Biochemical characterization and mutational analysis of a mismatch glycosylase from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5.

Mismatch glycosylase (MIG) can excise thymine and uracil from mutagenic T:G and U:G mispairs, which arise from cytosine and 5-methylcytosine deamination, respectively. Here, we present evidence that a thermostable MIG from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 (Tb-MIG) can remove thymine and uracil from T:G and U:G mispairs at high temperature, albeit at a low efficiency for U:G mispair. The enzyme displays maximum efficiency at 70 C - 75 °C and pH 7.0-8.0. Tb-MIG is extremely thermostable, retaining 50% activity after heating at 100 C for 2 hrs. In addition, Tb-MIG is a bifunctional glycosylase with an AP lyase activity, then resembles the MIG from the hyperthermopilic crenarchaeon Pyrobaculum aerophilium, but contrasts with the MIG from the hyperthermopilic crenarchaeon Aeropyrum pernix. Importantly, we show that residues Y133 and D151 in Tb-MIG are essential for thymine removal, and that residues A58, N153 and R156 are involved in thymine removal. Compared with the wild-type protein, the A58D and Y133K mutants display the increased AP lyase activity, confirming the essential roles played by the correspondingly conserved Asp and Lys in endonuclease III for AP site cleavage. Overall, our work is the first biochemical characterization of a hypthermophilic euryarchaeal MIG, augmenting our understanding on archaeal MIG function.