Mutational analysis of conserved residues in HhaI DNA methyltransferase

Hhal DNA methyltransferase belongs to the G5-cytosine methyltransferase family, which is characterized by the presence of a set of highly conserved amino acids and motifs present In an Invariant order. Hhal DNA methyltransferase has been subjected to a lot of biochemical and crystallographic studies. A number of issues, especially the role of the conserved amino acids In the methyltransferase activity, have not been addressed. Using sequence comparison and structural data, a structure-guided mutagenesis approach was undertaken, to assess the role of conserved amino acids in catalysis. Site-directed mutagenesis was performed on amino acids Involved in cofactor S-adenosyl-L-methionine (AdoMet) binding (Phe18, Trp41, Asp60 and Leu-100). Characterization of these mutants, by in vitro \in vivo restriction assays and DNA/AdoMet binding studies, Indicated that most of the residues present In the AdoMet-binding pocket were not absolutely essential. This study Implies plasticity In the recognition of cofactor by Hhal DNA methyltransferase.