Significance of the conserved amino acid sequence for human MTH1 protein with antimutator activity.
8-Oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP) is produced during normal cellular metabolism, and incorporation into DNA causes transversion mutation. Organisms possess an enzyme, 8-oxo-dGTPase, which catalyzes the hydrolysis of 8-oxo-dGTP to the corresponding nucleoside monophosphate, thereby preventing the occurrence of mutation. There are highly conserved amino acid sequences in prokaryotic and eukaryotic proteins containing this and related enzyme activities. To elucidate the significance of the conserved sequence, amino acid substitutions were introduced by site- directed mutagenesis of the cloned cDNA for human 8-oxo-dGTPase, and the activity and stability of mutant forms of the enzyme were examined. When lysine-38 was replaced by other amino acids, all of the mutants isolated carried the 8-oxo-dGTPase-negative phenotype. 8-Oxo-dGTPase-positive revertants, isolated from one of the negative mutants, carried the codon for lysine. Using the same procedure, the analysis was extended to other residues within the conserved sequence. At the glutamic acid-43, arginine-51 and glutamic acid-52 sites, all the positive revertants isolated carried codons for amino acids identical to those of the wild type protein. We propose that Lys-38, Glu-43, Arg-51 and Glu-52 residues in the conserved region are essential to exert 8-oxo-dGTPase activity.
Cai
J P
JP
Department of Biochemistry, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-82, Japan.
Kawate
H
H
Ihara
K
K
Yakushiji
H
H
Nakabeppu
Y
Y
Tsuzuki
T
T
Sekiguchi
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001419:Bacteria / Q000201:enzymology; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D003062:Codon; D017124:Conserved Sequence; D017931:DNA Primers; D045643:DNA Repair Enzymes; D004926:Escherichia coli; D006801:Humans; D007700:Kinetics; D008239:Lysine; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D003062:Codon; D017931:DNA Primers; D011994:Recombinant Proteins; D010744:Phosphoric Monoester Hydrolases; C078207:8-oxodGTPase; D045643:DNA Repair Enzymes; D008239:Lysine