-throughput sensitive sequence searching of over 1000 genomes from across the tree of life, in combination with phylogenetic analyses to consolidate previous ad hoc identification of diverse RSHs in different organisms and provide a much-needed unifying terminology for the field. We classify RSHs into 30 subgroups comprising three groups: long RSHs, small alarmone synthetases (SASs), and small alarmone hydrolases (SAHs). Members of nineteen previously unidentified RSH subgroups can now be studied experimentally, including previously unknown RSHs in archaea, expanding the "stringent response" to this domain of life. We have analyzed possible combinations of RSH proteins and their domains in bacterial genomes and compared RSH content with available RSH knock-out data for various organisms to determine the rules of combining RSHs. Through comparative sequence analysis of long and small RSHs, we find exposed sites limited in conservation to the long RSHs that we propose are involved in transmitting regulatory signals. Such signals may be transmitted via NTD to CTD intra-molecular interactions, or inter-molecular interactions either among individual RSH molecules or among long RSHs and other binding partners such as the ribosome.

Changes in E-NTPDase 3 expression and extracellular nucleotide hydrolysis during the myofibroblast/lipocyte differentiation.

Hepatic stellate cells (HSC) play a critical role in the development and maintenance of liver fibrosis. HSC are lipocytes that displayed the capacity to develop into myofibroblast-like cells. Ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) regulate the concentration of extracellular nucleotides, signaling molecules that play a role in the pathogenesis of hepatic fibrosis. In the present study, we identified and compared the expressions of E-NTPDase family members in two different phenotypes of the mouse hepatic stellate cell line (GRX) and evaluated the nucleotide hydrolysis by these cells. We show that both phenotypes of GRX cell line expressed NTPDase 3 and 5. However, only activated cells expressed NTPDase 6. In quiescent-like cells, the hydrolysis of triphosphonucleosides was significantly higher, and was related to an increase in Entpd3 mRNA expression. The diphosphonucleosides were hydrolyzed at a similar rate by two phenotypes of GRX cells. We suggest that up-regulation of Entpd3 mRNA expression modulates the extracellular concentration of nucleotides/nucleosides and affect P2-receptor signaling differently in quiescent-like cells and may play a role in the regulation of HSC functions.

Characterization of mouse UDP-glucose pyrophosphatase, a Nudix hydrolase encoded by the Nudt14 gene.

Recombinant mouse UDP-glucose pyrophosphatase (UGPPase), encoded by the Nudt14 gene, was produced in Escherichia coli and purified close to homogeneity. The enzyme catalyzed the conversion of [beta-(32)P]UDP-glucose to [(32)P]glucose-1-P and UMP, confirming that it hydrolyzed the pyrophosphate of the nucleoside diphosphate sugar to generate glucose-1-P and UMP. The enzyme was also active toward ADP-ribose. Activity is dependent on the presence of Mg(2+) and was greatest at alkaline pH above 8. Kinetic analysis indicated a K(m) of approximately 4mM for UDP-glucose and approximately 0.3mM for ADP-ribose. Based on V(max)/K(m) values, the enzyme was approximately 20-fold more active toward ADP-ribose. UGPPase behaves as a dimer in solution and can be cross-linked to generate a species of M(r) 54,000 from a monomer of 30,000 as judged by SDS-PAGE. The dimerization was not affected by the presence of glucose-1-P or UDP-glucose. Using antibodies raised against the recombinant protein, Western analysis indicated that UGPPase was widely expressed in mouse tissues, including skeletal muscle, liver, kidney, heart, lung, fat, heart and pancreas with a lower level in brain. It was generally present as a doublet when analyzed by SDS-PAGE, suggesting the occurrence of some form of post-translational modification. Efforts to interconvert the species by adding or inhibiting phosphatase activity were unsuccessful, leaving the nature of the modification unknown. Sequence alignments and database searches revealed related proteins in species as distant as Drosophila melanogaster and Caenorhabditis elegans.

Evolutionary conservation supports ancient origin for Nudt16, a nuclear-localized, RNA-binding, RNA-decapping enzyme.

