Cleavage of oxidized guanine nucleotide and ADP sugar by human NUDT5 protein

MutT-related proteins, including Escherichia coli MutT and the human MTH1 (NUDT1), degrade 8-oxo-7, 8-dihydrodeoxyguanosine triphosphate (8-oxo-dGTP) to 8-oxo-dGMP and thereby prevent mutations caused by the misincorporation of 8-oxoguanine into DNA. The human NUDT5, which has an intrinsic activity to cleave ADP sugars to AMP and sugar phosphate, possesses the ability to degrade 8-oxo-dGDP to the monophosphate. Since 8-oxo-dGDP and 8-oxo-dGTP are interconvertible by cellular enzymes, NUDT5 has the potential to prevent errors during DNA replication. The two activities associated with NUDT5 exhibit different pH dependencies; the optimum for the cleavage of ADP ribose is pH 7-9, while that for 8-oxo-dGDPase is around pH 10. The kinetic parameters for the two types of reactions indicated that ADP ribose is a better substrate for NUDT5 compared with oxidized guanine nucleotides. The 8-oxo-dGDP cleavage was competitively inhibited by ADP ribose and its reaction product, AMP, and in reverse, the cleavage of ADP ribose was inhibited by 8-oxo-dGDP. These results imply that the two types of substrates may share the same binding site for catalysis.     

NDX-1 protein hydrolyzes 8-oxo-7, 8-dihydrodeoxyguanosine-5'-diphosphate to sanitize oxidized nucleotides and prevent oxidative stress in Caenorhabditis elegans

8-oxo-dGTP is generated in the nucleotide pool by direct oxidation of dGTP or phosphorylation of 8-oxo-dGDP. It can be incorporated into DNA during replication, which would result in mutagenic consequences. The frequency of spontaneous mutations remains low in cells owing to the action of enzymes degrading such mutagenic substrates. Escherichia coli MutT and human MTH1 hydrolyze 8-oxo-dGTP to 8-oxo-dGMP. Human NUDT5 as well as human MTH1 hydrolyze 8-oxo-dGDP to 8-oxo-dGMP. These enzymes prevent mutations caused by misincorporation of 8-oxo-dGTP into DNA. In this study, we identified a novel MutT homolog (NDX-1) of Caenorhabditis elegans that hydrolyzes 8-oxo-dGDP to 8-oxo-dGMP. NDX-1 did not hydrolyze 8-oxo-dGTP, 2-hydroxy-dATP or 2-hydroxy-dADP. Expression of NDX-1 significantly reduced spontaneous A:T to C:G transversions and mitigated the sensitivity to a superoxide-generating agent, methyl viologen, in an E. coli mutT mutant. In C. elegans, RNAi of ndx-1 did not affect the lifespan of the worm. However, the sensitivity to methyl viologen and menadione bisulfite of the ndx-1-RNAi worms was enhanced compared with that of the control worms. These facts indicate that NDX-1 is involved in sanitization of 8-oxo-dGDP and plays a critical role in defense against oxidative stress in C. elegans.     

A novel mechanism for preventing mutations caused by oxidation of guanine nucleotides

MutT-related proteins, including the Escherichia coli MutT and human MutT homologue 1 (MTH1) proteins, degrade 8-oxo- 7,8-dihydrodeoxyguanosine triphosphate (8-oxo-dGTP) to a monophosphate, thereby preventing mutations caused by the misincorporation of 8-oxoguanine into DNA. Here, we report that human cells have another mechanism for cleaning up the nucleotide pool to ensure accurate DNA replication. The human Nudix type 5 (NUDT5) protein hydrolyses 8-oxo-dGDP to monophosphate with a K_m of 0.77 µM, a value considerably lower than that for ADP sugars, which were originally identified as being substrates of NUDT5. NUDT5 hydrolyses 8-oxo-dGTP only at very low levels, but is able to substitute for MutT when it is defective. When NUDT5 is expressed in E. coli mutT⁻ cells, the increased frequency of spontaneous mutations is decreased to normal levels. Considering the enzymatic parameters of MTH1 and NUDT5 for oxidized guanine nucleotides, NUDT5 might have a much greater role than MTH1 in preventing the occurrence of mutations that are caused by the misincorporation of 8-oxoguanine in human cells.     

