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Inositol pyrophosphate 1,5-IP8 regulates expression of a fission yeast phosphate homeostasis regulon, comprising phosphate acquisition genes pho1, pho84, and tgp1, via its action as an agonist of precocious termination of transcription of the upstream lncRNAs that repress PHO mRNA synthesis. 1,5-IP8 levels are dictated by a balance between the Asp1 N-terminal kinase domain that converts 5-IP7 to 1,5-IP8 and three inositol pyrophosphatases-the Asp1 C-terminal domain (a histidine acid phosphatase), Siw14 (a cysteinyl-phosphatase), and Aps1 (a Nudix enzyme). In this study, we report the biochemical and genetic characterization of Aps1 and an analysis of the effects of Asp1, Siw14, and Aps1 mutations on cellular inositol pyrophosphate levels. We find that Aps1's substrate repertoire embraces inorganic polyphosphates, 5-IP7, 1-IP7, and 1,5-IP8. Aps1 displays a ~twofold preference for hydrolysis of 1-IP7 versus 5-IP7 and aps1∆ cells have twofold higher levels of 1-IP7 vis-à-vis wild-type cells. While neither Aps1 nor Siw14 is essential for growth, an aps1∆ siw14∆ double mutation is lethal on YES medium. This lethality is a manifestation of IP8 toxicosis, whereby excessive 1,5-IP8 drives derepression of tgp1, leading to Tgp1-mediated uptake of glycerophosphocholine. We were able to recover an aps1∆ siw14∆ mutant on ePMGT medium lacking glycerophosphocholine and to suppress the severe growth defect of aps1∆ siw14∆ on YES by deleting tgp1. However, the severe growth defect of an aps1∆ asp1-H397A strain could not be alleviated by deleting tgp1, suggesting that 1,5-IP8 levels in this double-pyrophosphatase mutant exceed a threshold beyond which overzealous termination affects other genes, which results in cytotoxicity. IMPORTANCE: Repression of the fission yeast PHO genes tgp1, pho1, and pho84 by lncRNA-mediated interference is sensitive to changes in the metabolism of 1,5-IP8, a signaling molecule that acts as an agonist of precocious lncRNA termination. 1,5-IP8 is formed by phosphorylation of 5-IP7 and catabolized by inositol pyrophosphatases from three distinct enzyme families: Asp1 (a histidine acid phosphatase), Siw14 (a cysteinyl phosphatase), and Aps1 (a Nudix hydrolase). This study entails a biochemical characterization of Aps1 and an analysis of how Asp1, Siw14, and Aps1 mutations impact growth and inositol pyrophosphate pools in vivo. Aps1 catalyzes hydrolysis of inorganic polyphosphates, 5-IP7, 1-IP7, and 1,5-IP8 in vitro, with a ~twofold preference for 1-IP7 over 5-IP7. aps1∆ cells have twofold higher levels of 1-IP7 than wild-type cells. An aps1∆ siw14∆ double mutation is lethal because excessive 1,5-IP8 triggers derepression of tgp1, leading to toxic uptake of glycerophosphocholine.

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Borneol, camphor, and bornyl acetate are highly promising monoterpenoids widely used in medicine, flavor, food, and chemical applications. Bornyl diphosphate (BPP) serves as a common precursor for the biosynthesis of these monoterpenoids. Although bornyl diphosphate synthase (BPPS) that catalyzes the cyclization of geranyl diphosphate (GPP) to BPP has been identified in multiple plants, the enzyme responsible for the hydrolysis of BPP to produce borneol has not been reported. Here, we conducted in vitro and in vivo functional characterization to identify the Nudix hydrolase WvNUDX24 from W. villosa, which specifically catalyzes the hydrolysis of BPP to generate bornyl phosphate (BP), and then BP forms borneol under the action of phosphatase. Subcellular localization experiments indicated that the hydrolysis of BPP likely occurs in the cytoplasm. Furthermore, site-directed mutagenesis experiments revealed that four critical residues (R84, S96, P98, and G99) for the hydrolysis activity of WvNUDX24. Additionally, the functional identification of phosphatidic acid phosphatase (PAP) demonstrated that WvPAP5 and WvPAP10 were able to hydrolyze geranylgeranyl diphosphate (GGPP) and farnesyl diphosphate (FPP) to generate geranylgeranyl phosphate (GGP) and farnesyl phosphate (FP), respectively, but could not hydrolyze BPP, GPP, and neryl diphosphate (NPP) to produce corresponding monophosphate products. These findings highlight the essential role of WvNUDX24 in the first step of BPP hydrolysis to produce borneol and provide genetic elements for the production of BPP-related terpenoids through plant metabolic engineering and synthetic biology.