Nudt16p is a nuclear RNA decapping protein initially identified in Xenopus (X29) and known to exist in mammals. Here, we identified putative orthologs in 57 different organisms ranging from humans to Cnidaria (anemone/coral). In vitro analysis demonstrated the insect ortholog can bind RNA and hydrolyze the m(7)G cap from the 5'-end of RNAs indicating the Nudt16 gene product is functionally conserved across metazoans. This study also identified a closely related paralogous protein, known as Syndesmos, which resulted from a gene duplication that occurred in the tetrapod lineage near the amniote divergence. While vertebrate Nudt16p is a nuclear RNA decapping protein, Syndesmos is associated with the cytoplasmic membrane in tetrapods. Syndesmos is inactive for RNA decapping but retains RNA-binding activity. This structure/function analysis demonstrates evolutionary conservation of the ancient Nudt16 protein suggesting the existence and maintenance of a nuclear RNA degradation pathway in metazoans.

Taenia solium dUTPase: A potential target for anti-human cysticercosis.

dUTPase plays an essential role in pyrimidine metabolism in many organisms. In this study we report the dUTPase-encoding gene (Tdut) from Taenia solium oncospheres and larvae. Alignment reveals that the putative protein contains five conserved motifs that are often found in many characterized dUTPases. The deduced amino acid sequence has only 65.2% identity with human dUTPases. This low identity encourages its use for the design of new drugs against cysticercosis.

YZGD from Paenibacillus thiaminolyticus, a pyridoxal phosphatase of the HAD (haloacid dehalogenase) superfamily and a versatile member of the Nudix (nucleoside diphosphate x) hydrolase superfamily.

YZGD from Paenibacillus thiaminolyticus is a novel bifunctional enzyme with both PLPase (pyridoxal phosphatase) and Nudix (nucleoside diphosphate x) hydrolase activities. The PLPase activity is catalysed by the HAD (haloacid dehalogenase) superfamily motif of the enzyme, and the Nudix hydrolase activity is catalysed by the conserved Nudix signature sequence within a separate portion of the enzyme, as confirmed by site-directed mutagenesis. YZGD's phosphatase activity is very specific, with pyridoxal phosphate being the only natural substrate, while YZGD's Nudix activity is just the opposite, with YZGD being the most versatile Nudix hydrolase characterized to date. YZGD's Nudix substrates include the CDP-alcohols (CDP-ethanol, CDP-choline and CDP-glycerol), the ADP-coenzymes (NADH, NAD and FAD), ADP-sugars, TDP-glucose and, to a lesser extent, UDP- and GDP-sugars. Regardless of the Nudix substrate, one of the products is always a nucleoside monophosphate, suggesting a role in nucleotide salvage. Both the PLPase and Nudix hydrolase activities require a bivalent metal cation, but while PLPase activity is supported by Co2+, Mg2+, Zn2+ and Mn2+, the Nudix hydrolase activity is Mn2+-specific. YZGD's phosphatase activity is optimal at an acidic pH (pH 5), while YZGD's Nudix activities are optimal at an alkaline pH (pH 8.5). YZGD is the first enzyme reported to be a member of both the HAD and Nudix hydrolase superfamilies, the first PLPase to be recognized as a member of the HAD superfamily and the first Nudix hydrolase capable of hydrolysing ADP-x, CDP-x and TDP-x substrates with comparable substrate specificity.

Inhibition of family II pyrophosphatases by analogs of pyrophosphate and phosphate.

Imidodiphosphate (the pyrophosphate analog containing a nitrogen atom in the bridge position instead of oxygen) is a potent inhibitor of family II pyrophosphatases from Streptococcus mutans and Streptococcus gordonii (inhibition constant Ki approximately 10 microM), which is slowly hydrolyzed by these enzymes with a catalytic constant of approximately 1 min(-1). Diphosphonates with different substituents at the bridge carbon atom are much less effective (Ki = 1-6 mM). The value of Ki for sulfate (a phosphate analog) is only 12 mM. The inhibitory effect of the pyrophosphate analogs exhibits only a weak dependence on the nature of the metal ion (Mn, Mg, or Co) bound in the active site.

