Nudix Hydrolase Family Research Articles

Unexpected Noncovalent Off-Target Activity of Clinical BTK Inhibitors Leads to Discovery of a Dual NUDT5/14 Antagonist.

Cofactor mimicry represents an attractive strategy for the development of enzyme inhibitors but can lead to off-target effects due to the evolutionary conservation of binding sites across the proteome. Here, we uncover the ADP-ribose (ADPr) hydrolase NUDT5 as an unexpected, noncovalent, off-target of clinical BTK inhibitors. Using a combination of biochemical, biophysical, and intact cell NanoBRET assays as well as X-ray crystallography, we confirm catalytic inhibition and cellular target engagement of NUDT5 and reveal an unusual binding mode that is independent of the reactive acrylamide warhead. Further investigation of the prototypical BTK inhibitor ibrutinib also revealed potent inhibition of the largely unstudied NUDIX hydrolase family member NUDT14. By exploring structure-activity relationships (SARs) around the core scaffold, we identify a potent, noncovalent, and cell-active dual NUDT5/14 inhibitor. Cocrystallization experiments yielded new insights into the NUDT14 hydrolase active site architecture and inhibitor binding, thus providing a basis for future chemical probe design.

Transcriptomic profile investigations highlight a putative role for NUDT16 in sepsis.

Sepsis is an aberrant systemic inflammatory response mediated by the acute activation of the innate immune system. Neutrophils are important contributors to the innate immune response that controls the infection, but harbour the risk of collateral tissue damage such as thrombosis and organ dysfunction. A better understanding of the modulations of cellular processes in neutrophils and other blood cells during sepsis is needed and can be initiated via transcriptomic profile investigations. To that point, the growing repertoire of publicly accessible transcriptomic datasets serves as a valuable resource for discovering and/or assessing the robustness of biomarkers. We employed systematic literature mining, reductionist approach to gene expression profile and empirical in vitro work to highlight the role of a Nudix hydrolase family member, NUDT16, in sepsis. The relevance and implication of the expression of NUDT16 under septic conditions and the putative functional roles of this enzyme are discussed.

PpNUDX8, a Peach NUDIX Hydrolase, Plays a Negative Regulator in Response to Drought Stress.

Drought stress is a serious abiotic stress source that affects the growth and fruit quality of peach trees. However, the molecular mechanism of the NUDIX hydrolase family in peaches in response to drought stress is still unclear. Here, we isolated and identified the PpNUDX8 (Prupe.5G062300.1) gene from the peach NUDIX hydrolase family, and found that PpNUDX8 has a typical NUDIX hydrolase domain. In this study, we performed 15% PEG6000 drought treatment on peach seedlings, and qRT–PCR analysis showed that 15% PEG6000 induced the transcription level of PpNUDX8. Overexpression of PpNUDX8 reduced the tolerance of calli to 4% PEG6000 treatment. Compared with wild-type apple calli, PpNUDX8 transgenic apple calli had a lower fresh weight and higher MDA content. After 15% PEG6000 drought treatment, PpNUDX8 transgenic tobacco had a greater degree of wilting and shorter primary roots than Under control conditions. The chlorophyll, soluble protein, and proline contents in the transgenic tobacco decreased, and the MDA content and relative conductivity increased. At the same time, PpNUDX8 negatively regulated ABA signal transduction and reduced the transcriptional expression of stress response genes. In addition, PpNUDX8 was not sensitive to ABA, overexpression of PpNUDX8 reduced the expression of the ABA synthesis-related gene NCED6 and increases the expression of the ABA decomposition-related gene CYP1 in tobacco, which in turn leads to a decrease in the ABA content in tobacco. In addition, Under control conditions, overexpression of PpNUDX8 destroyed the homeostasis of NAD and reduced nicotinamide adenine dinucleotide (NADH) in tobacco. After 15% PEG6000 drought treatment, the changes in NAD and NADH in PpNUDX8 transgenic tobacco were more severe than those in WT tobacco. In addition, PpNUDX8 also interacted with PpSnRk1γ (Prupe.6G323700.1).

