Characterization of the large subunit of EcoHK31I methyltransferase by structural modeling and mutagenesis.
M.EcoHK31I is a naturally occurring mC5-methyltransferase with a large alpha polypeptide and a small beta polypeptide. Polypeptide alpha contains conserved motifs I-VIII and X, and polypeptide beta contains motif IX. To understand how polypeptide alpha carries out its function, a molecular model of the large domain of polypeptide alpha was generated using M.HhaI and M.HaeIII as templates. The large domain is a mixed alpha/beta structure. Residues 15-19 in motif I (Phe-Naa-Gly-Naa) are conserved for cofactor binding. The key catalytic residue Cys-79 in motif IV is also conserved in comparison with other C-5 MTases. Comparing polypeptide alpha with M.HhaI and M.HaeIII revealed a unique region upstream of motif X. To understand the role of this region, 14 charged residues between R224 and E271 in the putative small domain were mutated. Activity assays indicated that most of these charges can be eliminated or changed conservatively. Among these charged residues, R224, E240, D245 and D251 may take part in proper interaction with DNA in the presence of polypeptide beta.
Mak
Amanda N-S
AN
Department of Biochemistry and Center for Protein Science and Crystallography, The Chinese University of Hong Kong, Shatin, Hong Kong, China.
Fung
Wai-To
WT
Kong
Kathy P-S
KP
Poon
Alice W-S
AW
Ngai
Sai-Ming
SM
Shaw
Pang-Chui
PC
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D015257:DNA-Cytosine Methylases / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D008780:Methyltransferases / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010455:Peptides / Q000737:chemistry / Q000235:genetics; D011487:Protein Conformation; D016415:Sequence Alignment
D029968:Escherichia coli Proteins; D010455:Peptides; C092246:DNA modification methylase EcoHK31I; D015257:DNA-Cytosine Methylases; D008780:Methyltransferases