Structure of human MTH1, a Nudix family hydrolase that selectively degrades oxidized purine nucleoside triphosphates

Oxygen radicals generated through normal cellular respiration processes can cause mutations in genomic and mitochondrial DNA. Human MTH1 hydrolyzes oxidized purine nucleoside triphosphates, such as 8-oxo-dGTP and 2-hydroxy-dATP, to monophosphates, thereby preventing the misincorporation of these oxidized nucleotides during replication. Here we present the solution structure of MTH1 solved by multidimensional heteronuclear NMR spectroscopy. The protein adopts a fold similar to that of Escherichia coli MutT, despite the low sequence similarity between these proteins outside the conserved Nudix motif. The substrate-binding pocket of MTH1, deduced from chemical shift perturbation experiments, is located at essentially the same position as in MutT; however, a pocket-forming helix is largely displaced in MTH1 (approximately 9 A) such that the shape of the pocket differs between the two proteins. Detailed analysis of the pocket-forming residues enabled us to identify Asn33 as one of the key residues in MTH1 for discriminating the oxidized form of purine, and mutation of this residue modifies the substrate specificity. We also show that MTH1 catalyzes hydrolysis of 8-oxo-dGTP through nucleophilic substitution of water at the beta-phosphate.     

Mouse MTH2 protein which prevents mutations caused by 8-oxoguanine nucleotides

MutT-related proteins degrade 8-oxo-7,8-dihydrodeoxyguanosine triphosphate (8-oxo-dGTP), a mutagenic substrate for DNA synthesis, in the nucleotide pool, thereby preventing DNA replication errors. During a search of GenBank EST database, we found a new member of MutT-related protein, MTH2, which possesses the 23-amino acid MutT module. The cloned mouse MTH2 (mMTH2) cDNA was expressed in Escherichia coli mutT(-) cells and the protein was purified. mMTH2 protein hydrolyzes 8-oxo-dGTP to 8-oxo-dGMP, with Km of 32 microM. Expression of cDNA for mMTH2 reduced significantly the elevated level of spontaneous mutation frequency of E. coli mutT(-) cells. Thus, MTH2 has a potential to protect the genetic material from the untoward effects of endogenous oxygen radicals. MTH2 could act as an MTH1 redundancy factor.     

Exploration of Novel MTH1 Inhibitors Using Fragment-Based De Novo Design,Virtual Screening,and Reverse Virtual Screening Methods

Russian Journal of Bioorganic Chemistry - MTH1 (MutT homologue 1, NUDT1), a member of the Nudix phosphohydrolase superfamily of enzymes, was speculated to contribute to hampering tumor growth based...     

Mammalian enzymes for preventing transcriptional errors caused by oxidative damage

8-Oxo-7,8-dihydroguanine (8-oxoGua) is produced in cells by reactive oxygen species normally formed during cellular metabolic processes. This oxidized base can pair with both adenine and cytosine, and thus the existence of this base in messenger RNA would cause translational errors. The MutT protein of Escherichia coli degrades 8-oxoGua-containing ribonucleoside di- and triphosphates to the monophosphate, thereby preventing the misincorporation of 8-oxoGua into RNA. Here, we show that for human the MutT-related proteins, NUDT5 and MTH1 have the ability to prevent translational errors caused by oxidative damage. The increase in the production of erroneous proteins by oxidative damage is 28-fold over the wild-type cells in E.coli mutT deficient cells. By the expression of NUDT5 or MTH1 in the cells, it is reduced to 1.4- or 1.2-fold, respectively. NUDT5 and MTH1 hydrolyze 8-oxoGDP to 8-oxoGMP with V(max)/K(m) values of 1.3 x 10(-3) and 1.7 x 10(-3), respectively, values which are considerably higher than those for its normal counterpart, GDP (0.1-0.5 x 10(-3)). MTH1, but not NUDT5, possesses an additional activity to degrade 8-oxoGTP to the monophosphate. These results indicate that the elimination of 8-oxoGua-containing ribonucleotides from the precursor pool is important to ensure accurate protein synthesis and that both NUDT5 and MTH1 are involved in this process in human cells.     