Versatile physiological functions of the Nudix hydrolase family in berry development and stress response in grapevine

Nudix hydrolases are widely distributed across all classes of organisms and provide the potential capacity to hydrolyze a wide range of organic pyrophosphates. Although Nudix hydrolases are involved in plant detoxification processes in response to abiotic and biotic stresses, the biological functions of Nudix hydrolases remain largely unclear in grapevine. In the present study, a total of 25 putative grapevine Nudix hydrolases (VvNUDXs) were identified by bioinformatics analysis and classified into eight subfamilies based to their preferred substrates. Both tandem and segmental duplications were responsible for the evolution and expansion of the NUDX gene family in grapevine. To investigate the regulatory roles of VvNUDX genes during growth and development, as well as in response to abiotic and biotic stresses in grapevine, the expression patterns were revealed in publicly available microarray data. The spatial and temporal expression patterns of the VvNUDX genes indicated that they might play important roles in multiple developmental processes. Transcriptome and qRT-PCR analyses showed that ten VvNUDX genes were specifically expressed in grapevine berries, suggesting potential roles in grapevine berry development. Expression and phylogenetic analyses demonstrated that VvNUDX1 and VvNUDX3 might be involved in terpenoid biosynthesis in grapevine. Furthermore, most VvNUDX genes active toward the ADP-ribose/NADH showed different patterns in response to various abiotic and biotic stresses, such as salinity and drought, as well as different types of biotic treatments, such as Erysiphe necator, Bois Noir phytoplasma and leaf-roll-associated virus-3 (GLRaV-3). These results indicated that VvNUDX genes were associated with plant detoxification processes in response to abiotic and biotic stresses, and regulate the disease immunity and resistance pathways. The information obtained here may provide good opportunities to explore the physiological functions of VvNUDX genes in berry development and stress response networks in grapevine.

Isopentenyl diphosphate/dimethylallyl diphosphate-specific Nudix hydrolase from the methanogenic archaeon Methanosarcina mazei.

Nudix hydrolases typically catalyze the hydrolysis of nucleoside diphosphate linked to moiety X and yield nucleoside monophosphate and X-phosphate, while some of them hydrolyze a terminal diphosphate group of non-nucleosidic compounds and convert it into a phosphate group. Although the number of Nudix hydrolases is usually limited in archaea comparing with those in bacteria and eukaryotes, the physiological functions of most archaeal Nudix hydrolases remain unknown. In this study, a Nudix hydrolase family protein, MM_2582, from the methanogenic archaeon Methanosarcina mazei was recombinantly expressed in Escherichia coli, purified, and characterized. This recombinant protein shows higher hydrolase activity toward isopentenyl diphosphate and short-chain prenyl diphosphates than that toward nucleosidic compounds. Kinetic studies demonstrated that the archaeal enzyme prefers isopentenyl diphosphate and dimethylallyl diphosphate, which suggests its role in the biosynthesis of prenylated flavin mononucleotide, a recently discovered coenzyme that is required, for example, in the archaea-specific modified mevalonate pathway.

Structural insights into dpCoA-RNA decapping by NudC

ABSTRACT Various kinds of cap structures, such as m7G, triphosphate groups, NAD and dpCoA, protect the 5′ terminus of RNA. The cap structures bond covalently to RNA and affect its stability, translation, and transport. The removal of the caps is mainly executed by Nudix hydrolase family proteins, including Dcp2, RppH and NudC. Numerous efforts have been made to elucidate the mechanism underlying the removal of m7G, triphosphate group, and NAD caps. In contrast, few studies related to the cleavage of the RNA dpCoA cap have been conducted. Here, we report the hydrolytic activity of Escherichia coli NudC towards dpCoA and dpCoA-capped RNA in vitro. We also determined the crystal structure of dimeric NudC in complex with dpCoA at 2.0 Å resolution. Structural analysis revealed that dpCoA is recognized and hydrolyzed in a manner similar to NAD. In addition, NudC may also remove other dinucleotide derivative caps of RNA, which comprise the AMP moieties. NudC homologs in Saccharomyces cerevisiae and Arabidopsis thaliana exhibited similar dpCoA decapping (deCoAping) activity. These results together indicate a conserved mechanism underpinning the hydrolysis of dpCoA-capped RNA in both prokaryotes and eukaryotes.

Characterizing Enzymes of the Diphosphoinositol Polyphosphate Phosphohydrolase (DIPP) Family.

