Characterization and overproduction of EcoO109I methyltransferase.
In order to characterize EcoO109I methyltransferase, a recombinant Escherichia coli clone that overproduces the enzyme was constructed. The coding region of M.EcoO109I was joined to the lac promoter of an expression vector, pUC118, and the resulting plasmid was introduced into E. coli HB101. M.EcoO109I was purified homogeneously from IPTG-induced cells, and was found to consist of a monomer subunit. M.EcoO109I uniquely methylates the inner deoxycytidylate residue in the sequence 5'-(A/G)GGNCC(C/T)-3' to produce 5-methylcytosine. The enzyme was most active at pH 8.0-8.5 and 50 degrees C. The enzyme activity was not affected by the addition of Mg2+ or EDTA.
Kita
K
K
Department of Biotechnology, Tottori University, Japan. kita@bio.tottori-u.ac.jp
Tsuda
J
J
Nishigaki
R
R
D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D019175:DNA Methylation; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D015252:Deoxyribonucleases, Type II Site-Specific; C045450:RGGNCCY-specific type II deoxyribonucleases