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Nucleic Acids Research, 2002, Vol. 30, No. 12 2628-2638
© 2002 Oxford University Press

Mutational analysis of conserved residues in HhaI DNA methyltransferase

Umesh T. Sankpal and Desirazu N. Rao*

Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India

HhaI DNA methyltransferase belongs to the C5-cytosine methyltransferase family, which is characterized by the presence of a set of highly conserved amino acids and motifs present in an invariant order. HhaI 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 Leu100). 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 HhaI DNA methyltransferase.

* To whom correspondence should be addressed. Tel: +91 80 309 2538; Fax: +91 80 360 0683; Email: dnrao{at}biochem.iisc.ernet.in


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