The diphosphoinositol polyphosphate phosphohydrolases are a subset of the Nudix hydrolase family of enzymes. As such, they metabolize a wide range of substrates, including diphosphoinositol polyphosphates (inositol diphosphates, inositol pyrophosphates), dinucleotide phosphates, nucleosides as well as 5-phosphoribosyl 1-pyrophosphate and inorganic polyphosphate. Here, we describe protocols to optimize these enzymes, with consideration to buffer composition and sample preparation and how to analyze the metabolism of these substrates using high-performance liquid chromatography, giving advice where pitfalls are commonly encountered.

Nudix hydrolases with Coenzyme A (CoA) and acyl-CoA pyrophosphatase activities confer growth advantage to Mycobacterium smegmatis.

Nudix hydrolase family proteins hydrolyse toxic by-products of cellular metabolism such as mutagenic nucleoside triphosphates, sugar nucleotides and signalling molecules. We studied the substrate specificities of Nudix hydrolases encoded by rv3672c and rv3040c from Mycobacterium tuberculosis and their respective homologues, msmeg_6185 and msmeg_2327 from M. smegmatis. The rv3672c- and msmeg_6185-encoded proteins (Rv3672 and MSMEG_6185, respectively) showed CoA pyrophosphatase (CoAse) activity that converted acyl-CoA to adenosine-3',5'-diphosphate (3', 5'-ADP) and 4-acyl phosphopantetheine. The efficiencies of Rv3672 and MSMEG_6185 in hydrolysing CoA derivatives were found to be higher than those of the Rv3040 and MSMEG_2327 (encoded by rv3040c and msmeg_2327, respectively). Further, amongst the substrates tested, Rv3672 and MSMEG_6185 used CoA and oxidized CoA as the most preferred substrates. Use of the M. smegmatis model showed that the expression of msmeg_6185 occurs in the log and stationary phases but declines during the late stationary phase and becomes undetectable during hypoxia. The co-culture competition experiments performed between the wild-type and Δmsmeg_6185 strains of M. smegmatis in different carbon sources revealed that the presence of msmeg_6185 provided growth fitness advantage to M. smegmatis, irrespective of the carbon source, implicating its function in regulation for the optimal physiological levels of acyl-CoAs in the cell.

植物の補酵素型ビタミン代謝における Nudix hydrolaseファミリーの生理的役割

植物の補酵素型ビタミン代謝における Nudix hydrolaseファミリーの生理的役割

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.

Abstract IA12: Poisoning cancer with oxidized nucleotides by targeting MTH1

Abstract DNA damaging agents, i.e., radio- and chemotherapy, constitute the backbone for treatment of a wide variety of cancers and may result in complete cure from the disease. Here, I will give an overview on how DNA repair can be targeted using completely novel inhibitors and more specifically how cancer cells may require a specific DNA repair pathway to mediate survival to the high load of endogenous DNA damage. Cancers have deregulated levels of reactive oxygen species (ROS), damaging both DNA and free dNTPs. The MTH1 protein sanitizes oxidized dNTP pools, converting 8-oxo-dGTP to 8-oxo-dGMP, to prevent incorporation of damaged bases during DNA replication. MTH1 overexpression reverses the mutator phenotype caused by mismatch repair defects and prevents Ras-induced senescence by suppressing the overall level of DNA damage. These data suggest that a majority of damage in cancer cells occur on the free dNTP pool and that this need sanitation for cancer cell survival. Here we show that cancer cells are dependent on MTH1 activity for survival, due to the effects of MTH1 in preventing incorporation of oxidized dNTPs into DNA to avoid ATM and p53 mediated apoptosis. As MTH1-/- mice are viable and MTH1 is not required for survival of non-transformed cells, targeting MTH1 may selectively cause DNA damage to cancer cells. We validate MTH1 as an anti-cancer target in vivo and describe small molecules, TH287 and TH588 that potently and selectively inhibit MTH1. Protein co-crystal structures demonstrate that the compounds bind as inhibitors in the enzymatic pocket of MTH1. These first-in-class inhibitors of the Nudix hydrolase family cause increased incorporation of oxidized dNTPs in cells subject to high ROS levels, causing DNA damage and cytotoxicity to cancer cells. This study exemplifies a new general therapeutic approach to convert oxidative stress to cytotoxic DNA damage and cancer cell death. Here, I will present the progression of MTH1 inhibitors from pre-clinical development into early clinical trials.