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Thiopurine is metabolized to 6-thio-(deoxy) guanosine triphosphate (6-thio-(d) GTP), which is then incorporated into DNA or RNA and causes cytotoxicity. Nudix hydrolase 15 (NUDT15) reduces the cytotoxic effects of thiopurine by converting 6-thio-(d) GTP to 6-thio-(d) guanosine monophosphate (6-thio-(d) GMP). NUDT15 polymorphisms like the Arg139Cys variant are strongly linked to thiopurine-induced severe leukocytopenia and alopecia. Therefore, measurement of NUDT15 enzymatic activity in individual patients can help predict thiopurine tolerability and adjust the dosage. We aimed to develop a quantitative assay for NUDT15 enzymatic activity in human blood samples. Blood samples were collected from donors whose NUDT15 genetic status was determined. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to assess the 6-thio-GTP metabolic activity in cell extracts. Because 6-thio-guanosine diphosphate (6-thio-GDP) and 6-thio-GMP were generated upon incubation of 6-thio-GTP with human blood cell extracts, the method detecting 6-thio-GTP, 6-thio-GDP, and 6-thio-GMP was validated. All three metabolites were linearly detected, and the lower limit of quantification (LLOQ) of 6-thio-GTP, 6-thio-GDP, and 6-thio-GMP were 5 µM, 1 µM, and 2 µM, respectively. Matrix effects of human blood cell extracts to detect 6-thio-GTP, 6-thio-GDP, and 6-thio-GMP were 99.0 %, 100.5 %, and 101.4 %, respectively, relative to the signals in the absence of blood cell extracts. The accuracy and precision of the method and the stability of the samples were also assessed. Using this established method, the genotype-dependent differences in NUDT15 activities were successfully determined using cell extracts derived from human blood cells with NUDT15 wild-type (WT) or Arg139Cys variant and 6-thio-GTP (100 µM) as a substrate (18.1, 14.9, and 6.43 µM/h/106 cells for WT, Arg139Cys heterozygous, and homozygous variant, respectively). We developed a method for quantifying intracellular NUDT15 activity in peripheral blood mononuclear cells (PBMCs), which we defined as the conversion of 6-thio-GTP to 6-thio-GMP. Although PBMCs preparation takes some time, its reproducibility in experiments makes it a promising candidate for clinical application. This method can tell the difference between WT and Arg139Cys homozygous blood samples. Even in patients with WT NUDT15, WT samples showed variations in NUDT15 activity, which may correlate with variations in thiopurine dosage.

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Nudix hydrolases comprise a large and ubiquitous protein superfamily that catalyzes the hydrolysis of a nucleoside diphosphate linked to another moiety X (Nudix). Sulfolobus acidocaldarius possesses four Nudix domain-containing proteins (SACI_RS00730/Saci_0153, SACI_RS02625/Saci_0550, SACI_RS00060/Saci_0013/Saci_NudT5, and SACI_RS00575/Saci_0121). Deletion strains were generated for the four individual Nudix genes and for both Nudix genes annotated to encode ADP-ribose pyrophosphatases (SACI_RS00730, SACI_RS00060) and did not reveal a distinct phenotype compared to the wild-type strain under standard growth conditions, nutrient stress or heat stress conditions. We employed RNA-seq to establish the transcriptome profiles of the Nudix deletion strains, revealing a large number of differentially regulated genes, most notably in the ΔSACI_RS00730/SACI_RS00060 double knock-out strain and the ΔSACI_RS00575 single deletion strain. The absence of Nudix hydrolases is suggested to impact transcription via differentially regulated transcriptional regulators. We observed downregulation of the lysine biosynthesis and the archaellum formation iModulons in stationary phase cells, as well as upregulation of two genes involved in the de novo NAD+ biosynthesis pathway. Furthermore, the deletion strains exhibited upregulation of two thermosome subunits (α, ß) and the toxin-antitoxin system VapBC, which are implicated in the archaeal heat shock response. These results uncover a defined set of pathways that involve archaeal Nudix protein activities and assist in their functional characterization.

