Mutational analysis of conserved residues in HhaI DNA methyltransferase.
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.
Sankpal
Umesh T
UT
Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India.
Rao
Desirazu N
DN
D001665:Binding Sites; D002384:Catalysis; D017124:Conserved Sequence; D004247:DNA / Q000378:metabolism; D004252:DNA Mutational Analysis; D015257:DNA-Cytosine Methylases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004795:Enzyme Stability; D007700:Kinetics; D008745:Methylation; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D012436:S-Adenosylmethionine / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D012436:S-Adenosylmethionine; D004247:DNA; C037664:DNA modification methylase HhaI; D015257:DNA-Cytosine Methylases