Cloning and characterization of mouse nucleoside triphosphate diphosphohydrolase-3.

We have cloned and characterized the nucleoside triphosphate diphosphohydrolase-3 (NTPDase3) from mouse spleen. Analysis of cDNA shows an open reading frame of 1587 base pairs encoding a protein of 529 amino acids with a predicted molecular mass of 58953Da and an estimated isoelectric point of 5.78. The translated amino acid sequence shows the presence of two transmembrane domains, eight potential N-glycosylation sites and the five apyrase conserved regions. The genomic sequence is located on chromosome 9F4 and is comprised of 11 exons. Intact COS-7 cells transfected with an expression vector containing the coding sequence for mouse NTPDase3 hydrolyzed P2 receptor agonists (ATP, UTP, ADP and UDP) but not AMP. NTPDase3 required divalent cations (Ca2+ > Mg2+) for enzymatic activity. Interestingly, the enzyme had two optimum pHs for ATPase activity (pH 5.0 and 7.4) and one for ADPase activity (pH 8.0). Consequently, the ATP/ADP and UTP/UDP hydrolysis ratios were two to four folds higher at pH 5.0 than at pH 7.4, for both, intact cells and protein extracts. At pH 7.4 mouse NTPDase3 hydrolyzed ATP, UTP, ADP and UDP according to Michaelis-Menten kinetics with apparent K(m)s of 11, 10, 19 and 27 microM, respectively. In agreement with the K(m) values, the pattern of triphosphonucleoside hydrolysis showed a transient accumulation of the corresponding diphosphonucleoside and similar affinity for uracil and adenine nucleotides. NTPDase3 hydrolyzes nucleotides in a distinct manner than other plasma membrane bound NTPDases that may be relevant for the fine tuning of the concentration of P2 receptor agonists.

Inorganic pyrophosphatase in the roundworm Ascaris and its role in the development and molting process of the larval stage parasites.

Inorganic pyrophosphatase (PPase) is an important enzyme that catalyzes the hydrolysis of inorganic pyrophosphate (PPi) into ortho-phosphate (Pi). We report here the molecular cloning and characterization of a gene encoding the soluble PPase of the roundworm Ascaris suum. The predicted A. suum PPase consists of 360 amino acids with a molecular mass of 40.6 kDa and a pI of 7.1. Amino acid sequence alignment and phylogenetic analysis indicates that the gene encodes a functional Family I soluble PPase containing features identical to those of prokaryotic, plant and animal/fungal soluble PPases. The Escherichia coli-expressed recombinant enzyme has a specific activity of 937 micro mol Pi.min-1.mg-1 protein corresponding to a kcat value of 638 s-1 at 55 degrees C. Its activity was strongly dependent on Mg2+ and was inhibited by Ca2+. Native PPases were expressed in all developmental stages of A. suum. A homolog was also detected in the most closely related human and dog roundworms A. lumbricoides and Toxocara canis, respectively. The enzyme was intensely localized in the body wall, gut epithelium, ovary and uterus of adult female worms. We observed that native PPase activity together with development and molting in vitro of A. suum L3 to L4 were efficiently inhibited in a dose-dependent manner by imidodiphosphate and sodium fluoride, which are potent inhibitor of both soluble- and membrane-bound H+-PPases. The studies provide evidence that the PPases are novel enzymes in the roundworm Ascaris, and may have crucial role in the development and molting process.

Rhodobacter sphaeroides has a family II pyrophosphatase: comparison with other species of photosynthetic bacteria.