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Contamination of foods and feeds with Ochratoxin A (OTA) is a global problem, and its detoxification is challenging. In this study, Bacillus velezensis IS-6 culture isolate supernatant degraded 1.5 g/mL OTA by 89% after 24 h of incubation at 37 °C, whereas viable cells and intra-cell extracts were less effective. The OTA degradation by B. velezensis IS-6 was an enzymatic process mediated by the culture supernatant. The degradation activity was optimal at 37 °C and pH 7.0, and Fe2+ and Cu2+ ions enhanced the OTA degradation. The LC-MS/MS analysis confirmed that structure of OTA was modified, resulting in the production of OTα that was less toxic than OTA. The transcriptomic analysis of B. velezensis IS-6 showed that 38 differentially expressed genes (DEGs) were significantly up-regulated, and 24 DEGs were down-regulated after treatment with OTA. A novel OTA degradation enzyme Nudix hydrolase Nh-9 was successfully cloned and characterized from the up-regulated genes. The recombinant Nh-9 enzyme was overexpressed in Escherichia coli BL21 and purified by affinity chromatography, exhibiting 68% degradation activity against 1.0 µg/mL OTA at 37 °C in 24 h. The degraded product by the Nh-9 enzyme was identified as the less toxic OTα by LC-MS/MS. According to the findings, it can be inferred that Nh-9 is the main OTA-degrading enzyme in B. velezensis IS-6. Furthermore, OTA may be co-degraded by Nh-9, carboxylesterase, signal peptidase, and other degrading agents that are yet to be discovered in this strain.

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NUDT15, also known as MTH2, is a member of the NUDIX protein family that catalyzes the hydrolysis of nucleotides and deoxynucleotides, as well as thioguanine analogues. NUDT15 has been reported as a DNA sanitizer in humans, and more recent studies have shown that some genetic variants are related to a poor prognosis in neoplastic and immunologic diseases treated with thioguanine drugs. Despite this, the role of NUDT15 in physiology and molecular biology is quite unclear, as is the mechanism of action of this enzyme. The existence of clinically relevant variants has prompted the study of these enzymes, whose capacity to bind and hydrolyze thioguanine nucleotides is still poorly understood. By using a combination of biomolecular modeling techniques and molecular dynamics, we have studied the monomeric wild type NUDT15 as well as two important variants, R139C and R139H. Our findings reveal not only how nucleotide binding stabilizes the enzyme but also how two loops are responsible for keeping the enzyme in a packed, close conformation. Mutations in α2 helix affect a network of hydrophobic and π-interactions that enclose the active site. This knowledge contributes to the understanding of NUDT15 structural dynamics and will be valuable for the design of new chemical probes and drugs targeting this protein.Communicated by Ramaswamy H. Sarma.

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Human MutT homolog 1 (MTH1), also known as Nudix-type motif 1 (NUDT1), hydrolyzes 8-oxo-dGTP and 2-oxo-dATP with broad substrate recognition and has attracted attention in anticancer therapeutics. Previous studies on MTH1 have proposed that the exchange of the protonation state between Asp119 and Asp120 is essential for the broad substrate recognition of MTH1. To understand the relationship between protonation states and substrate binding, we determined the crystal structures of MTH1 at pH 7.7-9.7. With increasing pH, MTH1 gradually loses its substrate-binding ability, indicating that Asp119 is deprotonated at pH 8.0-9.1 in 8-oxo-dGTP recognition and Asp120 is deprotonated at pH 8.6-9.7 in 2-oxo-dATP recognition. These results confirm that MTH1 recognizes 8-oxo-dGTP and 2-oxo-dATP by exchanging the protonation state between Asp119 and Asp120 with higher pKa .

