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.
Fujii
Y
Y
Department of Biochemistry, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi Higashi-ku, Fukuoka 812-8582, Japan.
Shimokawa
H
H
Sekiguchi
M
M
Nakabeppu
Y
Y
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D017124:Conserved Sequence; D017931:DNA Primers; D018076:DNA, Complementary; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011755:Pyrophosphatases; D029701:Saccharomyces cerevisiae Proteins
D048868:Adaptor Proteins, Signal Transducing; D001426:Bacterial Proteins; D017931:DNA Primers; D018076:DNA, Complementary; D029968:Escherichia coli Proteins; D005656:Fungal Proteins; C118806:MTH1 protein, S cerevisiae; D008565:Membrane Proteins; D029701:Saccharomyces cerevisiae Proteins; D010744:Phosphoric Monoester Hydrolases; D011755:Pyrophosphatases; C052211:mutT protein, E coli