PMID:15381107
| Citation |
Kamiya, H, Iida, E and Harashima, H (2004) Important amino acids in the phosphohydrolase module of Escherichia coli Orf135. Biochem. Biophys. Res. Commun. 323:1063-8 |
|---|---|
| Abstract |
The Escherichia coli Orf135 protein, a MutT-type enzyme, hydrolyzes 2-hydroxy-dATP and 8-hydroxy-dGTP, in addition to dCTP and 5-methyl-dCTP, and its deficiency causes increases in both the spontaneous and H(2)O(2)-induced mutation frequencies. In this study, the Gly-36, Gly-37, Lys-38, Glu-43, Arg-51, Glu-52, Leu-53, Glu-55, and Glu-56 residues of Orf135, which are conserved in the three MutT-type proteins (Orf135, MutT, and MTH1), were substituted, and the enzymatic activity of these mutant proteins was examined. The mutant proteins with a substitution at the 36th, 37th, 52nd, and 56th amino acid residues completely lost their activity. On the other hand, the mutant proteins with a substitution at the 38th, 43rd, 51st, 53rd, and 55th residues could hydrolyze 5-methyl-dCTP. Some mutants with detectable activity for 5-methyl-dCTP did not hydrolyze dCTP. Activities for known substrates (5-methyl-dCTP, dCTP, 2-hydroxy-dATP, and 8-hydroxy-dGTP) were examined in detail with the four mutants, K38R, E43A, L53A, and E55Q. These results indicate the essential residues for the activity of the Orf135 protein. |
| Links |
PubMed Online version:10.1016/j.bbrc.2004.08.201 |
| Keywords |
Amino Acid Sequence; Amino Acids/chemistry; Amino Acids/metabolism; Deoxyribonucleotides/chemistry; Enzyme Activation; Escherichia coli/enzymology; Escherichia coli/genetics; Hydrolases/chemistry; Hydrolases/metabolism; Hydrolysis; Kinetics; Molecular Sequence Data; Mutagenesis, Site-Directed; Phosphorus Compounds/chemistry; Phosphorus Compounds/metabolism; Protein Engineering/methods; Pyrophosphatases/chemistry; Pyrophosphatases/metabolism; Recombinant Proteins/biosynthesis; Recombinant Proteins/chemistry; Structure-Activity Relationship; Substrate Specificity |
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