Molecular genetics and structural biology of human MutT homolog, MTH1

The human MTH1 gene located on chromosome 7p22 consists of 5 major exons. MTH1 gene produces seven types of mRNAs and the B-type mRNAs with exon 2b-2c segments direct synthesis of three forms of MTH1 polypeptides (p22, p21, and p18) by alternative initiation of translation, while the others encode only p18. In human cells, p18, the major form is mostly localized in the cytoplasm with some in the mitochondria. A single nucleotide polymorphism (SNP) in exon 2, which is tightly liked to another SNP (GTG83/ATG83), creates an additional alternative in-frame AUG in B-type MTH1 mRNAs yielding the fourth MTH1 polypeptide, p26 that possesses an additional mitochondrial targeting signal. These SNPs are likely to be one of the risk factors for cancer or for neuronal degeneration. The 30 amino acid residues are identical between MTH1 and MutT, and there is a highly conserved region consisting of 23 residues (MTH1: Gly36 to Gly58), with 14 identical residues. A chimeric protein in which the 23 residue sequence of MTH1 was replaced with that of MutT, retains the capability to hydrolyze 8-oxo-dGTP, indicating that the 23 residue sequences of MTH1 and MutT are functionally and structurally equivalent, and constitute a functional phosphohydrolase module. Saturated mutagenesis of the module in MTH1 indicated that an amphipathic property of the alpha-helix I consisting of 14 residues of the module (Thr44 to Gly58) is essential to maintain the stable catalytic surface for 8-oxo-dGTPase. MTH1 but not MutT efficiently hydrolyzes two forms of oxidized dATP, 2-hydroxy-dATP and 8-oxo-dATP, as well as 8-oxo-dGTP and 8-oxo-GTP. Thus, MTH1 is designated as the oxidized purine nucleoside triphosphatase and has a much wider substrate specificity than MutT. There is a significant homology between MTH1 protein and the C-terminal half of human MYH protein, which may be involved in the recognition of 8-oxoguanine and 2-hydroxyadenine.     

Functional significance of the conserved residues for the 23-residue module among MTH1 and MutT family proteins

Human MTH1 and Escherichia coli MutT proteins hydrolyze 7, 8-dihydro-8-oxo-dGTP (8-oxo-dGTP) to monophosphate, thus avoiding the incorporation of 8-oxo-7,8-dihydroguanine into nascent DNA. Although only 30 amino acid residues (23%) are identical between MTH1 and MutT, there is a highly conserved region consisting of 23 residues (MTH1, Gly(36)-Gly(58)) with 14 identical residues. A chimeric protein MTH1-Ec, in which the 23-residue sequence of MTH1 was replaced with that of MutT, retains its capability to hydrolyze 8-oxo-dGTP, thereby indicating that the 23-residue sequences of MTH1 and MutT are functionally and structurally equivalent and constitute functional modules. By saturation mutagenesis of the module in MTH1, 14 of the 23 residues proved to be essential to exert 8-oxo-dGTPase activity. For the other 9 residues (40, 42, 44, 46, 47, 49, 50, 54, and 58), positive mutants were obtained, and Arg(50) can be replaced with hydrophobic residues (Val, Leu, or Ile), with a greater stability and higher specific activity of the enzyme. Indispensabilities of Val(39), Ile(45), and Leu(53) indicate that an amphipathic property of alpha-helix I consisting of 14 residues of the module (Thr(44)-Gly(58)) is essential to maintain the stable catalytic surface for 8-oxo-dGTPase.     

Inhibition of 8-oxo-2'-deoxyguanosine 5'-triphosphate pyrophosphohydrolase (8-oxo-dGTPase) activity of the antimutagenic human MTH1 protein by nucleoside 5'-diphosphates

The hMTH1 protein, a human homologue of E. coli MutT protein, is an enzyme converting 8-oxo-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP) to 8-oxo-2'-deoxyguanosine 5'-monophosphate (8-oxo-dGMP) and inorganic pyrophosphate. It is thought to play an antimutagenic role by preventing the incorporation of promutagenic 8-oxo-dGTP into DNA. As found in our previous investigations, 8-oxo-2'-deoxyguanosine 5'-diphosphate (8-oxo-dGDP) strongly inhibited 8-oxo-dGTPase activity of MTH1. Following this finding, in the present study we have tested the canonical ribo- and deoxyribonucleoside 5'-diphosphates (NDPs and dNDPs) for possible inhibition of 8-oxo-dGTP hydrolysis by hMTH1 extracted from CCRF-CEM cells (a human leukemia cell line). Among them, the strongest inhibitors appeared to be dGDP (Ki=74 microM), dADP (Ki=147 microM), and GDP (Ki=502 microM). Other dNDPs and NDPs, such as dCDP, dTDP, ADP, CDP, and UDP were much weaker inhibitors, with Ki in the millimolar range. Based on the present results and published data, we estimate that the strongest inhibitors, dGDP and dADP, at physiological concentrations not exceeding 5 microM and GDP at mean concentration of 30 microM, taken together, can decrease the cellular hMTH1 enzymatic activity vs. 8-oxo-dGTP (expected to remain below 500 pM) by up to 15%. The other five NDPs and dNDPs tested cannot markedly affect this activity.     