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To infect plants, pathogenic fungi secrete small proteins called effectors. Here, we describe the catalytic activity and potential virulence function of the Nudix hydrolase effector AvrM14 from the flax rust fungus (Melampsora lini). We completed extensive in vitro assays to characterise the enzymatic activity of the AvrM14 effector. Additionally, we used in planta transient expression of wild-type and catalytically dead AvrM14 versions followed by biochemical assays, phenotypic analysis and RNA sequencing to unravel how the catalytic activity of AvrM14 impacts plant immunity. AvrM14 is an extremely selective enzyme capable of removing the protective 5' cap from mRNA transcripts in vitro. Homodimerisation of AvrM14 promoted biologically relevant mRNA cap cleavage in vitro and this activity was conserved in related effectors from other Melampsora spp. In planta expression of wild-type AvrM14, but not the catalytically dead version, suppressed immune-related reactive oxygen species production, altered the abundance of some circadian-rhythm-associated mRNA transcripts and reduced the hypersensitive cell-death response triggered by the flax disease resistance protein M1. To date, the decapping of host mRNA as a virulence strategy has not been described beyond viruses. Our results indicate that some fungal pathogens produce Nudix hydrolase effectors with in vitro mRNA-decapping activity capable of interfering with plant immunity.

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INTRODUCTION: Mercaptopurine (6-MP) is the backbone of the consolidation and maintenance therapy for paediatric acute lymphoblastic leukaemia (ALL). Nevertheless, it can cause critical myelosuppression. Predicting adverse reactions to 6-MP often involves the investigation of pharmacogenetic variants; in particular thiopurine S-methyltransferase (TPMT) and nudix hydrolase 15 (NUDT15). Lately, NUDT15 variants have been shown to play a significant pharmacogenetic role in predicting 6-MP intolerance in children of Asian descent. CASE REPORT: We present a six-year-old male child of Indian origin with persistent cytopenia after treatment. This prompted targeted sequencing of the genes TPMT and NUD15. The results revealed two copies of the variant of NUD15 rs116855232, that is, NUDT15*2 genotype. MANAGEMENT AND OUTCOME: Since the NUDT15*2 allele classified the patient as a poor metabolizer, he was restarted on a low dose of 6-MP, which he tolerated. DISCUSSION: Individuals with the NUDT15*2allele (*2/*2 genotype) are poor metabolizers of thiopurines which results in an adverse reaction to 6-MP. About 3.5% of Indians show variations in the TPMT gene as compared to 19.4% variations observed in NUDT15, which makes the latter a more reliable disease marker.

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Pediatric inflammatory bowel disease (PIBD) is rising rapidly in many industrialised and affluent areas in the Asia Pacific region. Current available guidelines, mainly from Europe and North America, may not be completely applicable to clinicians caring for children with PIBD in this region due to differences in disease characteristics and regional resources constraints. This position paper is an initiative from the Asian Pan-Pacific Society for Pediatric Gastroenterology, Hepatology and Nutrition (APPSPGHAN) with the aim of providing an up-to-date, evidence-based approach to PIBD in the Asia Pacific region, taking into consideration the unique disease characteristics and financial resources available in this region. A group of pediatric gastroenterologists with special interest in PIBD performed an extensive literature search covering epidemiology, disease characteristics and natural history, management and monitoring. Gastrointestinal infections, including tuberculosis, need to be excluded before diagnosing IBD. In some populations in Asia, the Nudix Hydrolase 15 (NUD15) gene is a better predictor of leukopenia induced by azathioprine than thiopurine-S-methyltransferase (TPMT). The main considerations in the use of biologics in the Asia Pacific region are high cost, ease of access, and potential infectious risk, especially tuberculosis. Conclusion: This position paper provides a useful guide to clinicians in the medical management of children with PIBD in the Asia Pacific region.

