Structure of Human MTH1, a Nudix Family Hydrolase That Selectively Degrades Oxidized Purine Nucleoside Triphosphates

Abstract

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-oxodGTP 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 (ϳ9 Å) such that the shape of the pocket differs between the two proteins. Detailed analysis of the pocket-forming residues enabled us to identify Asn 33 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 ␤-phosphate.Cellular DNA continually suffers assault from exogenous and endogenous agents that cause a wide variety of DNA modifications. Such modifications are often detrimental to the cell, leading to mutagenesis and carcinogenesis. Numerous enzymes have the important task of maintaining the integrity of DNA. These enzymes are generally well conserved from bacteria to humans.Oxygen radicals, which are spontaneously generated during normal cellular metabolism or by ionizing radiation or various chemicals, often attack nucleic acids, thereby generating modified bases in DNA (1, 2). Among these modified bases, the most abundant species, 8-oxo-7,8-dihydroguanine (8-oxo-G), 1 can pair with both cytosine and adenine with almost equal efficiency and consequently can induce A:T to C:G and G:C to T:A transversion mutations (3-5).Organisms are equipped with elaborate mechanisms to counteract the mutagenic effects of 8-oxo-G. In Escherichia coli, two glycosylases encoded by the mutM and mutY genes function to prevent mutation caused by the presence of 8-oxo-G in DNA. MutM protein removes 8-oxo-G paired with cytosine, whereas MutY protein removes adenine paired with 8-oxo-G (6 -9). To prevent further mutation through the presence of 8-oxo-dGTP, MutT hydrolyzes 8-oxo-dGTP to its monophosphate form, thereby preventing the oxidized purine from being misincorporated into genomic DNA. The importance of this enzyme has been underscored by the observation that deficiency of the mutT gene increases the occurrence of A:T to C:G transversion mutations 1000-fold (10, 11). The mechanism concerning the coordinated action of MutM, MutY, and MutT, which constitute the so-called "GO system," has been well characterized in prokaryotes. Protein factors with enzymatic activities similar to those of MutM, MutY, and MutT have been identified ...

Crystallization and preliminary X-ray analysis of human MTH1 complexed with two oxidized nucleotides, 8-oxo-dGMP and 2-oxo-dATP

“…SeMet-labelled hMTH1 was overexpressed under conditions of methionine-pathway inhibition (Doublié, 1997) in LeMaster Broth (LeMaster & Richards, 1985). Gel-filtration chromatography was performed using a HiLoad 16/60 Superdex 75 pg column (Amersham Biosciences) instead of a Sephacryl S-100 column (Mishima et al, 2004). The purified native protein and the SeMet derivative were concentrated to 6-12 mg ml À1 in 20 mM TrisHCl pH 7.5 and 5% glycerol and to 2-4 mg ml À1 in 20 mM Tris-HCl pH 7.5, respectively.…”

“…The full-length hMTH1 protein with no tags was expressed and purified as described previously (Mishima et al, 2004) with minor modifications. Native hMTH1 was overexpressed by transforming the pET8c/hMTH1 plasmid into E. coli strain BL21(DE3) cells cultured in LB broth.…”

“…Mutational analyses and NMR structures of hMTH1 suggest that Phe27, Trp117, Asp119 and Asn33 are the active-site residues associated with the recognition of 8-oxo-dGTP and 2-oxo-dATP; however, their mutants have different effects on the catalysis of 8-oxo-dGTP and 2-oxo-dATP (Sakai et al, 2002;Yoshimura et al, 2003;Mishima et al, 2004). For example, Asp119 is probably a key residue for the selective recognition of 2-oxo-dATP, because the D119A mutant completely abolishes the 2-oxo-dATPase activity but retains approximately 50% of the activity of the wild type with respect to 8-oxo-dGTPase (Yoshimura et al, 2003).…”

Differential expression profile analysis of DNA damage repair genes in CD133+/CD133− colorectal cancer cells

“…Three genes are involved in the modulation of nucleotide pools during the process of DNA damage repair in human cells (45). As an antimutagen, nucleoside diphosphate linked moiety X-type motif 1 (NUDT1) hydrolyzes oxidized purine nucleoside triphosphates, preventing the misincorporation of nucleotides (46). Downregulation of NUDT1 expression levels in CD133…”

Mycobacterium tuberculosis MutT1 (Rv2985) and ADPRase (Rv1700) Proteins Constitute a Two-stage Mechanism of 8-Oxo-dGTP and 8-Oxo-GTP Detoxification and Adenosine to Cytidine Mutation Avoidance

“…In humans, biochemical activities of several Nudix box proteins (NUDTs) have been identified (6,(15)(16)(17). NUDT1 (MTH1) converts 8-oxo-dGTP to 8-oxo-dGMP but does not utilize 8-oxo-dGDP as substrate.…”