MTH1, an oxidized purine nucleoside triphosphatase, prevents the cytotoxicity and neurotoxicity of oxidized purine nucleotides

In human and rodent cells, MTH1, an oxidized purine nucleoside triphosphatase, efficiently hydrolyzes oxidized dGTP, GTP, dATP and ATP such as 2'-deoxy-8-oxoguanosine triphosphate (8-oxo-dGTP) and 2'-deoxy-2-hydroxyadenosine triphosphate (2-OH-dATP) in nucleotide pools, thus avoiding their incorporation into DNA or RNA. MTH1 is expressed in postmitotic neurons as well as in proliferative tissues, and it is localized both in the mitochondria and nucleus, thus suggesting that MTH1 plays an important role in the prevention of the mutagenicity and cytotoxicity of such oxidized purines as 8-oxoG which are known to accumulate in the cellular genome. Our recent studies with MTH1-deficient mice or cells revealed that MTH1 efficiently minimizes accumulation of 8-oxoG in both nuclear and mitochondrial DNA in the mouse brain as well as in cultured cells, thus contributing to the protection of the brain from oxidative stress.     

An oxidized purine nucleoside triphosphatase,MTH1, suppresses cell death caused by oxidative stress

MTH1 hydrolyzes oxidized purine nucleoside triphosphates such as 8-oxo-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP) and 2-hydroxy-2'-deoxyadenosine 5'-triphosphate (2-OH-dATP) and thus protects cells from damage caused by their misincorporation into DNA. In the present study, we established MTH1-null mouse embryo fibroblasts that were highly susceptible to cell dysfunction and death caused by exposure to H2O2, with morphological features of pyknosis and electron-dense deposits accumulated in mitochondria. The cell death observed was independent of both poly(ADP-ribose) polymerase and caspases. A high performance liquid chromatography tandem mass spectrometry analysis and immunofluorescence microscopy revealed a continuous accumulation of 8-oxo-guanine both in nuclear and mitochondrial DNA after exposure to H2O2. All of the H2O2-induced alterations observed in MTH1-null mouse embryo fibroblasts were effectively suppressed by the expression of wild type human MTH1 (hMTH1), whereas they were only partially suppressed by the expression of mutant hMTH1 defective in either 8-oxo-dGTPase or 2-OH-dATPase activity. Human MTH1 thus protects cells from H2O2-induced cell dysfunction and death by hydrolyzing oxidized purine nucleotides including 8-oxo-dGTP and 2-OH-dATP, and these alterations may be partly attributed to a mitochondrial dysfunction.     

Diverse substrate recognition and hydrolysis mechanisms of human NUDT5

Human NUDT5 (hNUDT5) hydrolyzes various modified nucleoside diphosphates including 8-oxo-dGDP, 8-oxo-dADP and ADP-ribose (ADPR). However, the structural basis of the broad substrate specificity remains unknown. Here, we report the crystal structures of hNUDT5 complexed with 8-oxo-dGDP and 8-oxo-dADP. These structures reveal an unusually different substrate-binding mode. In particular, the positions of two phosphates (α and β phosphates) of substrate in the 8-oxo-dGDP and 8-oxo-dADP complexes are completely inverted compared with those in the previously reported hNUDT5–ADPR complex structure. This result suggests that the nucleophilic substitution sites of the substrates involved in hydrolysis reactions differ despite the similarities in the chemical structures of the substrates and products. To clarify this hypothesis, we employed the isotope-labeling method and revealed that 8-oxo-dGDP is attacked by nucleophilic water at Pβ, whereas ADPR is attacked at Pα. This observation reveals that the broad substrate specificity of hNUDT5 is achieved by a diversity of not only substrate recognition, but also hydrolysis mechanisms and leads to a novel aspect that enzymes do not always catalyze the reaction of substrates with similar chemical structures by using the chemically equivalent reaction site.     