Identification of the RNA Pyrophosphohydrolase RppH of Helicobacter pylori and Global Analysis of Its RNA Targets

RNA degradation is crucial for regulating gene expression in all organisms. Like the decapping of eukaryotic mRNAs, the conversion of the 5'-terminal triphosphate of bacterial transcripts to a monophosphate can trigger RNA decay by exposing the transcript to attack by 5'-monophosphate-dependent ribonucleases. In both biological realms, this deprotection step is catalyzed by members of the Nudix hydrolase family. The genome of the gastric pathogen Helicobacter pylori, a Gram-negative epsilonproteobacterium, encodes two proteins resembling Nudix enzymes. Here we present evidence that one of them, HP1228 (renamed HpRppH), is an RNA pyrophosphohydrolase that triggers RNA degradation in H. pylori, whereas the other, HP0507, lacks such activity. In vitro, HpRppH converts RNA 5'-triphosphates and diphosphates to monophosphates. It requires at least two unpaired nucleotides at the 5' end of its substrates and prefers three or more but has only modest sequence preferences. The influence of HpRppH on RNA degradation in vivo was examined by using RNA-seq to search the H. pylori transcriptome for RNAs whose 5'-phosphorylation state and cellular concentration are governed by this enzyme. Analysis of cDNA libraries specific for transcripts bearing a 5'-triphosphate and/or monophosphate revealed at least 63 potential HpRppH targets. These included mRNAs and sRNAs, several of which were validated individually by half-life measurements and quantification of their 5'-terminal phosphorylation state in wild-type and mutant cells. These findings demonstrate an important role for RppH in post-transcriptional gene regulation in pathogenic Epsilonproteobacteria and suggest a possible basis for the phenotypes of H. pylori mutants lacking this enzyme.

The Obesity-Linked Gene Nudt3 Drosophila Homolog Aps Is Associated With Insulin Signaling.

Several genome-wide association studies have linked the Nudix hydrolase family member nucleoside diphosphate-linked moiety X motif 3 (NUDT3) to obesity. However, the manner of NUDT3 involvement in obesity is unknown, and NUDT3 expression, regulation, and signaling in the central nervous system has not been studied. We performed an extensive expression analysis in mice, as well as knocked down the Drosophila NUDT3 homolog Aps in the nervous system, to determine its effect on metabolism. Detailed in situ hybridization studies in the mouse brain revealed abundant Nudt3 mRNA and protein expression throughout the brain, including reward- and feeding-related regions of the hypothalamus and amygdala, whereas Nudt3 mRNA expression was significantly up-regulated in the hypothalamus and brainstem of food-deprived mice. Knocking down Aps in the Drosophila central nervous system, or a subset of median neurosecretory cells, known as the insulin-producing cells (IPCs), induces hyperinsulinemia-like phenotypes, including a decrease in circulating trehalose levels as well as significantly decreasing all carbohydrate levels under starvation conditions. Moreover, lowering Aps IPC expression leads to a decreased ability to recruit these lipids during starvation. Also, loss of neuronal Aps expression caused a starvation susceptibility phenotype while inducing hyperphagia. Finally, the loss of IPC Aps lowered the expression of Akh, Ilp6, and Ilp3, genes known to be inhibited by insulin signaling. These results point toward a role for this gene in the regulation of insulin signaling, which could explain the robust association with obesity in humans.

Surprise Route To Rose Scent

Taking the time to stop and smell the roses can help you appreciate the simple beauty of life. Recently, a team of French researchers took the time to uncover how roses produce these sweet odors, and found that the plants do so through an unexpected route. The findings could help rose breeders develop flowers with more powerful scents. Geraniol, a monoterpene, makes up much of rose oil’s signature scent. In many plants, geraniol synthase, an enzyme that belongs to the terpene synthase family, is responsible for producing the fragrant molecule. So researchers were surprised to find that, in roses, geraniol biosynthesis is mediated instead by a completely different enzyme. Through genetic mapping analysis, the team found that highly scented roses expressed the gene, RhNUDX1, at greater levels than their less fragrant counterparts (Science 2015, DOI: 10.1126/science.aab0696). RhNUDX1 codes for a new enzyme in the Nudix hydrolase family. The enzyme cleaves ...

Versatile physiological functions of the Nudix hydrolase family in Arabidopsis.

