M.EcoHK31I is a naturally occurring mC5-methyltrans-ferase with a large a polypeptide and a small β polypeptide. Polypeptide a contains conserved motifs I-VIII and X, and polypeptide β contains motif IX. To understand how polypeptide a carries out its function, a molecular model of the large domain of polypeptide a was generated using M.HhaI and M.HaeIII as templates. The large domain is a mixed α/β 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 a 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 β.