The cytoplasmic pyrophosphatase from Rhodobacter sphaeroides was purified and characterized. The enzyme is a homodimer of 64 kDa. The N-terminus was sequenced and used to obtain the complete pyrophosphatase sequence from the preliminary genome sequence of Rba. sphaeroides, showing extensive sequence similarity to family II or class C pyrophosphatases. The enzyme hydrolyzes only Mg-PP(i) and Mn-PP(i) with a K(m) of 0.35 mM for both substrates. It is not activated by free Mg (2+), in contrast to the cytoplasmic pyrophosphatase from Rhodospirillum rubrum, and it is not inhibited by NaF, methylendiphosphate, or imidodiphosphate. This work shows that Rba. sphaeroides and Rhodobacter capsulatus cytoplasmic pyrophosphatases belong to family II, in contrast to Rsp. rubrum, Rhodopseudomonas palustris, Rhodopseudomonas gelatinosa, and Rhodomicrobium vannielii cytoplasmic pyrophosphatases which should be classified as members of family I. This is the first report of family II cytoplasmic pyrophosphatases in photosynthetic bacteria and in a gram-negative organism.

Rhodospirillum rubrum has a family I pyrophosphatase: purification, cloning, and sequencing.

The cytoplasmic pyrophosphatase of the photosynthetic bacterium Rhodospirillum rubrum was purified to electrophoretic homogeneity. The enzyme is a homohexamer of 20-kDa monomers. The gene was cloned and sequenced. Alignment of the deduced 179-amino-acid protein with known bacterial pyrophosphatases revealed conservation of all residues in the active site. Attempts to obtain an insertion mutant of the cytoplasmic pyrophosphatase gene did not yield any cell completely devoid of cytoplasmic pyrophosphatase activity. The mutants obtained showed 50% of the enzymatic activity and grew in twice the generation time of wild-type cells. This suggests that the membrane-bound pyrophosphatase of Rsp. rubrum is not sufficient for a normal growth rate, whereas the cytoplasmic enzyme is essential for growth. The characteristics of the gene and the encoded protein fit those of prokaryotic family I pyrophosphatases.

Suppression of spontaneous and hydrogen peroxide-induced mutations by a MutT-type nucleotide pool sanitization enzyme, the Escherichia coli Orf135 protein.

BACKGROUND: We recently found that the Escherichia coli Orf135 protein, a MutT-type enzyme, hydrolysed 2-hydroxy-dATP (2-OH-dATP), and less efficiently, 8-hydroxy-dGTP. RESULTS: In this study, we examined the effects of the absence of the orf135 gene. Frequencies of spontaneous and H2O2-induced mutations were two- to three-fold higher in the orf135- strain than in the wild-type strain. These mutations include various mutations involving a G:C-->T:A transversion, the same type of mutation elicited by 2-OH-dATP. Over-expression of the Orf135 protein suppressed mutations even in the wild-type strain, as well as in the orf135- strain. CONCLUSIONS: The mutator phenotype of bacteria lacking the Orf135 protein suggests that this protein is involved in the suppression of mutations induced by oxidized deoxynucleotides in vivo and that various MutT-type enzymes contribute to nucleotide pool sanitization.

The scavenger mRNA decapping enzyme DcpS is a member of the HIT family of pyrophosphatases.

We recently demonstrated that the major decapping activity in mammalian cells involves DcpS, a scavenger pyrophosphatase that hydrolyzes the residual cap structure following 3' to 5' decay of an mRNA. The association of DcpS with 3' to 5' exonuclease exosome components suggests that these two activities are linked and there is a coupled exonucleolytic decay-dependent decapping pathway. We purified DcpS from mammalian cells and identified the cDNA encoding a novel 40 kDa protein possessing DcpS activity. Consistent with purified DcpS, the recombinant protein specifically hydrolyzed methylated cap analog but did not hydrolyze unmethylated cap analog nor did it function on intact capped RNA. Sequence alignments of DcpS from different organisms revealed the presence of a conserved hexapeptide, containing a histidine triad (HIT) sequence with three histidines separated by hydrophobic residues. Mutagenesis analysis revealed that the central histidine within the DcpS HIT motif is critical for decapping activity and defines the HIT motif as a new mRNA decapping domain, making DcpS the first member of the HIT family of proteins with a defined biological function.