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DNA damage leads to rapid synthesis of poly(ADP-ribose) (pADPr), which is important for damage signaling and repair. pADPr chains are removed by poly(ADP-ribose) glycohydrolase (PARG), releasing free mono(ADP-ribose) (mADPr). Here, we show that the NUDIX hydrolase NUDT5, which can hydrolyze mADPr to ribose-5-phosphate and either AMP or ATP, is recruited to damage sites through interaction with PARG. NUDT5 does not regulate PARP or PARG activity. Instead, loss of NUDT5 reduces basal cellular ATP levels and exacerbates the decrease in cellular ATP that occurs during DNA repair. Further, loss of NUDT5 activity impairs RAD51 recruitment, attenuates the phosphorylation of key DNA-repair proteins, and reduces both H2A.Z exchange at damage sites and repair by homologous recombination. The ability of NUDT5 to hydrolyze mADPr, and/or regulate cellular ATP, may therefore be important for efficient DNA repair. Targeting NUDT5 to disrupt PAR/mADPr and energy metabolism may be an effective anti-cancer strategy.

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Nudix hydrolase (NUDX) hydrolyzes 8-oxo-(d)GTP to reduce the levels of oxidized nucleotides in the cells. 8-oxo-(d)GTP produced by reactive oxygen species (ROS) is incorporated into DNA/RNA and mispaired with adenine, causing replicational and transcriptional errors. Here, we identified a rice OsNUDX2 gene, whose expression level was increased 15-fold under UV-C irradiation. The open reading frame of the OsNUDX2 gene, which encodes 776 amino acid residues, was cloned into Escherichia coli cells to produce the protein of 100 kDa. The recombinant protein hydrolyzed 8-oxo-dGTP, in addition to dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP), as did Arabidopsis AtNUDX1; whereas the amino acid sequence of OsNUDX2 had 18% identity with AtNUDX1. OsNUDX2 had 14% identity with barley HvNUDX12, which hydrolyzes 8-oxo-dGTP and diadenosine tetraphosphates. Suppression of the lacZ amber mutation caused by the incorporation of 8-oxo-GTP into mRNA was prevented to a significant degree when the OsNUDX2 gene was expressed in mutT-deficient E. coli cells. These results suggest that the different substrate specificity and identity among plant 8-oxo-dGTP-hydrolyzing NUDXs and OsNUDX2 reduces UV stress by sanitizing the oxidized nucleotides.

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Plants accumulate reactive oxygen species (ROS) that may damage the cells under prolonged stress conditions. Reduction of the excessive ROS production can alleviate oxidative damage and enhance the survival rates under stress. TLDc-containing protein (TLD) was reported to confer tolerance to oxidative stress, but the regulatory mechanism of TLD remains unclear. In this study, we ectopically overexpressed the Ipomoea batatas TLDc gene (IbTLD) in tobacco and characterized its functions. RNA-sequencing analysis and Gene Ontology term enrichment analysis revealed that IbTLD up-regulates auxin-responsive genes in response to oxidative stress. Under salinity stress, the IbTLD transgenic lines showed higher germination rates, chlorophyll contents, and root lengths than wild type (W38). In addition, the IbTLD transgenic lines showed higher expression of ROS scavenging genes, nudix hydrolases, ROS scavenging enzyme activity, and lesser DNA damage compared to W38 under salinity stress. Therefore, our results suggest that IbTLD activates the expression of ROS scavenging genes and confers tolerance to salinity stress in planta.