Analysis of MTH1 gene function in mice with targeted mutagenesis

Oxidative DNA damage is thought to contribute to carcinogenesis, ageing, and neurological degeneration. Further, the cumulative risk of cancer increases dramatically with age in humans. In general terms, cancer can be regarded as a degenerative disease of ageing. There is evidence for the accumulation of oxidative DNA damage with age based on studies mainly measuring an increase in 8-oxoguanine. 8-Oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP) is formed in the nucleotide pool of a cell during normal cellular metabolism. When 8-oxoguanine is incorporated into DNA causes mutation. Organisms possess 8-oxo-dGTPase, an enzyme that specifically degrades 8-oxo-dGTP to 8-oxo-dGMP. To analyze the function of MTH1 with 8-oxo-dGTPase activity in vivo, we generated a mouse line carrying a mutant MTH1 allele created by targeted gene disruption. MTH1 homozygous mutant mice were found to have a physically normal appearance, but seemed to have lost 8-oxo-dGTPase activity in liver extracts. When we examined the susceptibility of the mutant mice to spontaneous tumorigenesis, no significant difference was observed in survival rate of MTH1+/+ and MTH1-/- mice. However, pathological examination revealed a statistically significant difference in the incidence of tumors. More tumors were formed in lungs, livers, and stomachs of MTH1-/- mice than in those of the wild type mice. These studies with MTH1-null mutant mice provided an important insight into the role of this nucleotide sanitization enzyme in terms of the spontaneous tumorigenesis as well as mutagenesis caused by the oxygen-induced DNA damage.     

Cellular 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate pyrophosphohydrolase activity of human and mouse MTH1 proteins does not depend on the proliferation rate

Mammalian MTH1 proteins, homologs of Escherichia coli MutT, are enzymes decomposing 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP) to 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-monophosphate and inorganic pyrophosphate. They play an antimutagenic role by preventing the incorporation of promutagenic 8-oxo-dGTP into DNA. MTH1 gene expression is higher in some physiological types of mammalian cells and in numerous cancer cells, but the mechanism of that upregulation still remains unclear. It has been hypothesized that MTH1 expression might be associated with a proliferation rate of the cells. Therefore, we tested this hypothesis by comparing the functional levels of MTH1 gene expression measured as the 8-oxo-dGTPase activity of its protein products in normal mouse livers and hepatectomized regenerating livers. Although the proliferation rate of the hepatocytes in the regenerating livers was much higher than that in control livers, as confirmed by immunohistochemical assay of proliferating cell nuclear antigen, the 8-oxo-dGTPase activity was not different. In a second approach, we used 57 lines of human cancer cells in which 8-oxo-dGTPase activity was measured and confronted with cell population doubling time. No significant correlations between 8-oxo-dGTPase activity and proliferation rate were observed within groups of six leukemia, eight melanoma, nine lung, seven colon, six central nervous system, six ovarian, eight renal, and seven breast cancer cell lines. Thus, we conclude that the MTH1 expression manifested as the 8-oxo-dGTPase activity of its protein products in mammalian cells is not associated with proliferation rate. Our results will help in further testing of the hypothesis that MTH1 overexpression may be a specific marker of carcinogenesis and/or oxidative stress.     

MTH1 Substrate Recognition—An Example of Specific Promiscuity

MTH1 (NUDT1) is an oncologic target involved in the prevention of DNA damage. We investigate the way MTH1 recognises its substrates and present substrate-bound structures of MTH1 for 8-oxo-dGTP and 8-oxo-rATP as examples of novel strong and weak binding substrate motifs. Investigation of a small set of purine-like fragments using 2D NMR resulted in identification of a fragment with weak potency. The protein-ligand X-Ray structure of this fragment provides insight into the role of water molecules in substrate selectivity. Wider fragment screening by NMR resulted in three new protein structures exhibiting alternative binding configurations to the key Asp-Asp recognition element of the protein. These inhibitor binding modes demonstrate that MTH1 employs an intricate yet promiscuous mechanism of substrate anchoring through its Asp-Asp pharmacophore. The structures suggest that water-mediated interactions convey selectivity towards oxidized substrates over their non-oxidised counterparts, in particular by stabilization of a water molecule in a hydrophobic environment through hydrogen bonding. These findings may be useful in the design of inhibitors of MTH1.     

NUDT5 hydrolyzes oxidized deoxyribonucleoside diphosphates with broad substrate specificity

The human NUDT5 protein catalyzes the hydrolysis of 8-hydroxy-dGDP. To examine its substrate specificity, four oxidized deoxyribonucleotides (2-hydroxy-dADP, 8-hydroxy-dADP, 5-formyl-dUDP, and 5-hydroxy-dCDP) were incubated with the NUDT5 protein. Interestingly, all of the nucleotides, except for 5-hydroxy-dCDP, were hydrolyzed with various efficiencies. The kinetic parameters indicated that 8-hydroxy-dADP was hydrolyzed as efficiently as 8-hydroxy-dGDP. The hydrolyzing activities for their triphosphate counterparts were quite weak. These results suggest that the NUDT5 protein eliminates various oxidized deoxyribonucleoside diphosphates from the nucleotide pool and prevents their toxic effects.