Kamiya Hiroyuki, Iida Emiko, Harashima Hideyoshi
Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan.
Biochem Biophys Res Commun. 2004 Oct 22;323(3):1063-8. doi: 10.1016/j.bbrc.2004.08.201.
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.
大肠杆菌的Orf135蛋白是一种MutT型酶，除了能水解dCTP和5-甲基-dCTP外，还能水解2-羟基-dATP和8-羟基-dGTP，其缺失会导致自发突变频率和H₂O₂诱导的突变频率均增加。在本研究中，对Orf135中在三种MutT型蛋白（Orf135、MutT和MTH1）中保守的Gly-36、Gly-37、Lys-38、Glu-43、Arg-51、Glu-52、Leu-53、Glu-55和Glu-56残基进行了替换，并检测了这些突变蛋白的酶活性。在第36、37、52和56位氨基酸残基处有替换的突变蛋白完全丧失了活性。另一方面，在第38、43、51、53和55位残基处有替换的突变蛋白能够水解5-甲基-dCTP。一些对5-甲基-dCTP有可检测活性的突变体不能水解dCTP。用四个突变体K38R、E43A、L53A和E55Q详细检测了对已知底物（5-甲基-dCTP、dCTP、2-羟基-dATP和8-羟基-dGTP）的活性。这些结果表明了Orf135蛋白活性所必需的残基。
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