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Geraniol is a potent tea odorant and exists mainly as geranyl glycoside in Camellia sinensis. Understanding the mechanisms of geraniol biosynthesis at molecular levels in tea plants is of great importance for practical improvement of tea aroma. In this study, geraniol and its glycosides from tea plants were examined using liquid chromatography coupled with mass spectrometry. Two candidate geraniol synthase (GES) genes (CsTPS) and two Nudix hydrolase genes (CsNUDX1-cyto and CsNUDX1-chlo) from the tea genome were functionally investigated through gene transcription manipulation and gene chemical product analyses. Our data showed that in tea leaves, levels of geranyl ß-primeveroside were dramatically higher than those of geranyl ß-glucoside, while free geraniol was undetectable in this study. A tempo-spatial variation of geranyl ß-primeveroside abundance in tea plants existed, with high levels in young and green tissues and low levels in mature or non-green tissues. Cytosolic CsNUDX1-cyto showed higher hydrolysis activity of geranyl-pyrophosphate to geranyl-monophosphate (GP) in vitro than did chloroplastidial CsNUDX1-chlo. A transgenic study revealed that expression of CsNUDX1-cyto resulted in significantly more geranyl ß-primeveroside in transgenic Nicotiana benthamiana compared with non-transgenic wild-type, whereas expression of CsNUDX1-chlo had no effect. An antisense oligo-deoxynucleotide study confirmed that suppression of CsNUDX1-cyto transcription in tea shoots led to a significant decrease in geranyl ß-primeveroside abundance. Additionally, CsNUDX1-cyto transcript levels and geranyl ß-primeveroside abundances shared the same tempo-spatial patterns in different organs in the tea cultivar "Shucha Zao," indicating that CsNUDX1-cyto is important for geranyl ß-primeveroside formation in tea plants. Results also suggested that neither of the two candidate GES genes in tea plants did not function as GES in transgenic N. benthamiana. All our data indicated that CsNUDX1-cyto is involved in geranyl ß-primeveroside production in tea plants. Our speculation about possible conversion from the chemical product of CsNUDX1-cyto to geranyl ß-primeveroside in plants was also discussed.

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Remdesivir and molnupiravir have gained considerable interest because of their demonstrated activity against SARS-CoV-2. These antivirals are converted intracellularly to their active triphosphate forms remdesivir-TP and molnupiravir-TP. Cellular hydrolysis of these active metabolites would consequently decrease the efficiency of these drugs; however, whether endogenous enzymes that can catalyze this hydrolysis exist is unknown. Here, we tested remdesivir-TP as a substrate against a panel of human hydrolases and found that only Nudix hydrolase (NUDT) 18 catalyzed the hydrolysis of remdesivir-TP with notable activity. The kcat/Km value of NUDT18 for remdesivir-TP was determined to be 17,700 s-1M-1, suggesting that NUDT18-catalyzed hydrolysis of remdesivir-TP may occur in cells. Moreover, we demonstrate that the triphosphates of the antivirals ribavirin and molnupiravir are also hydrolyzed by NUDT18, albeit with lower efficiency than Remdesivir-TP. Low activity was also observed with the triphosphate forms of sofosbuvir and aciclovir. This is the first report showing that NUDT18 hydrolyzes triphosphates of nucleoside analogs of exogenous origin, suggesting that NUDT18 can act as a cellular sanitizer of modified nucleotides and may influence the antiviral efficacy of remdesivir, molnupiravir, and ribavirin. As NUDT18 is expressed in respiratory epithelial cells, it may limit the antiviral efficacy of remdesivir and molnupiravir against SARS-CoV-2 replication by decreasing the intracellular concentration of their active metabolites at their intended site of action.

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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.

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Nudix hydrolases are conserved enzymes ubiquitously present in all kingdoms of life. Recent research revealed that several Nudix hydrolases are involved in terpenoid metabolism in plants. In modern roses, RhNUDX1 is responsible for formation of geraniol, a major compound of rose scent. Nevertheless, this compound is produced by monoterpene synthases in many geraniol-producing plants. As a consequence, this raised the question about the origin of RhNUDX1 function and the NUDX1 gene evolution in Rosaceae, in wild roses or/and during the domestication process. Here, we showed that three distinct clades of NUDX1 emerged in the Rosoidae subfamily (Nudx1-1 to Nudx1-3 clades), and two subclades evolved in the Rosa genus (Nudx1-1a and Nudx1-1b subclades). We also showed that the Nudx1-1b subclade was more ancient than the Nudx1-1a subclade, and that the NUDX1-1a gene emerged by a trans-duplication of the more ancient NUDX1-1b gene. After the transposition, NUDX1-1a was cis-duplicated, leading to a gene dosage effect on the production of geraniol in different species. Furthermore, the NUDX1-1a appearance was accompanied by the evolution of its promoter, most likely from a Copia retrotransposon origin, leading to its petal-specific expression. Thus, our data strongly suggest that the unique function of NUDX1-1a in geraniol formation was evolved naturally in the genus Rosa before domestication.

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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.

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