Interaction of the Tylosin-resistance Methyltransferase RlmAII at its rRNA Target Differs from the Orthologue RlmAI

RlmAII methylates the N1-position of nucleotide G748 in hairpin 35 of 23 S rRNA. The resultant methyl group extends into the peptide channel of the 50 S ribosomal subunit and confers resistance to tylosin and other mycinosylated macrolide antibiotics. Methylation at G748 occurs in several groups of Gram-positive bacteria, including the tylosin-producer Streptomyces fradiae and the pathogen Streptococcus pneumoniae. Recombinant S. pneumoniae RlmAII was purified and shown to retain its activity and specificity in vitro when tested on unmethylated 23 S rRNA substrates. RlmAII makes multiple footprint contacts with nucleotides in stem–loops 33, 34 and 35, and does not interact elsewhere in the rRNA. Binding of RlmAII to the rRNA is dependent on the cofactor S-adenosylmethionine (or S-adenosylhomocysteine). RlmAII interacts with the same rRNA region as the orthologous enzyme RlmAI that methylates at nucleotide G745. Differences in nucleotide contacts within hairpin 35 indicate how the two methyltransferases recognize their distinct targets.