The active site of the Escherichia coli MutY DNA adenine glycosylase

Escherichia coli MutY is an adenine DNA glycosylase active on DNA substrate s containing A/G, AIC, or A/8-oxoG mismatches. Although MutY can form a cov alent intermediate with its DNA substrates, its possession of 3' apurinic l yase activity is controversial. To study the reaction mechanism of MutY, th e conserved Asp-138 was mutated to Asn and the reactivity of this mutant Mu tY protein determined. The glycosylase activity was completely abolished in the D138N MutY mutant. The D138N mutant and wild-type MutY protein also po ssessed different DNA binding activities with various mismatches. Several l ysine residues were identified in the proximity of the active site by analy zing the imino covalent MutY-DNA intermediate. Mutation of Lys-157 and Lys- 158 both individually and combined, had no effect on MutY activities but th e K142A mutant protein was unable to form Schiff base intermediates with DN A substrates. However, the MutY K142A mutant could still bind DNA substrate s and had adenine glycosylase activity. Surprisingly, the K142A mutant MutY , but not the wild-type enzyme, could promote a beta/delta-elimination on a purinic DNA. Our results suggest that Asp-138 acts as a general base to dep rotonate either the epsilon-amine group of Lys-142 or to activate a water m olecule and the resulting apurinic DNA then reacts with Lys-142 to form the Schiff base intermediate with DNA. With the H142A mutant, Asp-138 activate s a water molecule to attack the C1' of the adenosine; the resulting apurin ic DNA is cleaved through beta/delta-elimination without Schiff' base forma tion.