The Escherichia coli Orf135 protein, a Muff-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 H2O2-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, Muff, 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. (C) 2004 Elsevier Inc. All rights reserved.