Nudix hydrolases are widely distributed in all kingdoms of life and have the potential to hydrolyze a wide range of organic pyrophosphates, including nucleoside di- and triphosphates, nucleotide coenzymes, nucleotide sugars, and RNA caps. However, except for E. coli MutT and its orthologs in other organisms that sanitize oxidized nucleotides to prevent DNA and RNA mutations, the functions of Nudix hydrolases had largely remained unclear until recently, because many members of this enzyme family exhibited broad substrate specificities. There is now increasing evidence to show that their functions extend into many aspects of the regulation of cellular responses. This review summarizes current knowledge on the molecular and enzymatic properties as well as physiological functions of Arabidopsis Nudix hydrolases. The information presented here may provide novel insights into the physiological roles of these enzymes in not only plant species, but also other organisms.

O6.5 - MTH1 inhibitors in cancer treatment

ABSTRACT DNA damaging agents, i.e., radio- and chemotherapy, constitute the backbone for treatment of a wide variety of cancers and may result in complete cure from the disease. Emerging data demonstrate that cancer cells harbour high level of endogenous DNA damage caused by replication, oxidative and other DNA damage stress. Consequently, cancer cells may require a specific DNA repair pathway to mediate survival to the high load of endogenous DNA damage. Cancers have deregulated levels of reactive oxygen species (ROS), damaging both DNA and free dNTPs. The MTH1 protein sanitises oxidized dNTP pools, converting 8-oxo-dGTP to 8-oxo-dGMP, to prevent incorporation of damaged bases during DNA replication. MTH1 overexpression reverses the mutator phenotype caused by mismatch repair defects and prevents Ras-induced senescence by suppressing the overall level of DNA damage. These data suggest that a majority of damage in cancer cells occur on the free dNTP pool and that this need sanitation for cancer cell survival. Here we show that cancer cells are dependent on MTH1 activity for survival, due to the effects of MTH1 in preventing incorporation of oxidized dNTPs into DNA to avoid ATM and p53 mediated apoptosis. As MTH1-/- mice are viable and MTH1 is not required for survival of non-transformed cells, targeting MTH1 may selectively cause DNA damage to cancer cells. We validate MTH1 as an anti-cancer target in vivo and describe small molecules, TH287 and TH588 that potently and selectively inhibit MTH1. Protein co-crystal structures demonstrate that the compounds bind as inhibitors in the enzymatic pocket of MTH1. These first-in-class inhibitors of the Nudix hydrolase family cause increased incorporation of oxidized dNTPs in cells subject to high ROS levels, causing DNA damage and cytotoxicity to cancer cells. This study exemplifies a new general therapeutic approach to convert oxidative stress to cytotoxic DNA damage and cancer cell death.

MTH1 inhibition eradicates cancer by preventing sanitation of the dNTP pool

Cancers have dysfunctional redox regulation resulting in reactive oxygen species production, damaging both DNA and free dNTPs. The MTH1 protein sanitizes oxidized dNTP pools to prevent incorporation of damaged bases during DNA replication. Although MTH1 is non-essential in normal cells, we show that cancer cells require MTH1 activity to avoid incorporation of oxidized dNTPs, resulting in DNA damage and cell death. We validate MTH1 as an anticancer target in vivo and describe small molecules TH287 and TH588 as first-in-class nudix hydrolase family inhibitors that potently and selectively engage and inhibit the MTH1 protein in cells. Protein co-crystal structures demonstrate that the inhibitors bind in the active site of MTH1. The inhibitors cause incorporation of oxidized dNTPs in cancer cells, leading to DNA damage, cytotoxicity and therapeutic responses in patient-derived mouse xenografts. This study exemplifies the non-oncogene addiction concept for anticancer treatment and validates MTH1 as being cancer phenotypic lethal.

Abstract CN05-04: Novel DNA repair targets for anticancer treatments.

