1887

Abstract

The gene encoding the LlaCI methyltransferase (M.LlaCI) from subsp. W15 was overexpressed in . The enzyme was purified to apparent homogeneity using three consecutive steps of chromatography on phosphocellulose, blue-agarose and Superose 12HR, yielding a protein of 31 300±1000 under denaturing conditions. The exact position of the start codon AUG was determined by protein microsequencing. This enzyme recognizes the specific palindromic sequence 5′-AAGCTT-3′. Purified M.LlaCI was characterized. Unlike many other methyltransferases, M.LlaCI exists in solution predominantly as a dimer. It modifies the first adenine residue at the 5′ end of the specific sequence to -methyladenine and thus is functionally identical to the corresponding methyltransferases of the HindIII ( Rd) and EcoVIII ( E1585-68) restriction–modification systems. This is reflected in the identity of M.LlaCI with M.HindIII and M.EcoVIII noted at the amino acid sequence level (50 % and 62 %, respectively) and in the presence of nine sequence motifs conserved among -adenine -class methyltransferases. However, polyclonal antibodies raised against M.EcoVIII cross-reacted with M.LlaCI but not with M.HindIII. Restriction endonucleases require Mg for phosphodiester bond cleavage. Mg was shown to be a strong inhibitor of the M.LlaCI enzyme and its isospecific homologues. This observation suggests that sensitivity of the M.LlaCI to Mg may strengthen the restriction activity of the cognate endonuclease in the bacterial cell. Other biological implications of this finding are also discussed.

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2003-11-01
2020-04-09
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