Abstract Mutations in DNA repair are sometimes found in cancer, such as mutations in mismatch repair or BRCA1 or BRCA2 genes. We previously demonstrated that PARP inhibitors can selectively kill BRCA2 defective tumours through synthetic lethality. Here, I describe efforts into translating the synthetic lethal approach into treatment of cancer in the clinic and the challenges. The overall conclusion is that the synthetic lethal approach offers significant advantages, but no real cancer cures and suffer from similar resistance problems as many other targeted therapies. I exemplify how DNA repair inhibitors to a novel DNA repair target, MTH1, can be used in a different context, to overload the high level of DNA damage that is present in cancer. The MTH1 protein sanitises oxidized dNTP pools, converting 8-oxo-dGTP to 8-oxo-dGMP, to prevent incorporation of damaged bases during DNA replication. Here we show that cancer cells are dependent on MTH1 activity for survival, due to the effects of MTH1 in preventing incorporation of oxidized dNTPs into DNA to avoid ATM and p53 mediated apoptosis. We validate MTH1 as an anticancer target in vivo and describe small molecules that potently and selectively inhibit MTH1. Protein co-crystal structures demonstrate that the compounds bind as inhibitors in the enzymatic pocket of MTH1. These first-in-class inhibitors of the Nudix hydrolase family cause increased incorporation of oxidized dNTPs in cells subject to high ROS levels, causing DNA damage and cytotoxicity to cancer cells. This study exemplifies a new general therapeutic approach by using enzyme inhibitors to increased overall DNA damage load in cancer. This strategy may offer a treatment for a broad spectrum of cancers, regardless of cancer genotype. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):CN05-04. Citation Format: Thomas Helleday. Novel DNA repair targets for anticancer treatments. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr CN05-04.

FAD Synthesis and Degradation in the Nucleus Create a Local Flavin Cofactor Pool

FAD is a redox cofactor ensuring the activity of many flavoenzymes mainly located in mitochondria but also relevant for nuclear redox activities. The last enzyme in the metabolic pathway producing FAD is FAD synthase (EC 2.7.7.2), a protein known to be localized both in cytosol and in mitochondria. FAD degradation to riboflavin occurs via still poorly characterized enzymes, possibly belonging to the NUDIX hydrolase family. By confocal microscopy and immunoblotting experiments, we demonstrate here the existence of FAD synthase in the nucleus of different experimental rat models. HPLC experiments demonstrated that isolated rat liver nuclei contain ∼300 pmol of FAD·mg(-1) protein, which was mainly protein-bound FAD. A mean FAD synthesis rate of 18.1 pmol·min(-1)·mg(-1) protein was estimated by both HPLC and continuous coupled enzymatic spectrophotometric assays. Rat liver nuclei were also shown to be endowed with a FAD pyrophosphatase that hydrolyzes FAD with an optimum at alkaline pH and is significantly inhibited by adenylate-containing nucleotides. The coordinate activity of these FAD forming and degrading enzymes provides a potential mechanism by which a dynamic pool of flavin cofactor is created in the nucleus. These data, which significantly add to the biochemical comprehension of flavin metabolism and its subcellular compartmentation, may also provide the basis for a more detailed comprehension of the role of flavin homeostasis in biologically and clinically relevant epigenetic events.

Multiple Nudix family proteins possess mRNA decapping activity

RNA decapping is an important contributor to gene expression and is a critical determinant of mRNA decay. The recent demonstration that mammalian cells harbor at least two distinct decapping enzymes that preferentially modulate a subset of mRNAs raises the intriguing possibility of whether additional decapping enzymes exist. Because both known decapping proteins, Dcp2 and Nudt16, are members of the Nudix hydrolase family, we set out to determine whether other members of this family of proteins also contain intrinsic RNA decapping activity. Here we demonstrate that six additional mouse Nudix proteins--Nudt2, Nudt3, Nudt12, Nudt15, Nudt17, and Nudt19--have varying degrees of decapping activity in vitro on both monomethylated and unmethylated capped RNAs. The decapping products from Nudt17 and Nudt19 were analogous to Dcp2 and predominantly generated m⁷GDP, while cleavage by Nudt2, Nudt3, Nudt12, and Nudt15 was more pleiotropic and generated both m⁷GMP and m⁷GDP. Interestingly, all six Nudix proteins as well as both Dcp2 and Nudt16 could hydrolyze the cap of an unmethylated capped RNA, indicating that decapping enzymes may be less constrained for the presence of the methyl moiety. Investigation of Saccharomyces cerevisiae Nudix proteins revealed that the yeast homolog of Nudt3, Ddp1p, also possesses decapping activity in vitro. Moreover, the bacterial Nudix pyrophosphohydrolase RppH displayed RNA decapping activity and released m⁷GDP product comparable to Dcp2, indicating that decapping is an evolutionarily conserved activity that preceded mammalian cap formation. These findings demonstrate that multiple Nudix family hydrolases may function in mRNA decapping and mRNA stability.